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Molecular Characterization of Hereditary Spherocytosis Mutants of the Cytoplasmic Domain of Anion Exchanger 1 and their Interaction with Protein 4.2 by Susan Pilar Bustos A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Biochemistry University of Toronto © Copyright by Susan Pilar Bustos 2011 Year of Convocation

Molecular Characterization of Hereditary Spherocytosis ... · ii Molecular Characterization of Hereditary Spherocytosis Mutants of the Cytoplasmic Domain of Anion Exchanger 1 and

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Page 1: Molecular Characterization of Hereditary Spherocytosis ... · ii Molecular Characterization of Hereditary Spherocytosis Mutants of the Cytoplasmic Domain of Anion Exchanger 1 and

Molecular Characterization of Hereditary Spherocytosis Mutants of the Cytoplasmic Domain of Anion Exchanger 1

and their Interaction with Protein 4.2

by

Susan Pilar Bustos

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy

Graduate Department of Biochemistry University of Toronto

© Copyright by Susan Pilar Bustos 2011 Year of Convocation

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Molecular Characterization of Hereditary Spherocytosis Mutants of the

Cytoplasmic Domain of Anion Exchanger 1 and their Interaction with

Protein 4.2

Susan Pilar Bustos

Doctor of Philosophy

Graduate Department of Biochemistry

University of Toronto

2011

Abstract

Anion exchanger 1 (AE1) is a red cell membrane glycoprotein that associates with cytoskeletal

protein 4.2 in a complex bridging the cell membrane to the cytoskeleton. Disruption of this

linkage results in unstable erythrocytes and hereditary spherocytosis (HS). Three HS mutations

(E40K, G130R and P327R) in the cytoplasmic domain of AE1 (cdAE1) result in a decreased

level of protein 4.2 in the red cell yet maintain normal amounts of AE1. Biophysical analyses

showed the HS mutations had little effect on the structure and conformational stability of the

isolated domain. However, the conformation of the cytoplasmic domain of the kidney anion

exchanger, lacking the first 65 amino acids including a central -strand, was thermally

destabilized relative to cdAE1 and had a more open structure. In transfected human embryonic

kidney (HEK)-293 cells the HS mutants had similar expression levels as wild-type AE1, and

protein 4.2 expression level was not dependent on the presence of AE1. Protein 4.2 localized to

the plasma membrane with wild-type AE1, the HS mutants of AE1, the membrane domain of

AE1 and kidney AE1, and to the ER with Southeast Asian ovalocytosis AE1. A fatty acylation

mutant of protein 4.2, G2A/C173A, could not localize to the plasma membrane in the absence of

AE1. Subcellular fractionation showed wild-type and G2A/C173A protein 4.2 were mostly

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associated with the cytoskeleton. Co-immunoprecipitation and Ni-NTA pull-down assays

revealed impaired binding of protein 4.2 to HS mutants compared to AE1, while the membrane

domain of AE1 was unable to bind protein 4.2. These studies show that HS mutations in cdAE1

cause impaired binding of protein 4.2, without causing gross structural changes in the domain.

The mutations change the binding surface on cdAE1 by the introduction of positive charges into

an otherwise acidic domain. This binding impairment may render protein 4.2 more susceptible to

degradation or loss during red cell development.

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Acknowledgements

I would like to thank Jing Li for her assistance in cDNA vector construction and in performing

replicates of some of the experiments in Chapter 3. I thank Dr. Walid Houry for the use of his

spectropolarimeter and fluorescence spectrophotometer and Dr. David Clarke for the use of his

differential scanning calorimeter. I would like to thank Dr. Avi Chakrabartty and Sylvia Ho for

the use of the analytical ultracentrifuge and collection of data. I would also like to thank Dr.

Lewis Kay and Dr. Ranjith Muhandiram for the use of the NMR facility and collection of NMR

spectra. I would like to thank Dr. Michael L. Jennings from the University of Arkansas for

Medical Sciences for the mouse anti-AE1 antibody. I would like to thank the Canadian Institutes

of Health Research for supporting this research in the form of a grant, and the Canadian Blood

Services for their support in the form of a Graduate Student Fellowship.

Lastly, and certainly not least, I would like to emphatically thank my supervisor, Dr.

Reinhart Reithmeier. As a supervisor, your encouragement and guidance during the research

lows were as crucial for my success as your praise and kudos during the research highs, which

were always marked with a cake or a celebratory lunch. As a mentor, your quiet confidence,

determination and integrity are things to which I aspire. You’ve sent me around the world to

present my work as a scientist, yet you’ve offered me unconditional support in the

unconventional path I have chosen to take with this degree. Your enthusiasm for your own work

has been an inspiration for my search for meaningful work I can be passionate about. Thank you,

Reinhart, for making my time in your lab a fun, challenging and rewarding experience. I leave

with fond memories of these last several years and anticipation for what comes next.

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Dedications

This degree is dedicated to my husband, Jim, and to my daughter, Sidney. Thank you, Jim, for

giving me the time and space to study all night when I needed to, and letting me sleep in the next

day. Thanks for all your understanding during the stressful times and for making sure I

celebrated all the milestones. You’ve been my biggest cheerleader all the way through, and I

share this success with you. Thank you, Sidney, for bringing me into your world every day

through your wild imagination and joyful, playful nature. Playing with you was, and continues to

be, the highlight of my day. Thank you for waiting so patiently while I went to the lab, or while I

studied in my ‘office’. Knowing I’d see your smiling face after I finished my work drove me to

work faster, and hoping to be a good model for you drove me to work better.

This work is also dedicated to my parents, Luis and Amelia, and to my mother-in-law,

Nuala. Thank you, Mom and Dad (Yaya and Dado), for always supporting whatever it was that I

wanted to study or pursue, finding ways to make it happen, yet letting me navigate my own

course. Thanks for your optimism, and for believing in me, even when I felt like an ‘imposter’.

Muchos besos y abrazos con ruido. Nuala (Nana), in supporting my whole, little family, you’ve

made it possible for me to get through some challenging times, both academically and

emotionally. Your dedication to us has helped us stay balanced and strong. Thanks for playing

such a crucial role in this journey.

I also dedicate this work to all my other cheerleaders; my siblings and siblings-in-law,

extended family (Bustos, Munroe, Calzato and Fell), and all of my awesome friends. Even when

you weren’t sure what I was doing, you were still there shaking the pom-poms and

congratulating me at the finish line.

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Table of contents

Abstract ........................................................................................................................................... ii

Acknowledgements ........................................................................................................................ iv Dedications ......................................................................................................................................v

Table of contents ............................................................................................................................ vi

List of tables ................................................................................................................................... ix List of figures ...................................................................................................................................x

List of appendices .......................................................................................................................... xi

List of abbreviations ..................................................................................................................... xii 1 Chapter 1: Introduction ...............................................................................................................1

1.1 Preamble ..............................................................................................................................1 1.2 Hereditary spherocytosis (HS) .............................................................................................2

1.2.1 Background ..............................................................................................................2 1.2.2 Red cell membrane and cytoskeleton ......................................................................5

1.2.3 Molecular basis of HS ..............................................................................................7 1.3 Anion exchanger 1 (AE1) ..................................................................................................10

1.3.1 Anion exchanger gene family ................................................................................10

1.3.2 AE1 in various species ...........................................................................................14

1.3.3 Structure and function ............................................................................................14 1.3.4 Oligomeric state .....................................................................................................15 1.3.5 Kidney AE1 (kAE1) ..............................................................................................15

1.3.6 Distal renal tubular acidosis (dRTA) .....................................................................16 1.3.7 Southeast Asian ovalocytosis AE1 (AE1SAO) .....................................................17

1.3.8 AE1 knock-outs .....................................................................................................18 1.3.9 HS mutations in AE1 .............................................................................................19

1.4 Membrane domain of AE1 (mdAE1) ................................................................................20

1.4.1 Physiological function ...........................................................................................20 1.4.2 Topology ................................................................................................................24

1.5 Cytoplasmic domain of AE1 (cdAE1) ...............................................................................27 1.5.1 Structure .................................................................................................................27 1.5.2 Physiological function ...........................................................................................30 1.5.3 HS mutations in cdAE1 affecting levels of protein 4.2 .........................................31

1.6 Protein 4.2 ..........................................................................................................................33 1.6.1 Background ............................................................................................................33 1.6.2 Transglutaminase family of enzymes ....................................................................33 1.6.3 Protein 4.2 isoforms ...............................................................................................36 1.6.4 Tissue distribution and expression in different species .........................................37

1.6.5 Structure and properties .........................................................................................38

1.6.6 Interactions with AE1, ankyrin and spectrin..........................................................40

1.6.7 Interactions with the Rh complex and CD47 .........................................................43 1.6.8 HS mutations in protein 4.2 ...................................................................................47

1.7 Thesis focus .......................................................................................................................49 1.7.1 Effect of HS mutations on the structure and stability of the cytoplasmic

domain of AE1 (Chapter 2) ....................................................................................49

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1.7.2 Protein 4.2 localization and interaction with wild-type and HS mutants of AE1

in HEK-293 cells (Chapter 3) ................................................................................49 2 Chapter 2: Structure and stability of hereditary spherocytosis mutants of the cytoplasmic

domain of the erythrocyte anion exchanger 1 protein ...............................................................51

2.1 Abstract ..............................................................................................................................51 2.2 Introduction ........................................................................................................................51 2.3 Materials and methods .......................................................................................................52

2.3.1 Materials ................................................................................................................52 2.3.2 Plasmid construction and mutagenesis ..................................................................53

2.3.3 Protein expression and purification .......................................................................53 2.3.4 Analytical ultracentrifugation ................................................................................54

2.3.5 Circular dichroism .................................................................................................54 2.3.6 pH dependence of intrinsic fluorescence ...............................................................55 2.3.7 Calorimetry ............................................................................................................55 2.3.8 Urea denaturation measured by intrinsic fluorescence ..........................................55 2.3.9 Limited tryptic digestion ........................................................................................56

2.4 Results ................................................................................................................................56

2.4.1 Expression and purification of cdAE1 and cdAE1 HS variants in Escherichia

coli..........................................................................................................................56 2.4.2 Analytical ultracentrifugation of wild-type and HS mutant cdAE1 proteins ........56

2.4.3 Secondary structure analysis of wild-type and HS mutant cdAE1 proteins ..........57 2.4.4 Effect of pH on the intrinsic fluorescence of the wild-type and HS mutant

cdAE1 proteins.......................................................................................................60 2.4.5 Thermal denaturation of wild-type and HS mutant cdAE1 proteins by circular

dichroism................................................................................................................60 2.4.6 Thermal denaturation of wild-type and HS mutant cdAE1 proteins by

calorimetry .............................................................................................................63 2.4.7 Urea denaturation of wild-type and HS mutant cdAE1 proteins ...........................66 2.4.8 Limited tryptic digestion of WT and HS mutant cdAE1 proteins .........................66

2.5 Discussion ..........................................................................................................................67 3 Chapter 3: Protein 4.2 interaction with hereditary spherocytosis mutants of the

cytoplasmic domain of human anion exchanger 1 ....................................................................72

3.1 Abstract ..............................................................................................................................72 3.2 Introduction ........................................................................................................................73 3.3 Materials and methods .......................................................................................................73

3.3.1 Materials ................................................................................................................73 3.3.2 Site-directed mutagenesis ......................................................................................74 3.3.3 Transient transfection and expression of AE1 and protein 4.2 in HEK-293

cells ........................................................................................................................75 3.3.4 SDS-PAGE and immunoblotting ...........................................................................75

3.3.5 Immunofluorescence and confocal microscopy.....................................................75 3.3.6 Ni-NTA pull-down.................................................................................................76

3.3.7 Co-immunoprecipitation ........................................................................................76 3.3.8 Subcellular fractionation of HEK-293 cells expressing wild-type or

G2A/C173A protein 4.2 .........................................................................................77 3.4 Results ................................................................................................................................78

3.4.1 Expression of protein 4.2 and AE1 proteins in transfected HEK-293 cells ...........78

3.4.2 Localization of AE1 proteins in HEK-293 cells ....................................................78 3.4.3 Co-localization of protein 4.2 and AE1 in HEK-293 cells ....................................80

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3.4.4 Interaction of protein 4.2 with AE1 proteins in HEK-293 cells ............................83

3.4.5 Co-localization of wild-type and G2A/C173A protein 4.2 and AE1 in HEK-

293 cells .................................................................................................................86 3.4.6 Co-localization of wild-type and G2A/C173A protein 4.2 and cell surface

glycans in the absence or presence of AE1 in HEK-293 cells ...............................88 3.4.7 Subcellular fractionation of HEK-293 cells expressing wild-type or

G2A/C173A protein 4.2 .........................................................................................88 3.5 Discussion ..........................................................................................................................90

4 Discussion and future directions ...............................................................................................95

4.1 Structure and conformational stability of the cytoplasmic domain of AE1.......................98 4.2 Protein 4.2 interaction with HS mutants of cdAE1..........................................................100

4.2.1 Quantitative in vitro binding analysis ..................................................................105 4.2.2 Structure determination ........................................................................................106 4.2.3 Interaction of protein 4.2 and AE1 during red cell development ........................108

4.3 Conclusions ......................................................................................................................108 References ....................................................................................................................................111

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List of tables

Table 1.1 SLC4 bicarbonate transporter family ..............................................................................11 Table 1.2 HS mutations in the AE1 protein ....................................................................................21 Table 1.3 HS mutations in protein 4.2 ............................................................................................48

Table 2.1 Effects of HS mutations on structure and stability of cdAE1 protein .............................62 Table 4.1 Mutant protein DNA constructs ..........................................................................................95

Table A1 Summary of biophysical properties of cdAE1, cdkAE1, and cdΔ54AE1 at pH 7.5 .....142

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List of figures

Figure 1.1 Healthy red blood cells and spherocytes ........................................................................3 Figure 1.2 Red cell membrane proteins separated by SDS-PAGE ..................................................4 Figure 1.3 Schematic model of the red cell membrane ...................................................................6 Figure 1.4 Diagrams of proteins expressed by the genes encoding human AE1, AE2 and AE3 ..12 Figure 1.5 Map of the human AE1 gene ........................................................................................13 Figure 1.6 Topographical model of human erythroid AE1 ............................................................26 Figure 1.7 Crystal structure of the cytoplasmic domain of AE1 (cdAE1) .....................................28 Figure 1.8 Crystal structure of the cytoplasmic domain of AE1 (cdAE1) and homology model

of protein 4.2 .............................................................................................................................41 Figure 1.9 Diagram of the protein 4.2 polypeptide with important regions mapped.....................44 Figure 1.10 Schematic model of AE1 and Rh protein complexes at the red cell membrane ........46 Figure 2.1 Analytical ultracentrifugation of wild-type cdAE1 ......................................................58 Figure 2.2 CD spectra of wild-type cdAE1 and HS mutants .........................................................59 Figure 2.3 Intrinsic fluorescence intensity of wild-type cdAE1 and HS mutants as a function

of pH ..........................................................................................................................................61 Figure 2.4 Thermal denaturation of wild-type cdAE1 and HS mutants monitored by CD ...........64 Figure 2.5 Thermal denaturation of wild-type cdAE1 and HS mutants by DSC ..........................65 Figure 2.6 Urea denaturation of wild-type and HS mutant cdAE1 proteins ..................................68 Figure 2.7 Trypsin digestion of wild-type and HS mutant cdAE1 proteins ..................................69 Figure 3.1 Expression of AE1 proteins and protein 4.2 in HEK-293 cells ....................................79 Figure 3.2 Immunofluorescence images of wild-type and mutant AE1 in HEK-293 cells ...........81 Figure 3.3 Immunofluorescence images of protein 4.2 and AE1 proteins in HEK-293 cells .......82

Figure 3.4 Ni-NTA pull-down of wild-type and mutant His6-tagged AE1 with protein 4.2 in

HEK-293 cells ...........................................................................................................................84 Figure 3.5 Co-immunoprecipitation (co-ip) of wild-type and mutant AE1 with protein 4.2 in

HEK-293 cells ...........................................................................................................................85 Figure 3.6 Immunofluorescence images of wild-type and G2A/C173A protein 4.2 and AE1

proteins in HEK-293 cells .........................................................................................................87 Figure 3.7 Immunofluorescence images of cell surface glycans using PNA and wild-type and

G2A/C173A protein 4.2 expressed in the absence or presence of AE1 in HEK-293 cells .......89 Figure 3.8 Subcellular fractionation of HEK-293 cells expressing wild-type or G2A/C173A

protein 4.2 ..................................................................................................................................91 Figure A1 Crystal structure of human cdAE1 .............................................................................135 Figure A2 Domain structure of AE1 isoforms and gel-separated AE1 constructs ......................136 Figure A3 Analytical ultracentrifugation of cdAE1 ....................................................................142 Figure A4 CD spectra of purified cdAE1, cdkAE1, and cdΔ54AE1 at pH 7.5 ...........................143 Figure A5 Thermal denaturation of purified cdAE1, cdkAE1, and cdΔ54AE1 by DSC at pH

7.5 ............................................................................................................................................145 Figure A6 Intrinsic tryptophan fluorescence emission spectra of (A) cdAE1, (B) cdkAE1, and

(C) cdΔ54AE1 at various pH values .......................................................................................147 Figure A7 Average emission wavelength of purified cdAE1, cdkAE1, and cdΔ54AE1 ............148 Figure A8 Urea denaturation of cdAE1 and cdkAE1 monitored by intrinsic tryptophan

fluorescence .............................................................................................................................149

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List of appendices

Appendix ......................................................................................................................................133

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List of abbreviations

2,3-DPG 2,3-diphosphoglycerate

3D three-dimensional

AE anion exchanger

AE1 anion exchanger 1

AE1SAO Southeast Asian ovalocytosis AE1

AE1HS HS mutants of AE1

AE2 anion exchanger 2

AE3 anion exchanger 3

AE4 anion exchanger 4

βME β-mercaptoethanol

BSA bovine serum albumin

BSSS bis(sulfosuccinimidyl)-suberate

CAII carbonic anhydrase II

CD circular dichroism

cdAE1 cytoplasmic domain of AE1

cdΔ54AE1 cytoplasmic domain of AE1 missing the first 54 amino acids

cdkAE1 cytoplasmic domain of kidney AE1

Cm apparent midpoint of the urea unfolding transition

CNX calnexin

co-ip co-immunoprecipitation

Cp excess heat capacity

CtAE1 carboxy-terminal AE1

C-terminal carboxy-terminal

C-terminus carboxy-terminus

DEER double electron-electron resonance

DMEM Dulbecco's modified Eagle's medium

DTT dithiothreitol

dRTA distal renal tubular acidosis

DSC differential scanning calorimeter/calorimetry

EE R34E/R35E protein 4.2

EPR electron paramagnetic resonance

ER endoplasmic reticulum

ESI-TOF electrospray ionization time-of-flight

GAPDH glyceraldehyde 3-phosphate dehydrogenase

GC G2A/C173A protein 4.2 mutant

GPA glycophorin A

GPB glycophorin B

GPC glycophorin C

GST glutathione S-transferase

H2DIDS 4,4'-diisothiocyanodihydrostilbene-2,2'-disulphonate

HA hemagglutinin

HEK-293 human embryonic kidney-293

HPLC high-performance liquid chromatography

HS hereditary spherocytosis

IOVs inside-out vesicles

IPTG isopropyl-β-D-thiogalactoside

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ITC isothermal calorimetry

kAE1 kidney AE1

LB Luria Bertani

LW intracellular adherence molecule 4

mdAE1 membrane domain of AE1

MWapp apparent molecular weight

MWseq sequence molecular weight

NBC Na+-coupled HCO3

- transporter

Ni-NTA nickel-nitrilotriacetic acid

NMR nuclear magnetic resonance

NMT N-myristoyl transferase

N-terminal amino-terminal

N-terminus amino-terminus

Ni-NTA nickel-nitrilotriacetic acid

PAT palmitoyl-acyl transferase

PBS phosphate buffered saline

PLC phospholipase C

PMSF phenylmethanesulfonyl fluoride

PNA peanut agglutinin

Rh Rhesus

RhAG Rh-associated glycoprotein

SAO Southeast Asian ovalocytosis

SCAM scanning cysteine accessibility mutagenesis

S.D. standard deviation

SDSL site-directed spin labeling

SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SE sedimentation equilibrium

SLC4 solute carrier 4

SV sedimentation velocity

tAE1 trout AE1

TG transglutaminase

Tm apparent midpoint of the thermal denaturation transition

TM transmembrane

Wrb Wright b

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1 Chapter 1: Introduction

1.1 Preamble

Protein-protein interactions and protein networks are fundamentally important in maintaining the

structural integrity and functionality of cells. The red blood cell is essentially a sac of

hemoglobin whose primary function is to deliver oxygen from the lungs to tissues and remove

carbon dioxide, a metabolic waste product, from the tissues to the lungs. The strength and

flexibility of the red cell allows it to travel through the circulatory system, including capillaries

half its diameter, without rupturing for its 120 day lifespan. The proteins that link the red cell

membrane to its cytoskeleton are responsible for these dynamic properties. Integral membrane

glycoprotein anion exchanger 1 (AE1) mediates the electroneutral exchange of chloride for

bicarbonate across the plasma membrane, while its N-terminal cytoplasmic domain (cdAE1)

binds a number of red cell proteins including ankyrin, a cytoskeletal protein. Ankyrin in turn,

binds to spectrin which, along with actin, creates the scaffolding of the red cell cytoskeleton.

Protein 4.2 binds to both AE1 and ankyrin, strengthening their interaction. Perturbation of this

membrane-cytoskeleton linkage caused by mutations in any of these four proteins (AE1, ankyrin,

spectrin and protein 4.2) can lead to membrane destabilization and hereditary spherocytosis

(HS), a hemolytic anemia.

While HS mutations in the membrane domain of AE1 typically cause misfolding of AE1 and

ER retention of the protein, three HS mutations in the cytoplasmic domain (E40K, G130R, and

P327R) are associated with a deficiency of protein 4.2 in the red cell, while maintaining normal

levels of AE1 at the membrane. The focus of this thesis is to characterize the effect of these three

HS mutations on the structure and conformational stability of cdAE1, and on the interaction of

AE1 with protein 4.2 in transfected cells. In a related study, the characterization of the

cytoplasmic domain of kidney AE1 (cdkAE1), a truncated AE1 isoform missing residues 1-65, is

included in the Appendix.

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1.2 Hereditary spherocytosis (HS)

1.2.1 Background

HS is a common hemolytic anemia and the most common human inherited red cell membrane

disorder. It occurs in about 1 to 2000 Caucasians, but is also common in the Japanese population

(Delaunay 2007). HS is characterized by osmotically fragile spherocytes (spherically-shaped

erythrocytes) that are selectively trapped and destroyed in the spleen (Iolascon et al. 1998). The

accumulation of defective erythrocytes causes enlargement of the spleen, or splenomegaly, and

the removal of the cells from circulation leads to anemia. The clinical severity of the disease

ranges from mild (asymptomatic) compensated hemolysis, where erythrocyte production and

destruction are balanced (Perrotta et al. 2008), to severe hemolytic anemia requiring frequent

blood transfusions and splenectomy. The variation in severity is due to the different molecular

defects that lead to HS and to different levels of bone marrow compensation.

HS symptoms can appear as early as one to two weeks following birth (Delaunay 2007). An

increase in hyperdense red cells with higher hemoglobin concentration is found. The osmotic

fragility occurs partly because of the spheroid shape of the cells, which are less deformable than

the normal discoid shape. HS has been described as resulting from defects of proteins connecting

the red cell membrane to the cytoskeleton (Eber and Lux 2004). Due to the weakened

interactions between the membrane and cytoskeleton, fragments of the membrane bleb off, the

surface area-to-volume ratio of the cell decreases, and the cell becomes spherical and unstable.

Blood smears from HS patients show variable numbers of spherocytes, which lack the central

pale area that is seen in healthy red cells. The images in Figure 1.1 show a comparison between

healthy red cells and spherocytes from cattle, but the effect is the same in humans.

When the red cell membrane proteins of HS patients are subjected to sodium dodecyl sulfate-

polyacrylamide gel electrophoresis (SDS-PAGE), the responsible protein can often be detected

by a decrease in its intensity on the gel. However, the deficiency observed for a certain protein

can also be caused indirectly by a defect in another protein. Figure 1.2 shows red cell membrane

proteins separated by SDS-PAGE following centrifugation of red cell ghosts. The numbering of

proteins based on their order on the gel and their common names are shown to the left.

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Figure 1.1: Healthy red blood cells and spherocytes.

Electron micrographs of wild-type bovine erythrocytes on the left and HS bovine spherocytes on

the right. Modified from Inaba et al. (1996).

HS

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Figure 1.2: Red cell membrane proteins separated by SDS-PAGE.

Red cell ghosts incubated in 5 mM sodium phosphate buffer, pH 8, were centrifuged, solubilized

with SDS sample buffer, run on SDS-PAGE and stained with Coomassie blue. Modified from

Steck (1974).

AE1, Band 3

Protein 4.2

GAPDH

Ankyrin

α-spectrin

β-spectrin

Actin

Protein 4.1

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1.2.2 Red cell membrane and cytoskeleton

When speaking of the red cell membrane, included are the bilayer surrounding the cell,

transmembrane proteins within the bilayer, and the underlying cytoskeleton (Tse and Lux 1999).

The interaction between proteins of the cytoskeleton and integral membrane proteins gives the

red cell its strength and flexibility. These components are shown in a diagram of the red cell

membrane in Figure 1.3. The cytoskeleton is a mesh-like network made up primarily of the

proteins spectrin and actin (Ursitti et al. 1991). Monomers of α-spectrin and β-spectrin intertwine

to form heterodimers (Bennett 1985). These dimers connect head-to-head to form long spectrin

heterotetramers. There are two main points of contact between the cytoskeleton and the

membrane. At one point of contact the tail end of the spectrin tetramer interacts with a junctional

complex, consisting of short actin filaments, tropomyosin, tropomodulin and adducin (Tse and

Lux 1999). Protein 4.1 interacts with the N-terminus of β-spectrin at the actin-binding domain in

the junctional complex and with integral membrane protein glycophorin C (GPC), thereby

linking the cytoskeleton to the membrane. Protein 4.1 also enhances spectrin-actin binding.

At the other major point of contact, the C-terminus of β-spectrin binds to ankyrin, which

binds to the cytoplasmic domain of the integral membrane protein anion exchanger 1 (AE1), or

Band 3. Protein 4.2 stabilizes the interaction between AE1 and ankyrin (Rybicki et al. 1996).

Only the tetrameric form of AE1 binds to ankyrin (Van Dort et al. 1998). Dimeric AE1 is found

associated with the protein 4.1-GPC junctional complex, or as a freely diffusing complex (van

den Akker et al. 2010b). These membrane-cytoskeletal interactions give the red cell the strength

and flexibility that allows it to travel through the circulatory system without rupturing for its 120

day lifespan. In a study using human red blood cell precursors (bone marrow erythroblasts) AE1,

spectrin and ankyrin were found to be expressed at the same time during erythropoiesis (Nehls et

al. 1993). The proteins appeared at the proerythroblast stage, which is the earliest erythroblast

stage. Another study using human erythroid precursors from peripheral blood showed the early

expression of spectrin followed by AE1. Protein 4.2 was not observed until the late erythroblast

stage (orthochromatic) even though protein 4.2 mRNA was seen in the early erythroblasts (Wada

et al. 1999). However, a more recent study of human erythroblast differentiation showed that

AE1 and protein 4.2 are not only expressed at the same time in basophilic erythroblasts, but they

also interact as soon as they appear (van den Akker et al. 2010a).

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Figure 1.3: Schematic model of the red cell membrane.

The interactions of the integral membrane proteins and cytoskeletal proteins are shown.

Modified from Tse and Lux (1999).

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1.2.3 Molecular basis of HS

HS is caused by defects in erythrocyte proteins that are involved in the major interaction between

the erythrocyte membrane and the cytoskeleton: spectrin, ankyrin, protein 4.2 and AE1.

Approximately 45 % of HS cases are due to mutations in ankyrin and about 30 % are due to

mutations in β-spectrin (Tse and Lux 1999). Mutations in AE1 account for about 20 % of HS

cases, while approximately 5 % are due to protein 4.2 mutations.

Most of the HS ankyrin mutations are nonsense or frameshift mutations that result in unstable

mRNA transcripts leading to ankyrin deficiencies or truncated peptides causing binding defects.

Several of the latter are truncated at the AE1-binding domain of ankyrin and others are affected

at the spectrin-binding domain (Tse and Lux 1999). The inability of ankyrin to properly bind to

either AE1 or spectrin would disrupt the important linkage between the membrane and

cytoskeleton. Ankyrin defects have been found in both dominant and recessive HS and the

clinical picture ranges from mild hemolysis to transfusion-dependent anemia. Defects lead to

deficiencies in ankyrin, as seen by SDS-PAGE, as well as secondary deficiencies in spectrin and

protein 4.2 (Delaunay 2007). Ankyrin defects can also have a greater effect on the levels of

interacting proteins than on ankyrin itself. Ankyrin from an HS patient with a missense mutation

in the AE1-binding domain (V463I) has decreased affinity for AE1 (Eber et al. 1996) and this

mutant results in a higher deficiency of AE1 than of ankyrin. Even though there is no AE1

molecular defect, the levels of AE1 are affected more than those of the defective ankyrin. A

group recently studied protein sorting in enucleating erythroblasts from ankyrin-deficient mice

(Salomao et al. 2010). They found that while AE1 sorted to the reticulocytes in wild-type mice,

AE1 sorted to both reticulocytes and expelling nuclei in the ankyrin-deficient mice. This shows

that one mechanism of deficiency of AE1 in HS caused by defects in ankyrin may be abnormal

sorting during nuclear extrusion. The same may be true for other proteins where a defect in one

protein results in the deficiency of another.

β-spectrin is the limiting component in the formation of the red cell cytoskeleton (Hanspal

and Palek 1987), while α-spectrin is made in excess. For this reason, HS associated with spectrin

is mostly caused by mutations in β-spectrin. Several null mutations are known to cause dominant

HS where only one allele is expressed (Tse and Lux 1999). No recessive mutations have been

found, nor any compound heterozygous states. Mutations in the β-spectrin gene often appear de

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novo and the associated HS is moderate to severe, but in general does not require transfusions

after the first year of life (Delaunay 2007). The prominent β-spectrin mutation Promissão results

in removal of the initiation codon since Met1 is mutated to Val. Mutations such as this one, and

others causing amino acid substitutions, result in β-chain deficiency. Because the β-chain is the

limiting step its deficiency results in a deficiency in spectrin assembly. Mutations in α-spectrin

causing HS show a recessive pattern and result in severe spectrin deficiency (Delaunay 2007).

However, no homozygous null alleles have been discovered since this would probably be lethal.

Since α-spectrin is made in three- to four-fold excess of what is used in the cytoskeleton,

defective α-spectrin production from only one allele does not cause HS (Tse and Lux 1999). In

this case, protein expression from the remaining wild-type allele is sufficient for normal

cytoskeletal construction.

HS associated with mutations in AE1 is for the most part dominantly inherited (Tse and Lux

1999), however there are some cases of recessive inheritance (Inoue et al. 1998, Ribeiro et al.

2000, Rybicki et al. 1993). Mutations are found throughout the gene, including regions coding

for the membrane domain (mdAE1) and the cytoplasmic domain (cdAE1). Details about the

specific domains of AE1 will be discussed in the AE1 section to follow. The clinical picture

ranges from mild to severe HS requiring splenectomy and transfusions. SDS-PAGE usually

shows a reduction of AE1 with a secondary reduction in protein 4.2. A homozygous null

mutation (Band 3 Coimbra), which is the equivalent of a human AE1 knock-out, is caused by a

point mutation, V488M (Ribeiro et al. 2000). In the patient with this mutation, both AE1 and

protein 4.2 were completely absent, and spectrin, ankyrin, glyceraldehyde 3-phosphate

dehydrogenase (GAPDH) and glycophorin A were significantly reduced. HS caused by this

mutation was severe, with transfusions required immediately after birth, splenectomy performed

at 3.5 years, and a regimen of regular blood transfusions thereafter.

Some nonsense mutations result in mRNA instability, resulting in mRNA degradation and

AE1 deficiency (Tse and Lux 1999). This results in fewer points of contact for the cytoskeleton

at the membrane. Missense mutations located in the transmembrane (TM) domain can cause

protein conformational changes leading to protein degradation and a decreased level of AE1 at

the membrane. More details about how these mutations in mdAE1 are associated with HS will be

discussed later. Two recessive HS cases caused by AE1 mutations, E40K and G130R, in the

cytoplasmic domain have normal AE1 content in red cells, but a deficiency in protein 4.2 (Inoue

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et al. 1998, Rybicki et al. 1993). A decrease in protein 4.2 with normal amounts of AE1 is also

seen in one dominant mutation, P327R, located in the cytoplasmic domain (Jarolim et al. 1992a).

Since these three mutations are in the cytoplasmic domain and only result in protein 4.2

deficiency, it is believed they result in impaired protein 4.2 binding. In fact, when the G130R

mutant exists in trans with another AE1 mutant (Band 3 Okinawa) that prevents its stable

incorporation into the membrane, there is a near total deficiency of protein 4.2 (Kanzaki et al.

1997). In the patient with these AE1 mutations, only the G130R AE1 protein is seen in the

mature erythrocyte. The authors reasoned that in red cell precursors virtually all of the protein

4.2 is bound to the Okinawa mutant and because of its inability to stably incorporate into the

membrane, the Okinawa-protein 4.2 complex became degraded, leaving only the G130R mutant

at the membrane and no protein 4.2 in the mature cell. In the homozygous G130R case, it may be

that protein 4.2 binds sufficiently to both copies of mutant AE1 to retain about half of the protein

4.2 content in the red cell.

Several mutations in the protein 4.2 gene result in red cells with no detectable protein 4.2, but

the resulting HS is not as severe as it is in patients with a complete absence of AE1. The clinical

picture resulting from protein 4.2 mutations in the homozygous state ranges from mild HS to

moderate, uncompensated hemolytic anemia, while heterozygotes are normal (Iolascon et al.

1998). These mutations result in decreased levels of protein 4.2 in the red cell, and some affect

binding to AE1 (Toye et al. 2005) and ankyrin (Su et al. 2007). Deficiency of protein 4.2 occurs

with a secondary reduction in CD47, an Rh complex protein believed to help anchor the

membrane to the cytoskeleton (Bruce et al. 2002). Two novel protein 4.2 mutations in an HS

patient, Chartres-1 (Tyr435STOP) and Chartres-2 (out of frame deletion in exon 9), resulted in

complete protein 4.2 deficiency (van den Akker et al. 2010a). In the protein 4.2-deficient

erythrocytes of this patient, the AE1-ankyrin interaction was weakened, supporting the role of

protein 4.2 as a stabilizer of the membrane-cytoskeleton interaction. As mentioned above, some

HS mutations in cdAE1 result in protein 4.2 deficiency with a normal AE1 content. The

mechanism of disease with these cdAE1 mutations may be indirect, whereby impaired protein

4.2 binding to cdAE1 causes a deficiency in protein 4.2, which then results in a weakened AE1-

ankyrin interaction.

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1.3 Anion exchanger 1 (AE1)

1.3.1 Anion exchanger gene family

Anion exchangers (AE) are integral membrane proteins encoded by members of the solute

carrier 4 (SLC4) gene family, also known as the bicarbonate transporter family (Romero et al.

2004). The human family of ten members includes three AE proteins (AE1-3) and five Na+-

coupled HCO3- transporters (NBC). The last two members, AE4 and BTR1, have unknown

functions, but based on sequence homology lie between the two major groups of exchangers and

co-transporters. Table 1.1 shows the names, functions and tissue distribution of the family

members, SLC4A1 (AE1, Band 3) being the subject of this thesis.

The AE proteins can be divided into three structural regions: a hydrophilic amino-terminal

(N-terminal) extension, a hydrophilic cytoplasmic core domain, and a carboxyl-terminal (C-

terminal) hydrophobic TM domain. The N-terminal extensions of AE2 and AE3 are homologous

and are about 300 and 200 residues longer, respectively, than the AE1 extension which is only 58

residues long. The conserved cytoplasmic core of AE1 begins at Val59 (Reithmeier et al. 1996).

The membrane domain of the AE proteins, which in AE1 begins at Gly361 (Tanner 1997) and

extends to the C-terminus, catalyzes the electroneutral exchange of Cl- for HCO3

- across the

plasma membrane. The membrane domains in the AE family have high sequence identity,

indicating that the anion exchange function has been conserved and operates by a similar

mechanism. AE1 (Band 3) is expressed abundantly in erythrocytes and a truncated kidney

isoform (kAE1) missing the first 65 amino acids, including the N-terminal extension, is

expressed in the α-intercalated cells of the collecting tubule of kidney. AE2 is ubiquitously

expressed and AE3 is expressed mainly in the heart and the brain. Figure 1.4 shows

diagrammatic comparisons of the protein domains of AE1, kAE1, AE2 and AE3.

The gene encoding human AE1 was localized to chromosome 17q21-qter (Showe et al.

1987). The complete protein-coding sequence of human AE1 was obtained from a fetal liver

library cDNA clone and was predicted to code for a protein 911 amino acids in length (Tanner et

al. 1988). It was later discovered that this AE1 sequence was not the most common sequence,

but contained the Memphis I variant, K56E (Jarolim et al. 1992b, Yannoukakos et al. 1991), a

widespread polymorphism with no effect on AE1 function. The only known molecular

characteristic of this variation is that it causes proteolytic fragments derived from the amino-

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terminus of the protein to run 3 kDa slower on SDS-PAGE (Mueller and Morrison 1977). The

wild-type sequence of AE1 was also cloned from a fetal liver cDNA library (Lux et al. 1989).

The AE1 gene is about 18 kb in length and contains 20 exons (Sahr et al. 1994, Schofield et al.

1994). Erythroid AE1 is transcribed from a promoter region immediately upstream of exon 1,

while the kAE1 mRNA lacks exons 1 to 3, and uses an initiator codon within exon 5. This gives

rise to a truncated protein product missing the first 65 residues from the N-terminus of the

erythroid version and begins at Met66 (Kollert-Jons et al. 1993). Figure 1.5 compares the exon

structures and transcriptional start sites of AE1 and kAE1.

Table 1.1: SLC4 bicarbonate transporter family

Gene Common name Function Tissue distribution

SLC4A1 AE1, Band 3 Cl-/HCO3

- exchange,

erythrocyte stability

Erythrocyte, kidney

SLC4A2 AE2 Cl-/HCO3

- exchange Ubiquitous distribution

SLC4A3 AE3 Cl-/HCO3

- exchange Brain, heart, retina,

kidney, GI tract

SLC4A4 NBCe1, NBC1 Na+/HCO3

- cotransport NBCe1-A: kidney, eye

NBCe1-B: ubiquitous

NBCe1-C: brain

SLC4A5 NBCe2, NBC4 Na+/HCO3

- cotransport Liver, testes, spleen

SLC4A7 NBCn1, NBC2, NBC3 Na+/HCO3

- cotransport Ubiquitous distribution

SLC4A8 NDCBE Na+-dependent Cl

-/

HCO3- exchange

Brain, testes, kidney,

ovary

SLC4A9 AE4 Cl-/HCO3

- exchange? Kidney

SLC4A10 NCBE Na+/HCO3

- cotransport? Brain

SLC4A11 BTR1 Unknown Kidney

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Figure 1.4: Diagrams of proteins expressed by the genes encoding human AE1, AE2 and

AE3. Predicted TM domains are blue, while cytoplasmic domains, including the N-terminal

extensions, are white. First and last amino acids are indicated. Modified from Alper (2009).

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Figure 1.5: Map of the human AE1 gene.

The positions of exons 1-20 are indicated. Protein coding regions are shown as black boxes, and

the 5’ and 3’ noncoding regions shown as white boxes. Erythroid (ATGE) and kidney (ATGK)

initiator codons are indicated, and exon K1 that forms the 5’ end of kidney AE1 mRNA is shown

as a blue box. Modified from Sahr et al. (1994).

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1.3.2 AE1 in various species

A wide range of vertebrate cells display electroneutral Cl-/HCO3

- exchange function. In

erythrocytes and specialized kidney cells, this function is assigned to AE1. AE1 was first cloned

from the mouse erythrocyte in 1985 (Kopito and Lodish 1985). The human erythroid AE1

sequence was deduced from fetal liver cDNA three years later (Tanner et al. 1988). The mouse

AE1 protein is 18 residues longer than human AE1 and their sequences are highly similar with

an 81 % sequence identity. Other mammalian AE1 sequences have been determined for bovine

(GenBank AF163826) and rat erythrocyte AE1 (NCBI NP_036783.2). Chicken is the only bird

whose erythroid AE1 sequence has been deduced (Cox and Lazarides 1988). AE1 has also been

sequenced from fish species, including trout (Hubner et al. 1992) and zebrafish (Paw et al.

2003). The transport activity of trout AE1 (tAE1) can be regulated by cell swelling, whereby

tAE1 releases taurine in order to regulate cell volume (Motais et al. 1997).

An anion transporter homologue was identified in the yeast Saccharomyces cerevisiae

(Philippsen et al. 1997) by sequence similarity to mammalian anion exchangers and Na+/HCO3

-

transporters. Expression of its cDNA in yeast produced a protein localized to the plasma

membrane that could bind anions including Cl- and HCO3

- (Zhao and Reithmeier 2001). Yeast

YNL275w, the AE1 homologue, is related to Bor1 of plants, a borate transporter (Jennings et al.

2007, Takano et al. 2002). AE gene family members are expressed in Caenorhabditis elegans

(Sherman et al. 2005), but not in bacteria. However, a 4,4'-diisothiocyanodihydrostilbene-2,2'-

disulphonate (H2DIDS)-sensitive membrane protein involved in bicarbonate transport into the

photosynthesizing cells of the marine alga sea lettuce (Ulva sp.) was discovered (Drechsler et al.

1993). Shortly after, a 95 kDa membrane protein in the same organism was recognized by

antibodies against human AE1 (Sharkia et al. 1994), indicating that a similar protein may have

evolved in marine algae for the purpose of bicarbonate transport, likely involved in CO2 fixation.

1.3.3 Structure and function

The human AE1 protein consists of two structurally and functionally distinct domains. The 43

kDa N-terminal cdAE1, from Met1 to Lys360 (Tanner 1997) binds to cytoskeletal proteins,

glycolytic enzymes and deoxyhemoglobin (Low 1986, Willardson et al. 1989). This region,

through its interaction with the cytoskeleton, is responsible for helping maintain the structural

integrity of the red cell. This domain can be cleaved from the red cell ghost membrane using

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mild trypsin or chymotrypsin treatment (Steck et al. 1976) and has been shown to maintain its

protein-binding function independent of the membrane domain (Low 1986, Wang et al. 1992).

The 52 kDa mdAE1, from Gly361 to Val911 (Lepke and Passow 1976, Tanner 1997) spans the

plasma membrane about 12 times and is responsible for the exchange of Cl- for HCO3

- (Jennings

1989b). mdAE1 has been shown to maintain its transport function in the absence of cdAE1

(Grinstein et al. 1978). The extreme C-terminal 33 residues of AE1 are cytoplasmic and contain

a region that binds carbonic anhydrase II (CAII) (Vince and Reithmeier 1998), which produces

the HCO3- that is transported out of the cell by AE1. The association of CAII with AE1 increases

the anion exchange efficiency of AE1 and is therefore believed to form a transport metabolon,

linking transport (AE1) and metabolism (CAII) (Sterling et al. 2001).

1.3.4 Oligomeric state

AE1 exists as a mixture of dimers and tetramers in the erythrocyte membrane and in detergent

solutions (Jennings 1989b). When examined by high-performance liquid chromatography

(HPLC) in solutions containing the nonionic detergent C12E8, the predominant species (70 %)

were dimers with the remainder being tetramers (Casey and Reithmeier 1991). Dissociation of

AE1 to monomers requires the use of denaturing detergents such as SDS (Salhany et al. 1997).

Ankyrin binds and stabilizes the tetrameric form of AE1 (Pinder et al. 1995, Thevenin and Low

1990). AE1 tetramers are dimers of dimers based on the cytoplasmic domain crystal structure

where only one subunit from each dimer is in contact within the tetramer (Zhang et al. 2000).

After the combined cross-linking of the membrane and cytoplasmic domains of AE1 in intact red

cells with the membrane impermeant active ester bis(sulfosuccinimidyl)-suberate (BSSS) and

then Cu2+

/o-phenanthroline to cross-link cytoplasmic sulfhydryls, respectively, a study showed

that mostly dimers were formed. These results indicate that membrane domains and cytoplasmic

domains of the same pair of subunits become cross-linked to each other within a stable dimer

(Jennings and Nicknish 1985).

1.3.5 Kidney AE1 (kAE1)

A kidney isoform of AE1 (kAE1) is expressed in the basolateral membrane of α-intercalated

acid-secreting cells in the collecting ducts of the distal nephron (Wagner et al. 1987). The kAE1

protein exchanges Cl- for HCO3

- across the basolateral membrane resulting in bicarbonate

reabsorption into the blood allowing acid excretion into the urine by a H+-ATPase (Karet 2002).

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The kAE1 protein lacks the first 65 residues of AE1 (Kollert-Jons et al. 1993) and is unable to

bind glycolytic enzymes and ankyrin (Ding et al. 1994, Wang et al. 1995). The acidic extreme

N-terminus of AE1 is missing in the kidney isoform. Since this region has been implicated as the

binding region for glycolytic enzymes and one of the regions of ankyrin binding in AE1, it is not

surprising that kAE1 is unable to bind to these proteins. Another region missing in kAE1 is the

first β-strand that runs through the centre of the globular protein-binding domain of cdAE1.

Biophysical studies carried out by Biochemistry project student Allison Pang under my

supervision (see Appendix) have revealed that the cytoplasmic domain of kAE1 (cdkAE1) is less

thermally stable than erythroid cdAE1 and exists in a more open structure (Pang et al. 2008).

Tryptophan residues that are buried in a hydrophobic environment in cdAE1 are more solvent-

exposed in the folded cdkAE1 protein.

A novel mutant we constructed that is missing the acidic N-terminus but retains the central

β-strand (cdΔ54AE1) had a similar folded structure and thermal stability as cdAE1, indicating

that the differently-folded structure of cdkAE1 could be attributed to the missing β-strand (Pang

et al. 2008). This altered structure of cdkAE1 may account for the impaired binding of kAE1 to

AE1-binding partners. Its ability to bind protein 4.2 has not been determined, but even with its

altered structure, cdkAE1 may have retained its protein 4.2-binding site. As well, protein 4.2 is

present in kidney cells, so it is possible it plays a similar role in kidney as it does in erythrocytes,

stabilizing the interaction between the membrane and the underlying cytoskeleton.

1.3.6 Distal renal tubular acidosis (dRTA)

Mutations in kAE1 can result in distal renal tubular acidosis (dRTA), a kidney disease

characterized by impaired acid secretion into the urine. This leads to metabolic acidosis,

hypokalaemia, bone disease, and nephrocalcinosis (Batlle et al. 2001, Rodriguez-Soriano 2000).

Several mutations in the membrane domain of kAE1 cause dRTA either because of mistargeting

of the protein to the apical membrane, or because of impaired exchanger function. Interestingly,

there are no reports of dRTA mutations in the cytoplasmic domain of kAE1. Normally,

mutations in AE1 cause either HS or dRTA, but not both. For example, the R589H mutation is

associated with dRTA, but not HS. Not surprisingly, when R589H AE1 and R589H kAE1 were

transiently expressed in HEK-293 cells, the mutation caused intracellular retention of kAE1, but

not of AE1 (Quilty et al. 2002). There are two known cases where a patient presents with both

diseases. The V488M mutation (Band 3 Coimbra) (Ribeiro et al. 2000), essentially results in a

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knock-out of AE1 in erythrocytes and kAE1 in kidney cells, as discussed above. It is not

surprising that a total absence of AE1 and kAE1 can cause HS and dRTA. The S667F mutation

(Band 3 Courcouronnes) (Toye et al. 2008) causes an erythrocyte AE1 reduction to about 35 %

and results in both HS and dRTA.

As with Southeast Asian ovalocytosis AE1 (AE1SAO), which is described below,

glycophorin A (GPA) can facilitate the cell surface expression in Xenopus oocytes of the dRTA

mutant G701D that is otherwise retained intracellularly (Tanphaichitr et al. 1998). Kidney cells

do not express GPA, nor do they express an equivalent protein behaving in the same way

(Kittanakom et al. 2004). Thus, no rescue of the dRTA trafficking mutants to the kidney cell

surface occurs as it does in red cell precursors expressing GPA. This accounts for the lack of

effect of dRTA mutations on red cell AE1 expression and trafficking.

1.3.7 Southeast Asian ovalocytosis AE1 (AE1SAO)

Southeast Asian ovalocytosis (SAO) is a condition whereby red blood cells become rigid and

oval-shaped (Amato and Booth 1977). As the name implies, SAO is found almost exclusively in

Southeast Asia, including the Malay archipelago, the Philippines, Indonesia, Thailand, and Papua

New Guinea (Nurse et al. 1992). This hematological condition is caused by a nine amino acid

deletion (Δ400-408) at the junction between the cytoplasmic and transmembrane domains of

AE1 (Jarolim et al. 1991). The SAO deletion mutant of AE1 (AE1SAO) causes the cell to be

extraordinarily rigid (Mohandas et al. 1992), conferring some protection against malaria since

the red cells become resistant to entry of the parasite (Kidson et al. 1981). This clinical

protection has allowed SAO to persist in the Southeast Asian population which has a high

incidence of Plasmodium falciparum malaria (Allen et al. 1999). AE1SAO is asymptomatic in

the heterozygous state, but the homozygous state is thought to be embryonic lethal since this

state has never been observed and because of a higher incidence of miscarriage when both

parents carry the deletion (Liu et al. 1994).

In ovalocytes the AE1SAO mutant is able to traffic to the plasma membrane to about 48 %

of the total (Sarabia et al. 1993) but has no anion exchange function (Schofield et al. 1992,

Tanner 1997) as a result of a misfolded membrane domain (Moriyama et al. 1992). The mutant

has an increased association with the cytoskeleton, decreased lateral (Liu et al. 1990) and

rotational (Liu et al. 1995) movement, and decreased extractability from red cell ghost

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membranes (Sarabia et al. 1993), which contribute to the rigidity of the SAO red cell. In HEK-

293 cells the AE1SAO mutant becomes retained in the ER during biosynthesis and is not seen at

the plasma membrane (Cheung et al. 2005a). The misfolded AE1SAO protein in HEK-293 cells

is most likely retained in the endoplasmic reticulum (ER) by the cellular quality control

machinery, whereas in the red cell erythroid-specific factors may allow for cell-surface

expression. In HEK cells the deletion disrupts the proper membrane integration of

transmembrane region 1, likely causing the polar amino acid region directly N-terminal to the

deletion to get pulled into the membrane (Cheung and Reithmeier 2005). This would account for

a less flexible link between the cytoplasmic and membrane domains and the rigidity seen in SAO

cells. Red cells express glycophorin A (GPA), which is known to associate with AE1 at the

membrane. The association of AE1 and GPA at the plasma membrane creates the Wright b (Wrb)

blood group antigen at the surface. Arg61 of GPA interacts with Glu658 in TM 8 of AE1 to

form the Wrb antigen (Bruce et al. 1995). In Xenopus oocytes, GPA can facilitate the cell surface

expression of wild-type AE1 (Groves and Tanner 1992) as well as that of the SAO deletion

mutant that is otherwise retained intracellularly (Groves et al. 1993). This shows that the region

of AE1 around TM 8 that associates with GPA remains intact in AE1SAO.

AE1SAO is associated with hemolytic anemia at birth, but disappears within the first three

years of life (Laosombat et al. 2010). The lack of hemolytic anemia in SAO individuals after

three years of age implies that the cytoskeletal protein interactions of its cytoplasmic domain are

intact. In fact, AE1SAO interacts more strongly with ankyrin than does wild-type AE1 (Liu et al.

1990). Circular dichroism (CD) and calorimetric data showed structural similarities between the

cytoplasmic domains of wild-type AE1 and AE1SAO (Moriyama et al. 1992, Sarabia et al.

1993).

1.3.8 AE1 knock-outs

As mentioned above, Band 3 Coimbra (V488M) in the homozygous state is the equivalent of an

AE1 knock-out in humans (Ribeiro et al. 2000). The mutation resulted in a membrane trafficking

defect and the absence of AE1 causing severe HS requiring splenectomy and regular blood

transfusions. Protein 4.2 was also absent, and significant reductions in spectrin, ankyrin, GAPDH

and glycophorin A were observed. The mutation also caused a lack of kAE1 in kidney, which

resulted in dRTA. In addition to regular transfusions to treat the HS, the patient required daily

bicarbonate supplements to treat the metabolic acidosis associated with dRTA. In the

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heterozygous state Band 3 Coimbra caused typical HS and was associated with partial deficiency

in AE1 and protein 4.2.

Studies in AE1 knock-out mice revealed that most AE1-/-

mice die within two weeks of birth

(Southgate et al. 1996). These mice have severe hemolytic anemia, no protein 4.2 or glycophorin

A in their red cells, but an assembled red cell cytoskeleton detached from the membrane.

Heterozygote mice are identical to normal mice in appearance and growth. A natural AE1

mutation in cattle, Arg646Stop, resulted in red cells with no AE1 in homozygous animals

(Inaba et al. 1996). This mutation created the equivalent of an AE1 knock-out in cattle and

occurred with deficiencies in red cell spectrin, ankyrin, actin, and protein 4.2. The red cells were

spherocytic and extremely unstable and the cattle had moderate uncompensated anemia with HS.

This indicated that AE1 provides membrane structural stability, but is not essential to the

survival of cows. Zebrafish with AE1 mutations (Retsina) equivalent to an AE1 knock-out

developed chronic anemia (Paw et al. 2003). In addition, the absence of AE1 caused a

cytokinetic defect at the erythroblast stage. This cytokinetic defect was also seen in mouse AE1

knock-outs, indicating a conserved role for AE1 in normal erythroid cytokinesis.

1.3.9 HS mutations in AE1

Since AE1 is expressed in erythrocytes and kidney cells, both of these tissues may be affected by

mutations in AE1. The affected tissue depends on the site of the mutation in the AE1 gene. The

kidney defect, dRTA, was discussed above in the kAE1 section. In erythrocytes, defective AE1

may lead to hemolytic HS. HS-associated mutations can occur in mdAE1 and cdAE1. Mutations

in mdAE1 have been shown to cause misfolding of the protein and retention in the ER in studies

using transfected mammalian cells (Quilty and Reithmeier 2000). ER retention of AE1 in

differentiating red cell precursors would result in a deficiency of AE1 at the plasma membrane of

mature cells, and fewer sites of membrane-cytoskeletal linkage since the ER is removed during

erythropoiesis. The decrease in AE1 at the plasma membrane accounts for the membrane

structural instability observed in HS patients with these mdAE1 mutations. Some mutations in

the cdAE1 create a premature stop codon in the gene, which codes for a truncated protein

product. This type of mutation also results in less AE1 at the plasma membrane of red cells, and

accounts for the membrane structural instability of these cells.

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Interestingly, three HS mutations in cdAE1 (E40K, G130R, and P327R) do not result in a

misfolded or truncated protein product (Inoue et al. 1998, Jarolim et al. 1992a, Rybicki et al.

1993). These mutant proteins are present at the red cell membrane in normal amounts, yet result

in HS nonetheless. The only known phenotype of these mutants is a deficiency of protein 4.2, a

peripheral membrane protein that binds to AE1 and ankyrin. Table 1.2 lists all known mutations

of AE1 associated with HS.

1.4 Membrane domain of AE1 (mdAE1)

1.4.1 Physiological function

The 52 kDa membrane domain of AE1 encompassing residues 361 to 911 is responsible for the

electroneutral exchange of Cl- for HCO3

- across the red cell membrane. AE1 works by an

electroneutral ping-pong mechanism (Furuya et al. 1984). The protein has only one anion

binding site but exists in two main conformations allowing the binding site to be exposed to

either the cytoplasmic side or the extracellular side (Pal et al. 2006). Interconversion between the

two conformational states occurs with anion binding, but this conformational change is very slow

in the absence of substrates. AE1 is a very fast transporter operating at 100 000 chloride ions per

second per molecule. AE1 is able to transport other anions, such as sulfate, but at a much slower

rate (Milanick and Gunn 1984). mdAE1 is able to carry out exchange function in the absence of

the cytoplasmic domain. A study using AE1 in red cell inside-out vesicles (IOVs) found that

trypsinization to release the cytoplasmic region had no effect on sulfate efflux showing that the

transport activity of the membrane domain was intact (Grinstein et al. 1978).

Some studies have uncovered clues regarding important residues involved in anion

transport. Anion transport can be inhibited by a variety of anionic compounds, most notably

stilbene disulfonates such as H2DIDS, which bind to one site per AE1 molecule accessible from

the cell exterior (Lepke et al. 1976). At low temperature (0 ºC) and short times, this binding is

reversible. At higher temperature (37 ºC) H2DIDS can react covalently with Lys539 of AE1, and

can react more slowly with Lys851 of the same AE1 protein, cross-linking two parts of AE1

(Okubo et al. 1994). Mutation of homologous lysine residues (Lys558Asn and Lys869Met)

in mouse AE1 prevented irreversible inhibition by H2DIDS but did not affect the transport

function of AE1 when expressed in Xenopus oocytes (Wood et al. 1992), showing that this Lys

was not essential for transport function.

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Table 1.2: HS mutations in the AE1 protein

AE1 variant Mutation Abnormal allele Reference

Cytoplasmic domain defects: protein 4.2 deficiency

Montefiore Glu40Lys Homozygous (Rybicki et al. 1993)

Fukuoka Gly130Arg Homozygous (Inoue et al. 1998)

Tuscaloosa Pro327Arg (Memphis II:

Lys56Glu in cis) Heterozygous (Jarolim et al. 1992a)

Cytoplasmic domain defects: affects ankyrin binding

Nachod (Hradec

Kralove II) Δcodons 117-121 Heterozygous (Jarolim et al. 1996)

Cytoplasmic domain defects: mRNA instability

Neapolis Δ of 1st 11 amino acids Heterozygous (Perrotta et al. 2005)

Genas G89A in exon 2 Heterozygous (Alloisio et al. 1996)

Foggia ΔACCCACACCAC in codon

54 or 55 Heterozygous

(Miraglia del Giudice

et al. 1997)

Bohain ΔT in codon 81 Heterozygous (Dhermy et al. 1997)

Hodinin (Prague IV) Trp81Stop Heterozygous (Jarolim et al. 1996)

Napoli I Insertion TCTTTCT in codon

100 Heterozygous

(Miraglia del Giudice

et al. 1997)

Osnabruck I (Lyon) Arg150Stop Heterozygous (Alloisio et al. 1996)

Worcester Insertion G into codons 170-172 Heterozygous (Jarolim et al. 1996)

Campinas Stop codon (+13) after exon 7 Heterozygous (Lima et al. 1997)

Princeton Insertion C into codons 273-275 Heterozygous (Jarolim et al. 1996)

Noirterre Gln330Stop Heterozygous (Jenkins et al. 1996)

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Table 1.2: HS mutations in the AE1 protein (continued)

AE1 variant Mutation Abnormal allele Reference

Cytoplasmic domain defects: AE1 deficiency

Cape Town Glu90Lys (Arg870Trp in trans)

Compound

heterozygous with

Band 3 Prague III

(Bracher et al. 2001)

Mondego Pro147Ser (E40K in cis; V488M in

trans)

Compound

heterozygous with

Band 3 Coimbra

(Alloisio et al. 1997)

Boston Ala285Asp Heterozygous (Jarolim et al. 1996)

Membrane domain defects: mRNA instability

Bruggen ΔC in codon 419 Heterozygous (Eber et al. 1996)

Bicêtre II ΔG in codons 454-456 Heterozygous (Dhermy et al. 1997)

Pribram (Prague VI) Stop codon (+7) after exon 12 Heterozygous (Jarolim et al. 1996)

Evry ΔT in codon 496 Heterozygous (Dhermy et al. 1997)

Smichov (Prague VII) ΔC in codon 616 Heterozygous (Jarolim et al. 1996)

Trutnov Tyr628Stop Heterozygous (Jarolim et al. 1996)

Hobart ΔG in codons 646-647 Heterozygous (Jarolim et al. 1996)

Osnabruck II Δ of codon 663 or 664 Heterozygous (Eber et al. 1996)

Membrane domain defects: AE1 deficiency

Benesov (Prague V) Gly455Glu Heterozygous (Jarolim et al. 1996)

Edmonton Cys479Trp (Gly701Asp in trans) Compound

heterozygous with

Gly701Asp

(Chu et al. 2010)

Coimbra Val488Met Heterozygous and

homozygous

(Alloisio et al. 1997)

Bicêtre I Arg490Cys Heterozygous (Dhermy et al. 1997)

Pinhal Arg490His Heterozygous (Lima et al. 1999)

Milano Insertion of 23 amino acids in codon 498 Heterozygous (Bianchi et al. 1997)

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Table 1.2: HS mutations in the AE1 protein (continued)

AE1 variant Mutation Abnormal allele Reference

Membrane domain defects: AE1 deficiency

Dresden Arg518Cys Heterozygous (Eber et al. 1996)

Tambaú Met663Lys Heterozygous (Lima et al. 2005)

Courcouronnes Ser667Phe Heterozygous (Toye et al. 2008)

Most (Prague VIII) Leu707Pro Heterozygous (Eber et al. 1996)

Okinawa Gly714Arg (Gly130Arg in trans) Compound

heterozygous with

Band 3 Fukuoka

(Kanzaki et al. 1997)

Kumamoto (Prague II) Arg760Gln Heterozygous (Jarolim et al. 1995)

Hradec Kralove Arg760Trp Heterozygous (Jarolim et al. 1995)

Chur Gly771Asp Heterozygous (Maillet et al. 1995)

Napoli II Ile783Asn Heterozygous (Miraglia del Giudice

et al. 1997)

Jablonec Arg808Cys Heterozygous (Jarolim et al. 1995)

Prague Duplication of nucleotides 2455-2464 Heterozygous (Jarolim et al. 1994)

Birmingham His834Pro Heterozygous (Jarolim et al. 1996)

Philadelphia Thr837Met Heterozygous (Jarolim et al. 1996)

Tokyo Thr837Ala Heterozygous (Iwase et al. 1998)

Prague III Arg870Trp Heterozygous (Jarolim et al. 1995)

Vesuvio ΔACCAC in codon 894 Heterozygous (Perrotta et al. 1999)

Conversion of Glu681 to an alcohol by reduction using Woodward’s reagent K and BH4-,

thereby removing its negative charge, inhibits chloride anion exchange, yet activates chloride-

sulfate exchange (Jennings 1995). The same thing happens when a low pH outside the cell

protonates a titratable carboxyl group, thought to be the same Glu681. This glutamate is believed

to be the binding site for the proton that is co-transported with sulfate during net chloride-sulfate

exchange (Jennings and Smith 1992). This residue is located at the end of TM 8 on the

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cytoplasmic side of the membrane, yet treatment yielding the alcohol is accessible only to the

extracellular side. This is a small region of the protein that is alternately accessible to the outside

and inside of the cell as a result of a conformational change.

The mutation of His721, His837, and His852 to glutamine, or of His752 to serine, inhibited

chloride transport mediated by mouse AE1 expressed in Xenopus oocytes (Muller-Berger et al.

1995). Mutation of Lys558 to asparagine (Lys558Asn) in combination with any of the

HisGln mutations restored chloride flux. This indicates a possible interaction between Lys558

and these histidine residues. The authors reasoned that the transmembrane helices carrying these

histidines, Lys558 and Glu699 (the residue necessary for chloride flux and homologous to

human Glu681) could form an access channel lined with histidine and glutamate residues. Even

with evidence for involvement of specific residues in the anion transport mechanism, an active

anion binding site is yet to be identified within mdAE1.

1.4.2 Topology

mdAE1 spans the red cell membrane up to 12 times (Popov et al. 1997) and is N-glycosylated at

Asn642 in extracellular loop 4 (Jay 1986, Tanner et al. 1988). Proteolysis studies have

determined protease-sensitive sites which are exposed to either the cytoplasmic or extracellular

side of the cell membrane (Kang et al. 1992). For example, cleavage at Lys360 helped determine

its intracellular location and boundary between the cytoplasmic and transmembrane domains.

Tyr553 was cleaved by chymotrypsin on the outside of the cell, positioning this residue on the

extracellular surface. Proteolysis studies also helped determine which residues were located in

surface-exposed loops and others which were part of TM domains (Hamasaki et al. 1997).

Chemical labeling studies with non-permeant reagents have also helped determine

localization of specific residues within the folded structure of AE1. The anion transport inhibitor

eosin-5-maleimide was found to react with Lys430 (Cobb and Beth 1990) which, according to

hydropathy plots, was localized to the first extracellular loop connecting TM domains 1 and 2.

As mentioned above, the membrane-impermeable inhibitor H2DIDS reacted with Lys539,

positioning this residue on the extracellular surface (Okubo et al. 1994). One consideration in

these studies is that the reagents are often quite small and could enter an access channel, reacting

with a residue close to the cytoplasmic side of the membrane. Glu681 is an example of such a

residue. The crystal structures of numerous polytopic membrane proteins (White 2010) have

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revealed that many channels and transporters contain long tilted helices, kinked helices and re-

entrant loops beyond the typical 20 amino acid hydrophobic span seen in proteins such as

rhodopsin. While valuable, topology models do not provide the necessary detail to illuminate the

mechanism of action of membrane proteins. Scanning N-glycosylation mutagenesis has been

used to accurately map the ends of the TM segments of AE1 (Popov et al. 1997). In this

technique, N-glycosylation acceptor sites are introduced throughout the protein followed by

detection of glycosylation. For an extracellular loop to become efficiently glycosylated, the loop

had to be larger than 25 residues in size. Acceptor sites had to be located more than 12 residues

away from the preceding TM segment and more than 14 residues away from the next TM

segment. This 12 + 14 rule was used to localize the ends of TM segments in combination with

hydropathy analysis. This technique has been used to define the extracellular limits of TMs 7 and

8 that limit the loop containing the endogenous N-glycosylation site (Groves and Tanner 1999),

the re-entrant loop between TMs 9 and 10 (Kanki et al. 2002) and the limits of TM 1 and 2 in

normal AE1 and AE1SAO (Cheung et al. 2005b).

Scanning cysteine accessibility mutagenesis (SCAM) has also been used to establish

membrane topology (Zhu and Casey 2007). In this technique, individual cysteine residues are

introduced by mutagenesis into a cysteineless mutant of AE1. The intracellular or extracellular

location of each new cysteine is determined based on its reactivity to sulfhydryl-directed

reagents, such as biotin maleimide. Using this technique, 80 cysteine residues were introduced

between Phe806 and Cys885 (Zhu et al. 2003). These residues were systematically localized to

extracellular loops, intracellular loops, and to the TM domains.

Natural variations or polymorphisms in the AE1 sequence create blood group antigens that

are exposed to the outside of the red cell (Tanner 1997) which have also assisted in establishing

membrane topology. For example, the Diego blood group is created by a P854L polymorphism

located in the extracellular loop connecting the last two TM segments (Salhany et al. 1996). The

results of the above studies to determine AE1 topology are displayed in the folding model of

AE1 in Figure 1.6.

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Figure 1.6: Topographical model of human erythroid AE1.

The kidney AE1 start site at Met66 is indicated with an arrow. Extracellular blood group

antigens are shown in blue. Mutations associated with HS and ovalocytosis (Δ400-408) are

shown in orange. Mutations associated with hereditary stomatocytosis and xerocytosis are shown

in red. Mutations associated with dRTA are shown in green and are only found in the C-terminal

domain. AE1 is N-glycosylated at Asn642. The probable site of limited trypsin digestion, K174,

is indicated. Modified from Alper (2009). HS: hereditary spherocytosis; HSt: hereditary

stomatocytosis; dRTA: distal renal tubular acidosis.

N-glycosylation

Diego antigen

Wright b antigen

Probable site of

limited trypsin digestion

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Electron microscopic analysis of two-dimensional crystals has shown that mdAE1 (residues

361 to 911) reconstituted with lipids exists as a dimer of two monomers related by two-fold

symmetry (Wang et al. 1993). This structure shows that the membrane domain alone maintains

the native dimeric structure of the intact protein and the cytoplasmic domain is not necessary for

dimer formation, confirming earlier biochemical studies (Casey and Reithmeier 1991). Three-

dimensional (3D) crystals of the mdAE1 have been grown that diffract to low resolution (14 Å)

(Lemieux et al. 2002). Deglycosylation increased homogeneity and inhibitor binding increased

the conformational stability of the protein. The types of detergents used and the amount of

phospholipids co-purifying with mdAE1 were critical to the formation of 3D crystals. More

recent work using electron microscopy has revealed some details of the angles of the α-helical

segments in mdAE1 (Yamaguchi et al. 2010). The three-dimensional structure of the outward-

open conformation of mdAE1 was solved at 7.5 Å resolution and several long and tilted helices

were recognized. V-shaped densities near the centre of the dimer were observed. Even with this

higher resolution mdAE1 structure, further investigation is needed to determine the mechanism

of anion exchange.

1.5 Cytoplasmic domain of AE1 (cdAE1)

1.5.1 Structure

The crystal structure of cdAE1 encompassing residues 1 to 379 was solved to 2.6 Å resolution at

pH 4.8 in the laboratory of Dr. P.S. Low (Zhang et al. 2000a) and is shown in Figure 1.7. A tight

symmetric dimer was formed by cdAE1 and was stabilized by interlocked dimerization arms,

which encompassed the C-terminal part of the domain comprising residues 304 to 357. Within

the dimerization domain, eight backbone-to-backbone hydrogen bonds formed between two

strands of the intermonomeric antiparallel β-sheet. As well, a hydrophobic core of nine

interacting leucine residues stabilized the dimer. A globular domain spanning residues 55 to 290,

which is the peripheral protein-binding domain, extended away from the dimerization arm of

each subunit. A short helix-and-loop segment, residues 291-303, connected the two domains.

Residues 1 to 54 (extreme N-terminus), 202 to 211, and 357 to 379 (C-terminus which links to

the membrane domain) were not observed in the crystal structure, presumably due to segmental

flexibility. A cysteine cluster, made up of Cys201 and Cys317 from each dimer subunit, was

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Figure 1.7: Crystal structure of the cytoplasmic domain of AE1 (cdAE1). The symmetric dimer of cdAE1 was crystallized and its structure determined by X-ray

diffraction (Zhang et al. 2000). The two subunits are coloured in grey and green. The locations

of the HS mutations G130R and P327R are indicated. Residues 1-54 (red dotted line) were not

resolved in the structure. The E40K HS mutation is located here and its approximate location is

indicated on the structure. Residues 1-65 (red) are missing in kAE1. Nt, amino-terminus; Ct,

carboxy-terminus.

G130R

P327R

E40K

Nt Ct

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located near the dimer interface. Interdomain and intradomain disulfide bond formation is

possible under experimental oxidizing conditions, but the cysteines in cdAE1 normally exist as

free thiols in red cells (Low 1986). The subunits of the dimer appear to swap dimerization arm

domains, such that the arm from one subunit becomes more associated with the globular domain

of the other subunit, and vice versa. This domain swapping may create protein-binding surfaces

that can only exist in the dimer, thereby necessitating dimer formation for proper cdAE1

function.

The cytoplasmic domain of AE1 purified from erythrocytes at physiological pH was found

to exist as a dimer in solution (Appell and Low 1981). This was determined after unmodified

cdAE1 and disulfide cross-linked cdAE1, both proteolytically released from erythrocyte

membrane vesicles, eluted in the same peak fraction from a gel filtration column. As well, the

molecular weight of the native fragment as determined by sedimentation velocity (SV)

experiments was approximately the same as that of the cdAE1 dimer. Recombinant cdAE1

expressed in, and purified from, E. coli was also shown to exist as a dimer in solution as

determined by calibrated gel filtration (Wang et al. 1992a), which supports the validity of the

dimeric form observed in the crystal structure solved at low pH. In fact, cdAE1 remains a dimer

over a large pH range, but exhibits a pH-dependent conformational change (Zhou and Low

2001). This is seen by a more-than-doubling of its intrinsic fluorescence as the pH is raised from

pH 6 to 11 and an increase in Stokes radius from 51 Å to 62 Å without a change in secondary

structure. Four tryptophan residues (Trp75, Trp81, Trp94 and Trp105) are located relatively

close to each other in the primary sequence of each subunit. A hydrogen bond between Trp105

of one subunit and Asp316 of the other subunit that is present in the lower pH conformation is

broken in a higher pH conformation of cdAE1 as the peripheral protein-binding domain moves

away from the dimerization arm. The crystal structure represents the low pH conformation and is

more compact, while increasing the pH opens up the structure.

Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance

(EPR) and double electron-electron resonance (DEER) spectroscopies was used to study the

solution structure of cdAE1 at neutral pH (Zhou et al. 2005). Single cysteine mutants of cdAE1

were constructed from a cysteineless mutant followed by SDSL of the single cysteines. Solvent

accessibility of labeled residues was performed using a paramagnetic broadening agent. This

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technique confirmed the α-helical structure and solvent accessibility of regions in the peripheral

protein-binding domain and in the dimer interface that were consistent with the crystal structure.

As well, intersubunit distances between spin labels on various residues in the dimerization arm

agreed with distances found between these residues in the crystal structure. These results indicate

that the compact crystal structure solved at low pH may, in fact, represent the physiological form

of cdAE1.

1.5.2 Physiological function

The cytoplasmic domain of AE1 functions as a site of protein interaction at the internal surface

of the red cell membrane. Protein binding functions to regulate glycolysis and oxygen binding to

hemoglobin, and to maintain the structure and stability of the red cell. The extreme N-terminus

of cdAE1 is extraordinarily acidic and the N-terminal methionine is N-acetylated (Drickamer

1978, Kaul et al. 1983). This region interacts directly with glycolytic enzymes,

deoxyhemoglobin, hemichromes, protein 4.1 and ankyrin (Low 1986, Willardson et al. 1989).

Two tyrosine residues present in this region (Tyr8 and Tyr21) become phosphorylated by

tyrosine kinases in red cells, and ankyrin binding to cdAE1 blocks the phosphorylation of Tyr8

(Willardson et al. 1989). Phosphorylation of Tyr8 regulates the binding and activity of glycolytic

enzymes, and consequently, glycolysis in the red cell (Harrison et al. 1991). This region of AE1

binds to the active sites of glycolytic enzymes like GAPDH, inhibiting their activity.

Phosphorylation of Tyr8 by the combined action of Syk and Lyn kinases releases glycolytic

enzymes, allowing glycolysis to proceed. Indeed, recent evidence has suggested that glycolytic

enzymes that bind to cdAE1 exist as a complex on the cytoplasmic surface of the red cell

membrane, anchored there by the association with AE1 (Campanella et al. 2005).

The extreme N-terminal 11 residues of cdAE1 bind to the central cavity between the β-

chains of deoxyhemoglobin (Walder et al. 1984). The binding site extends deep into the cavity

and includes most of the basic residues within the 2,3-diphosphoglycerate (2,3-DPG) binding

site. Binding of cdAE1 to deoxyhemoglobin lowers its oxygen affinity and increases the Bohr

effect. This has the same effect that binding of 2,3-DPG has on hemoglobin, which is to lower

the affinity for oxygen and thereby transfer it from red blood cells to respiring tissues. In the

lungs, the opposite occurs where cdAE1 affinity for hemoglobin decreases and oxygen inhaled

from the air must bind to hemoglobin to be delivered to the tissues for oxidative metabolism.

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An important function of cdAE1 is to help stabilize and strengthen the red cell membrane. It

does this through its interaction with the cytoskeleton. cdAE1 binds directly to ankyrin and

protein 4.2, which in turn bind to spectrin, creating a physical link between the red cell

membrane and the cytoskeleton. Details of the protein 4.2 interaction with cdAE1 will be

discussed in Section 1.6. The ankyrin binding site on AE1 has been localized to two regions of

cdAE1: the extreme N-terminus and a β-hairpin loop in a hinge region made up of residues 175

to 185 (Chang and Low 2003, Willardson et al. 1989). Binding of ankyrin to AE1 induces the

oligomerization of AE1 to a tetramer. The AE1 tetramer’s rotational mobility is restricted as a

result of its association with the cytoskeleton (Van Dort et al. 1998). The association between

ankyrin and AE1 has been reported to occur in the ER or in the first compartment of the Golgi

apparatus. In addition to the role of ankyrin in anchoring AE1 to the cytoskeleton, it also appears

to be involved in the exit of AE1 out of the ER (Gomez and Morgans 1993). An interesting

model has been proposed for the association of chicken AE1 with the cytoskeleton using

circulating erythroid cells from chicken embryos (Ghosh et al. 1999). AE1 dimers assemble in

the ER and traffic out to the plasma membrane. Then they are endocytosed and traffic back to the

Golgi where they form tetramers, bind ankyrin and return to the plasma membrane to lock into

the cytoskeleton.

1.5.3 HS mutations in cdAE1 affecting levels of protein 4.2

As mentioned in Section 1.2, three mutations in cdAE1 are associated with HS and a decrease in

protein 4.2 in the red cell while maintaining a normal amount of AE1. These mutations are E40K

(Band 3 Montefiore), G130R (Band 3 Fukuoka) and P327R (Band 3 Tuscaloosa). The mutations

are associated with 88 % (homozygous state), 55 % (homozygous state), and 29 % (heterozygous

state) decreases in the amount of protein 4.2 in the red cell, respectively, as determined from one

HS patient each (Inoue et al. 1998, Jarolim et al. 1992a, Rybicki et al. 1993). The Band 3

Tuscaloosa mutation has been found to occur in conjunction with the Memphis I mutation

(K56E) in one individual with HS (Jarolim et al. 1992a). Since Band 3 Memphis I is an

asymptomatic variant that represents a widespread polymorphism found in hematologically

normal subjects (Jarolim et al. 1992b) it is unlikely that this mutation contributes to the

phenotype seen in this patient. Studies where these three mutations were first characterized

revealed that AE1 in IOVs prepared from patient red cells containing these mutations have a

lower binding capacity for protein 4.2. However, denaturing conditions were used to purify

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protein 4.2 and to strip peripheral proteins from IOVs. As well, there were large variations in the

binding results of some of the experiments, perhaps due to the harsh purification conditions. For

these reasons, a true comparison of protein 4.2 binding by these HS mutants and wild-type AE1

has yet to be performed.

The three HS mutations are included in Table 1.2 and are indicated in the crystal structure of

cdAE1 in Figure 1.7. Interestingly, the three mutations do not cluster into a small region of

cdAE1, but rather are quite far apart in the structure. The E40K mutation is located in the

unstructured N-terminal region of the protein so its approximate location is indicated. The

G130R mutation is located in a prominent α-helix in the globular protein-binding domain. The

P327R mutation is located at the N-terminus of the α-helix of the dimerization arm.

As mentioned earlier in this section, the X-ray structure of cdAE1 reveals a domain swap

between monomer subunits, where the dimerization arms intertwine and become associated with

the globular domain from the other subunit. In fact, Pro327 (the site of the HS mutation P327R)

from one subunit occurs on the same face of the dimer as Glu40 and Gly130 (sites of HS

mutations E40K and G130R) from the other subunit. It may be that these three sites make up part

of the large binding surface for protein 4.2, which would explain why mutations at these sites

cause deficiency of protein 4.2 in the red cell.

AE1 species sequence alignment shows that Glu40 is conserved in mouse and rat while the

conservative substitution of aspartate, retaining the negative charge, occurs in chicken, trout and

zebrafish. Another conservative substitution of glutamine occurs in cattle, but with a loss of the

negative charge. The high conservation of this negative charge probably reflects the importance

of this acidic region in the binding of several red cell proteins, including glycolytic enzymes,

hemoglobin and protein 4.2. Gly130 is conserved in mouse and rat but is replaced with alanine in

chicken and trout, and by serine in zebrafish, thereby retaining a small amino acid side chain in

this position. Pro327 is perfectly conserved in mouse, rat, cow and chicken, but a glutamine

occurs in this position for trout and zebrafish. The conservation in mammals and birds reflects

this residue’s importance as well. The protein 4.2 binding site on cdAE1 has not been

determined, but the deficiency of protein 4.2 in red cells that have these mutations in AE1

suggests these three residues may be directly involved in the interaction. Alternatively, these

mutations could cause a conformational change in cdAE1 resulting in loss of protein 4.2 binding.

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1.6 Protein 4.2

1.6.1 Background

Protein 4.2 is a 72 kDa cytoskeletal protein that resides on the inner surface of the plasma

membrane of red cells. It makes up 5 % of the total membrane protein of the red cell and is

present at about 200 000 copies per cell (Branton et al. 1981). The main role of protein 4.2 is in

maintaining the structural integrity of the red cell membrane through interactions with plasma

membrane and cytoskeletal proteins (Tanner 2002). The gene for mouse protein 4.2 was found to

co-localize on chromosome 2 with the gene for the mouse pallid mutation, which serves as a

mouse model for human platelet storage pool deficiencies (White et al. 1992). Pallid was

believed to be a mutation in the mouse protein 4.2 gene, and for this reason protein 4.2 was given

the name pallidin. The mouse pallid mutation and the gene for mouse protein 4.2 have since been

discovered to reside at distinct loci (Gwynn et al. 1997).

Full-length human protein 4.2 cDNA was cloned and sequenced from a human reticulocyte

expression library (Korsgren et al. 1990, Sung et al. 1990). The complete amino acid sequence

of protein 4.2 was derived from the nucleotide sequence and was found to be homologous to

transglutaminase (TG) protein sequences (Korsgren et al. 1990, Sung et al. 1990).

1.6.2 Transglutaminase family of enzymes

Transglutaminases are calcium-dependent cross-linking enzymes that carry out various

biological functions, including blood coagulation, skin-barrier formation and extracellular-matrix

assembly (Lorand and Graham 2003). TGs are found in many different types of species from

humans and other mammals, chicken and fish, all the way down to insects, invertebrates and

slime mould. TGs catalyze the formation of covalent γ-glutamyl-ε-lysine cross-links between

glutamine acyl-donors and lysine acyl-acceptors. These cross-links result in either the

polymerization of proteins or the covalent linking of proteins which were already reversibly

bound by non-covalent bonds. TGs are also involved in other reactions, such as the mediation of

post-translational modifications by deamidation and amine incorporation, and protein

esterification.

There are eight catalytically active members of the human TG family (TG1 – TG7, and

factor XIIIa) and protein 4.2 is considered to be the catalytically inactive ninth member. Protein

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4.2 shares sequence identities with TG family members from 23 % (TG1) to 33 % (TG2). A

critical cysteine in the active site of TGs, necessary for catalysis, is substituted by alanine in

protein 4.2 (Korsgren et al. 1990). When TG binds calcium, a large conformational change takes

place exposing the active site (Bergamini 1988). GTP binding by TG negatively regulates its

cross-linking activity (Murthy et al. 1999). ATP has a similar, but less pronounced, effect.

Protein 4.2 was shown to lack cross-linking activity in red blood cell ghosts and IOVs in the

absence or presence of calcium (Korsgren et al. 1990). Also, no calcium-stimulated cross-linking

activity was detected by protein 4.2 in solution. Protein 4.2 has two highly conserved glutamate

residues (Glu439, Glu444) that are known to bind calcium in other TG family proteins

(Satchwell et al. 2009). The spectrin-binding tryptic peptide of protein 4.2 encompassing

residues 380-462, which contains the conserved glutamate residues, has been shown to bind

calcium in vitro (Korsgren et al. 2010), but the effect of calcium on full-length protein 4.2 is not

known.

Protein 4.2 shares over 30 % sequence identity with the majority of the TG family members,

which suggests a clear evolutionary relationship (Murzin et al. 1995). In fact, the genes for

protein 4.2, TG5 and TG7 are arranged in tandem on the 15q15.2 region of human chromosome

15 (Grenard et al. 2001). The genes encoding TG2, TG3 and TG6 are found on the 20q11

segment of human chromosome 20. In mouse these six genes are all found on distal chromosome

2. These findings suggest that these genes were duplicated from a single gene followed by

redistribution to two distinct chromosomes in the human genome.

Interestingly, human protein 4.2 shares the highest amino acid sequence identity with TG2

(33 %) which is found, among many other tissues, in human erythrocytes (Chen and Mehta 1999,

Lee et al. 1986). The role of TG2 in red blood cells is still not clear, but there is evidence that it

may be involved in programmed cell death (Sarang et al. 2007). Calcium-activated TG2 in

erythrocytes has been found to catalyze the formation of high-molecular weight polymers which

contain AE1, ankyrin, spectrin, protein 4.1, catalase and hemoglobin (Lorand and Graham 2003).

The main TG2-reactive site on AE1 is Gln30 in the cytoplasmic domain (Murthy et al. 1994).

These polymers have been isolated from the red blood cells of sickle cell anemia (Lorand et al.

1980) and Hb-Köln (Lorand 2007, Lorand et al. 1987) patients. It is believed that TG2 may

contribute to the decreased life span of these erythrocytes by exposing cell surface epitopes that

are recognized by macrophages (Lorand and Graham 2003).

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The presence of protein 4.2 in these polymers was not examined, but a study using the

purified recombinant mouse proteins cdAE1, protein 4.2 and reticulocyte TG2 found a

relationship between the three proteins (Gutierrez and Sung 2007). In vitro assays revealed that

as calcium concentration increased, TG2-mediated cross-linking of cdAE1 increased, but TG2

binding to cdAE1 decreased. This suggests that the conformational change associated with

calcium-binding by TG2, and necessary for cross-linking activity, causes it to dissociate from

cdAE1. Protein 4.2 stabilized binding of TG2 to cdAE1 and inhibited TG2-mediated cross-

linking. It is not known what the reason or mechanism for this inhibition could be in

erythrocytes. As mentioned above, it is not known whether or not full-length protein 4.2 binds

calcium in vivo or undergoes a conformational change in its presence. Protein 4.2 may prevent

TG2 calcium binding or dissociation from cdAE1, thereby inhibiting cross-linking, by some

other mechanism. The observations offer the intriguing possibility that protein 4.2 could regulate

the mechanical properties of the red blood cell membrane, partly through regulation of TG2-

mediated cross-linking of AE1.

Another suspected role of TG2, independent of its cross-linking activity, is in signaling

(Lorand and Graham 2003). TG2 is able to bind, hydrolyze, and be inhibited by GTP (Lee et al.

1993). TG2 is also able to bind and hydrolyze ATP (Iismaa et al. 1997). Protein 4.2 has a

nucleotide-binding P-loop that binds ATP, but not GTP (Azim et al. 1996), but whether or not it

hydrolyses ATP is not known. It has been proposed that protein 4.2 may assist in creating a

membrane reservoir of ATP needed for membrane transport proteins in the erythrocyte.

TG2 from rat liver was discovered to be identical to a high-molecular weight G-protein,

Ghα, which was shown to mediate the activation of phospholipase C (PLC) by the α1B-

adrenergic receptor in transfected COS-1 cells (Nakaoka et al. 1994). TG2 (Ghα) exists in a

heterodimeric complex with Ghβ (Mhaouty-Kodja 2004), which was shown to be calreticulin

when isolated from rat liver (Feng et al. 1999). Ghβ purified from human heart was also

discovered to be calreticulin by amino acid sequencing. TG2 maintains its signaling function

even when its cross-linking activity has been inactivated by mutation (Lorand and Graham

2003). However, binding of calreticulin to TG2 inhibits both the transglutaminase and signaling

functions of the enzyme (Feng et al. 1999).

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Calreticulin is a calcium-binding protein that plays an important role in calcium storage in

the ER (Nash et al. 1994). The protein has also been found in the nuclear envelope, the nucleus

and the cytoplasm (Rojiani et al. 1991), where it was found to regulate integrins by interacting

with the cytoplasmic tails of their α-subunits in Jurkat cells (human T-lymphoblastoid cells)

(Coppolino et al. 1995). Since mature red blood cells no longer have the ER, which contains the

majority of the cellular calreticulin, it is unclear as to the existence or purpose of a calreticulin-

TG2 interaction in these cells. Evidence of cytoplasmic calreticulin in the mature erythrocyte

was shown in a study of chaperones in differentiating human CD34+ erythroid progenitor cells.

Terminally differentiated cells with expelled nuclei maintained levels of calreticulin, while its

paralog calnexin, a resident ER protein, was lost (Patterson et al. 2009). TG2 has also been found

to accelerate the differentiation of K562 cells (an erythroleukemia cell line) through activation of

Akt phosphorylation and inactivation of ERK1/2 phosphorylation (Kang et al. 2004), suggesting

a possible role for TG2 in erythrocyte differentiation.

1.6.3 Protein 4.2 isoforms

The human protein 4.2 gene consists of 13 exons and 12 introns (Korsgren and Cohen 1991).

Two cDNA sequences were obtained from human reticulocytes which were 2.4 and 2.5 kilobases

(kb) in length (Sung et al. 1990). The minor, longer isoform (Type I) contains a 90-base pair (bp)

in-frame insertion encoding an extra 30 amino acids near the N-terminus (Sung et al. 1992) and

codes for a 74 kDa protein (Korsgren and Cohen 1991). The major, shorter isoform (Type II) is

created by alternative splicing within exon 1, where 90 nucleotides are removed, resulting in

translation of a 72 kDa protein. The first exon contains the 5′-untranslated cDNA sequence and

the first 10 nucleotides of the coding region, which code for the first three amino acids of both

isoforms (Sung et al. 1992). The 90-bp insert in the longer isoform is adjacent to the first exon

and is followed by the rest of intron 1. The splicing donor site for the short isoform matches the

splice site consensus sequence (Ohshima and Gotoh 1987) better than that of the longer isoform,

and may help explain the greater abundance of short isoform mRNA in reticulocytes and

expressed protein in erythrocytes. The function of the 30 amino acid insert in the long protein

isoform is not known, but there is some speculation that it may affect phosphorylation levels of

membrane skeleton proteins (Sung et al. 1992).

Two other protein 4.2 mRNA splicing isoforms have been described where nucleotides

comprising exon 3 are removed from the Type I and Type II isoforms (Bouhassira et al. 1992,

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Korsgren and Cohen 1991). These additional isoforms, designated Type III and IV, have not

been detected at the protein level. Interestingly, only one protein 4.2 mRNA and one protein

isoform have been detected in mouse reticulocytes, which correspond to the Type II isoform in

human (Rybicki et al. 1994). Amino acid numbering of protein 4.2 in this thesis is according to

the Type II isoform unless otherwise indicated.

1.6.4 Tissue distribution and expression in different species

Immunoreactive forms of protein 4.2 have been detected in association with the plasma

membranes of non-erythroid cells and tissues in human and pig, including platelets, brain and

kidney (Friedrichs et al. 1989). In these non-erythroid human tissues, the short 72 kDa isoform,

which is the most common in erythrocytes, was detected. These cells also contain isoforms of the

erythrocyte cytoskeleton proteins spectrin, protein 4.1 and ankyrin (Bennett 1979, Bennett 1985,

Moon and McMahon 1987). This indicates that protein 4.2 may have similar roles in membrane

stabilization in non-erythroid cells.

In another study, immunoreactive protein 4.2 was detected in human lymphocytes, platelets,

spinal cord and bovine brain tissue (Schwartz et al. 1987). In lymphocytes and platelets, protein

4.2 was detected as the shorter isoform and was present mostly in cell membranes. In spinal cord

and brain, protein 4.2 was found in the cell cytosol and membrane fractions, and brain protein

4.2 was detected as a doublet of the short and long isoforms. Protein 4.2 was immunodetected in

bovine and chicken eye lenses and erythrocytes (Sung and Lo 1997). The protein 4.2 detected

corresponded to the minor, long isoform found in human erythrocytes. The lack of detection of

the major, short isoform was attributed to the species-specific nature of the anti-protein 4.2

antibody used. Other major proteins involved in the erythrocyte membrane-cytoskeletal linkage –

AE1, ankyrin, spectrin, actin and protein 4.1 – have been immunodetected in human eye lens

tissue (Allen et al. 1987). Because these proteins appear and disappear together during lens

maturation, it is suggested that an erythrocyte-like membrane structural organization may exist in

these cells.

In contrast to studies where protein 4.2 was detected in various tissues of different species,

another study suggested that protein 4.2 in the developing mouse is restricted to erythroid cells

(Zhu et al. 1998). The authors detected protein 4.2 mRNA in the erythroid cell-producing organs

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and circulating erythrocytes during embryonic development and in adult mice, but not in other

tissues.

While TGs are found in a wide range of species, from humans to invertebrates, protein 4.2

appears to be the younger, catalytically inactive cousin detected only in mammals and birds. A

Brazilian study collected and lysed red cells from 44 different mammalian species (Guerra-

Shinohara and Barretto 1999). Red cell ghosts were solubilized and run on SDS-PAGE gels

followed by Coomassie Blue staining. Protein 4.2 was detected in human, monkey, gorilla, seal,

members of the cat and dog families, raccoon, mouse, rat, hamster, rabbit, sloth, elephant, camel,

giraffe, deer, sheep, cattle, goat, tapir, dolphin, bat, manatee and opossum. Protein 4.2 was not

detected in guinea pig, swamp rat, or horse. However, this staining method may not have been

sensitive enough to detect low levels of protein 4.2, which a more sensitive immunodetection

technique might have done.

In addition to the human and mouse cDNA sequences discussed previously, the bovine

erythrocyte cDNA sequence for protein 4.2 has been determined (NCBI Reference Sequence:

NP_776737.1). As well, cDNA sequences similar to erythrocyte protein 4.2 have been

determined for chimpanzee (XP_001156126.1), Norway rat (XP_342504), dog (XP_851181),

horse (XP_001500600), opossum (XP_001364606), and chicken (XP_417393).

Studies in protein 4.2 knock-out mice showed the mice to have mild HS, about 30 %

decrease in AE1 (probably due to loss of membrane by vesiculation), normal spectrin and

ankyrin content, and an intact cytoskeleton (Peters et al. 1999). AE1-mediated ion transport was

decreased in these mouse erythrocytes by about 40 %, a value close to the decrease in AE1

protein, which indicates that the decrease in ion transport may be a function of fewer AE1

proteins.

1.6.5 Structure and properties

Human protein 4.2 is a 691 amino acid protein with unusual solubility and membrane-binding

properties. It’s a peripheral membrane protein, versus an integral membrane protein, yet requires

harsh conditions for removal from IOVs, such as pH of 11 or higher (Steck and Yu 1973). It is

also conformationally unstable in physiological salt solutions and requires low concentrations of

detergent to stay in solution (Dotimas et al. 1993). Protein 4.2 has an N-terminal glycine

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following the initiating methionine, the latter being removed co-translationally (Korsgren et al.

1990, Sung et al. 1990). The N-terminal glycine has been shown to be N-myristoylated (Risinger

et al. 1992). This modification may partially account for the protein’s unusual solubility. N-

myristoylation is an irreversible, co-translational modification important for protein association

with cell membranes, which is required for proper protein localization or function (Wright et al.

2009).

N-myristoylated protein 4.2 (Type II) was found to localize to the plasma membrane when

expressed in Sf9 insect cells (Risinger et al. 1996). No myristoylation was detected in the G2A

mutant of Type II protein 4.2, Gly2 being the site of the modification. The G2A mutant was

detected in an intracellular compartment, indicating its inability to associate with the plasma

membrane. Myristoylation of the minor, unspliced protein 4.2 isoform (Type I) was barely

detectable and this protein was also localized intracellularly. The fifth residue of a protein

substrate helps determine the likelihood of myristoylation by N-myristoyl transferase (NMT),

with small, uncharged residues being more favorable. Type II protein 4.2 has a glycine in this

position, while Type I has a proline here, which results in it being a poor substrate (Towler et al.

1988). Mouse protein 4.2, which corresponds to the shorter human isoform, was myristoylated to

similar levels as the human Type II protein 4.2, and was also found at the plasma membrane. The

mouse protein has a serine at the fifth position, making myristoylation favorable. Similar results

were seen in COS 7 cells, except for the G2A mutant, which was not able to be expressed in

these cells. In subcellular fractionation experiments, homogenized Sf9 cells were spun to

separate the soluble and particulate fractions. Human protein 4.2 (Type II) and the G2A mutant

were both found to be associated with the particulate fraction, which contains the cytoskeleton.

This indicates that the myristoylation defect in the G2A mutant does not affect its ability to

interact with proteins present in the particulate fraction.

Protein 4.2 is also palmitoylated at Cys173 (Das et al. 1994). S-palmitoylation is a reversible

post-translational modification where a palmitoyl group is added to a cytoplasmic cysteine

residue by palmitoyl-acyl transferases (PATs) (Bijlmakers and Marsh 2003). Other proteins that

are palmitoylated in human erythrocytes include AE1, ankyrin, protein 4.1, and a subpopulation

of spectrin (Das et al. 1994). Palmitoylation, like myristoylation, adds an extra hydrophobic

moiety to a protein, important for hydrophobic protein-protein or protein-membrane interactions

(Bijlmakers and Marsh 2003). There is no known consensus sequence for palmitoylation, since a

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variety of amino acids can influence the modification. However, it may be the recognition of a

common structural feature on a protein that allows it to be palmitoylated. In peripheral

membrane proteins, such as protein 4.2, cysteines that are close to membrane-associated domains

seem to be preferred for palmitoylation, possibly because of their proximity to membrane-

localized PATs. Protein 4.2 could be depalmitoylated using hydroxylamine, which indicated a

thioester linkage (Das et al. 1994). Depalmitoylated protein 4.2 displayed decreased binding to

protein 4.2-stripped IOVs compared to native protein 4.2.

The three-dimensional structures of several TGs have been solved and can be used as

templates in creating a homology model of protein 4.2. A homology model of protein 4.2 is seen

in Figure 1.8 next to the crystal structure of cdAE1 to show their relative sizes. This homology

model of protein 4.2 was created using human transglutaminase 2 (TG2) as a template instead of

sea bream TG, as was previously done (Toye et al. 2005), since it is a human homologue, is

found in erythrocytes, has the greatest sequence similarity to protein 4.2 and the mouse TG2

interacts with mouse cdAE1 and protein 4.2 (Gutierrez and Sung 2007). The orientation of our

model is turned 180° relative to the previous one so that the palmitoyl group at Cys173 points

upwards towards the conventional placement of the plasma membrane. The published crystal

structure of cdAE1 was also oriented this way so our protein 4.2 model was made to match this

orientation. The orientation of the model is such that the palmitoyl group that attaches to Cys173

would point upwards towards the plasma membrane. Cys173 is located at the top of a predicted

β-hairpin loop that has been found to be important for binding to cdAE1.

1.6.6 Interactions with AE1, ankyrin and spectrin

Protein 4.2’s main point of interaction at the membrane is cdAE1 (Korsgren and Cohen 1986). A

fraction of AE1 extracted from red cells is associated with protein 4.2 (Yu and Steck 1975).

Protein 4.2 purified from red cells bound to protein 4.2-depleted IOVs, and purified cdAE1

competed for this binding. As well, when the cytoplasmic domain was cleaved from AE1 in

these IOVs by mild trypsin treatment, protein 4.2 binding was almost completely abolished.

Purified protein 4.2 was also found to bind strongly to cdAE1 in vitro (Korsgren and Cohen

1988). The presence of AE1 is crucial for the maintenance of protein 4.2 in the red cell, since

AE1-deficient red cells in human (Ribeiro et al. 2000), mouse (Peters et al. 1996), and cow

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Figure 1.8: Crystal structure of the cytoplasmic domain of AE1 (cdAE1) and homology

model of protein 4.2.

The symmetric dimer of cdAE1 (left) was crystallized and its structure determined by X-ray

diffraction (Zhang et al. 2000a). The sites of the HS mutations are indicated on the structure and

the residues missing in kAE1 are shown in red. Residues 1-54 (red dotted line) were not resolved

in the structure. The structure of cdAE1 is shown alongside the homology model of protein 4.2

(right). The protein 4.2 structure was created with SWISS-MODEL (Arnold et al. 2006) using

human TG2 (Liu et al. 2002) as the template. The regions in blue and green of the protein 4.2

model represent the 23 kDa N-terminal cdAE1-binding region. Residues 33-45 are coloured in

light green and are predicted to form a β-strand. Residues 157-181 are coloured in dark green,

and encompass the predicted β-hairpin loop containing the palmitoylatable Cys173 at the top.

Structures of cdAE1 and protein 4.2 are on the same scale. aa: amino acids; Nt: amino-terminus;

Ct: carboxy-terminus.

cdAE1 protein 4.2 homology

model

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(Inaba et al. 1996) are also deficient in protein 4.2. This relationship is not interdependent, since

the absence of protein 4.2, either in mouse knock-outs or HS patients with protein 4.2 mutations,

does not always cause a deficiency in AE1. However, in protein 4.2-deficient red cells, AE1 has

a weaker interaction with the cytoskeleton since it is more easily extracted from cells and has

increased lateral diffusion (Rybicki et al. 1996). This observation points to the role of protein 4.2

as a strengthener of membrane-cytoskeleton linkage, via AE1 interaction.

In a study to determine its cdAE1-binding region, partial digestion of protein 4.2 with

staphylococcal V8 protease and blot-overlay using biotinylated cdAE1 was performed

(Bhattacharyya et al. 1999). The N-terminal region of protein 4.2 encompassing residues 1-238

was found to represent the cdAE1-binding domain. The interaction of protein 4.2 glutathione S-

transferase (GST)-fusion proteins with purified cdAE1 was then performed by blot-overlay.

Residues 157-181 of protein 4.2, which contain the palmitoylatable Cys173, were found to be

critical for the interaction. This binding region is predicted to form a β-hairpin in the centre of

folded protein 4.2 with Cys173 located at the tip of the hairpin shown in dark green in Figure

1.8. Another study used biotinylated protein 4.2 peptides in binding assays with cdAE1 isolated

from red blood cells (Rybicki et al. 1995). Amino acids 33-45 of protein 4.2 were found to

contain a binding site. Arg34 and Arg35 were essential for the interaction and the authors

reasoned that this basic Arg-Arg motif may bind to an acidic region, such as the extreme acidic

N-terminus of cdAE1. Since this region of cdAE1 contains the HS mutation E40K, it is possible

that going from an acidic glutamate residue to a basic lysine residue is enough to repel the basic

Arg-Arg motif of protein 4.2 that may bind there. This Arg-Arg motif is also perfectly conserved

in all nine human TGs, as well as across species from human to mouse, dog and cow. The

conservation of this motif in protein 4.2 suggests it may have an important role, possibly

influencing the protein 4.2-AE1 interaction.

There is also evidence that protein 4.2 associates with ankyrin in red cells. Protein 4.2,

ankyrin and AE1 can be co-immunoprecipitated in a complex from red cells (Bennett and

Stenbuck 1980). As with AE1 deficiencies, ankyrin deficiencies in red blood cells also cause a

decrease in protein 4.2 (Yawata 1994). Su et al. (2006) used far-western blots and pull-down

assays using GST-fusion proteins derived from protein 4.2 to reveal an ankyrin-binding site

located within residues 157-170 of protein 4.2 (Su et al. 2006), which is the same region found to

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bind to cdAE1 in the blot-overlay experiments performed by Bhattacharyya et al. (1999) above,

and the one predicted to form a β-hairpin loop. However, the study by Su et al. (2006) did not

see an obvious interaction between the fusion protein containing these residues of protein 4.2 and

cdAE1. Korsgren and Cohen (1988) also showed an interaction between protein 4.2 and ankyrin,

both purified from red blood cells (Korsgren and Cohen 1988).

Another protein involved in the main linkage of the red cell membrane to the cytoskeleton is

spectrin. Protein 4.2 was found to bind spectrin in solution and to promote the binding of spectrin

to ankyrin-stripped IOVs (Golan et al. 1996), which suggests an additional mode of membrane-

cytoskeleton stabilization by this protein. In another study using blot overlays, Mandal et al..

(2002) showed that biotinylated spectrin was able to bind to full-length protein 4.2 and a 30 kDa

fragment of proteolysed protein 4.2 whose N-terminus began at Gly239 (Mandal et al. 2002).

Spectrin was found to interact with a protein 4.2 peptide comprising residues 440-462 in

solution, and this peptide was able to inhibit the interaction between spectrin and full-length

protein 4.2. This region of protein 4.2 is a highly charged stretch which is predicted to form an α-

helix.

The AE1-ankyrin-spectrin-protein 4.2 complex has been long accepted to comprise the

major link between the membrane and cytoskeleton, with the complex located at one end of the

spectrin tetramer. However, it was recently found that protein 4.2 was able to bind to the other

end of the spectrin tetramer, the end associated with actin. Actin binds to the N-terminus of β-

spectrin, and the adjacent C-terminal end of α-spectrin has a calmodulin-like domain called the

EF-domain. Korsgren et al.. (2010) found that the EF-domain of spectrin bound native and

recombinant protein 4.2 in pull-down assays (Korsgren et al. 2010). This study also showed

inhibition of full-length protein 4.2 binding to the EF-domain of spectrin using a protein 4.2

peptide encompassing residues 380-462. Figure 1.9 shows a diagram of the protein 4.2

polypeptide with important regions of known locations mapped.

1.6.7 Interactions with the Rh complex and CD47

The Rh (Rhesus) complex is a group of membrane proteins that interact with red cell cytoskeletal

proteins and provide another linkage point between the membrane and cytoskeleton (Van Kim et

al. 2006). This linkage helps maintain the structural integrity of the cell. The RH system is a

highly immunogenic and polymorphic blood group system. The complex is composed of the Rh

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Figure 1.9: Diagram of the protein 4.2 polypeptide with important regions mapped. Protein 4.2 is shown from Met1 to A691, with the dotted line denoting a break in the sequence

between residue 462 and the C-terminal amino acids. Important regions with known sequence

locations are indicated. The region of protein 4.2 homologous to the TG active site (266-270) is

inactive in protein 4.2. The region of protein 4.2 homologous to the TG Ca2+

-binding domain

(423-432) is shown, but Ca2+

-binding by full-length protein 4.2 has not been demonstrated.

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protein (D, CcEe), Rh-associated glycoprotein (RhAG), LW (also known as Intracellular

Adherence Molecule 4, or ICAM-4), CD47 and glycophorin B (GPB). Functional studies in

yeast (Marini et al. 2000) and Xenopus oocytes (Westhoff et al. 2002) have implicated RhAG as

an ammonium transporter. Studies in human red blood cells indicate that RhAG mediates

transfer across the membrane of ammonia (Ripoche et al. 2004) and possibly CO2 (Bruce 2008).

CD47 (Integrin Associated Protein, IAP) is a highly glycosylated, five-TM domain protein with

an IgV-like domain at its N-terminus (Van Kim et al. 2006). The integrin-CD47 complex

couples to G proteins to form a signaling complex (Brown 2001). However, integrins are not

expressed in mature erythrocytes (Lindberg et al. 1994), indicating other roles for CD47 in red

cells. One proposed role has been as a marker for self in mature red blood cells, preventing their

clearance by macrophages (Oldenborg et al. 2000).

Evidence for an interaction between protein 4.2 and CD47 comes from studies where red

cells from HS patients deficient in protein 4.2, due to protein 4.2 frameshift deletions, also have a

deficiency in CD47 (Bruce et al. 2002, Mouro-Chanteloup et al. 2003). As well, in red cells with

the AE1 mutation S667F (Band 3 Courcouronnes), both AE1 and protein 4.2 are reduced to

about 35 % of normal levels, while CD47, Rh polypeptides and RhAG are reduced to about 60 %

(Toye et al. 2008). A strong association at the membrane between the Rh complex and the AE1

complex, through CD47 interaction with protein 4.2, creates a strong antigenic entity at the red

cell surface. This may allow for the prolonged survival of red cells by allowing them to evade

macrophages in the immune system by serving as a marker of self. Direct evidence of association

between the AE1 and Rh protein complexes comes from a study where Rh proteins co-

immunoprecipitated with AE1 from red blood cells (Bruce et al. 2003). The authors speculated

that there is a functional role for the association of these complexes into a macrocomplex, such

as a CO2/O2 gas exchange unit (metabolon) in the red cell. Figure 1.10 shows a model of the

AE1 and Rh protein complexes linking the plasma membrane and cytoskeleton. Protein 4.2 is

shown as the protein linking the two complexes through its interactions with cdAE1 and CD47.

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Figure 1.10: Schematic model of AE1 and Rh protein complexes at the red cell membrane. The integral membrane and cytoskeletal proteins of the AE1 and Rh protein complexes are

shown, as well as those proteins involved in linking the two complexes. Modified from Bruce et

al. (2003).

(AE1) (AE1)

P cdAE1 cdAE1

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1.6.8 HS mutations in protein 4.2

Eleven protein 4.2 mutations associated with HS have been discovered so far in humans

(Satchwell et al. 2009, van den Akker et al. 2010a). These mutations are summarized in Table

1.3. All of these mutations, in the homozygous state or compound heterozygous state, result in a

complete, or near complete, absence of protein 4.2 in red cells. As mentioned above, deficiency

of protein 4.2 occurs with a concomitant deficiency in CD47. Most of the protein 4.2 mutations

result in premature termination of translation leading to truncated protein products. The

deficiency of protein 4.2 seen is either due to degradation of these protein products or to mRNA

instability, since a reduced number of transcripts is often observed. The rest of the defects are

due to missense mutations resulting in protein products with an amino acid substitution. Protein

4.2 Nippon (see Table 1.3) was the first variant discovered (Bouhassira et al. 1992). This

mutation results in less than 1 % of normal protein 4.2 expression in red blood cells. However,

when this mutant was co-expressed with AE1 in Xenopus oocytes it displayed similar binding to

AE1 compared to wild-type protein 4.2 (Toye et al. 2005). Protein 4.2 Nippon was also able to

localize to the plasma membrane in the absence of AE1 in these cells, similar to wild-type

protein 4.2.

The mechanism of loss of Protein 4.2 Nippon in the red cell, for example, by mRNA or

protein degradation, is unknown. It may be that with successful expression and localization in

red cells, Protein 4.2 Nippon would be functional in that context. In four out of five compound

heterozygotes, Protein 4.2 Nippon is one of the defective alleles. Protein 4.2 Komatsu

(homozygous) and Protein 4.2 Tozeur (compound heterozygous with p4.2 Nippon) (see Table

1.3) displayed impaired binding to AE1 and did not localize to the plasma membrane when

expressed alone or when co-expressed with AE1 in Xenopus oocytes (Toye et al. 2005). This

shows that impaired protein 4.2 binding to AE1 can be associated with HS.

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Table 1.3: HS mutations in protein 4.2

Protein 4.2

variant

Mutation Abnormal allele Reference

Lisboa ΔG at base 174 or 175 in exon 2

introduces Stop codon

Homozygous (Hayette et al. 1995)

Fukuoka Trp119Stop (Type I) Compound heterozygous with

p4.2 Nippon

(Takaoka et al. 1994)

Nippon Ala142Thr (Type I) Homozygous (Bouhassira et al. 1992)

Komatsu Asp175Tyr (Type I) Homozygous (Kanzaki et al. 1995b)

Notame GA in intron 6 splice donor

site introduces Stop codon

Compound heterozygous with

p4.2 Nippon

(Matsuda et al. 1995)

Tozeur Arg310Gln (Type I) Compound heterozygous with

p4.2 Nippon

(Hayette et al. 1995)

Shiga Arg317Cys (Type I) Compound heterozygous with

p4.2 Nippon

(Kanzaki et al. 1995a)

Chartres I Tyr435 Stop Compound heterozygous with

p4.2 Chartres II

(van den Akker et al.

2010a)

Chartres II ΔA1176-T1177 in exon 9

introduces Stop codon

Compound heterozygous with

p4.2 Chartres I

(van den Akker et al.

2010a)

Hammersmith Arg593Stop (Type I) Homozygous (Bruce et al. 2002)

Nancy ΔG in codon 287 in exon 7

introduces Stop codon

Homozygous (Beauchamp-Nicoud et

al. 2000)

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1.7 Thesis focus

The hypothesis of this thesis is that HS mutations in the cytoplasmic domain of AE1 cause

impaired protein 4.2 binding. This occurs either because of changes in cdAE1 conformation

resulting in misfolded binding regions, or because of changes in the cdAE1 interaction surface of

the properly folded protein.

1.7.1 Effect of HS mutations on the structure and stability of the cytoplasmic

domain of AE1 (Chapter 2)

The cause of the deficiency of protein 4.2 in red cells carrying the three HS mutations, E40K,

G130R and P327R located in cdAE1 is unknown. I hypothesize that these cdAE1 mutations

cause either gross conformational changes in the domain or changes in the direct binding surface

of cdAE1. Either would result in impaired binding of protein 4.2, leading to the loss of protein

4.2 during red cell differentiation. In Chapter 2, the structure and conformational stability of

purified cdAE1 with these mutations was studied using a variety of biophysical methods. The

results of a similar study, carried out by Biochemistry project student Allison Pang under my

supervision, comparing the structure and conformational stability of cdAE1 with kidney cdAE1

are presented in the Appendix.

1.7.2 Protein 4.2 localization and interaction with wild-type and HS mutants of

AE1 in HEK-293 cells (Chapter 3)

Mutations in protein 4.2 and three mutations in cdAE1 have been shown to cause protein 4.2

deficiency and HS. In the case of Protein 4.2 Tozeur and Komatsu, part of the molecular basis of

HS can be attributed to impaired interaction with cdAE1. I hypothesize that the three cdAE1 HS

mutations result in impaired binding to protein 4.2 leading to HS. This binding impairment may

be caused by conformational changes of cdAE1 with these HS mutations, as addressed in

Chapter 2, or by a change in the interaction surface of properly folded cdAE1. In Chapter 3, the

interaction of protein 4.2 with AE1 and the three HS mutants (E40K, G130R and P327R) was

studied in HEK-293 cells using pull-down assays. Protein 4.2 interaction with kAE1 and

AE1SAO was also examined. The localization of protein 4.2 in the absence or presence of wild-

type and HS mutant AE1 proteins was also studied in transfected HEK-293 cells using

immunofluorescence and confocal microscopy. The plasma membrane localization of wild-type

protein 4.2 and an acylation mutant of protein 4.2, G2A/C173A (GC), were compared in the

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absence or presence of AE1. The degree of cytoskeletal attachment of wild-type and GC protein

4.2 were compared by subcellular fractionation studies.

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2 Chapter 2: Structure and stability of hereditary

spherocytosis mutants of the cytoplasmic domain of the

erythrocyte anion exchanger 1 protein

Adapted with permission from Bustos, S.P. & Reithmeier, R.A.F. Structure and stability of

hereditary spherocytosis mutants of the cytosolic domain of the erythrocyte anion exchanger 1

protein. Biochemistry, 45, 1026-1034. Copyright 2006 American Chemical Society

(http://pubs.acs.org/doi/abs/10.1021/bi051692c) (Bustos and Reithmeier, 2006). Jing Li

contributed to this work by the generation of the cdAE1 HS mutants. Jing Li also assisted in the

design of the purification protocol of the His6-tagged cdAE1 proteins. I designed and conducted

the experiments and analyzed all of the results.

2.1 Abstract

The N-terminal cytoplasmic domain of AE1 anchors the cytoskeleton to the membrane. Several

proteins bind to cdAE1, including protein 4.2, a cytoskeletal protein. Three mutations in cdAE1

are associated with HS and decreased levels of protein 4.2 in erythrocytes. In this study these

cdAE1 mutants (E40K, G130R and P327R) were expressed and purified from Escherichia coli.

Sedimentation equilibrium (SE) experiments using the analytical ultracentrifuge showed that the

wild-type and mutant proteins are dimers. No difference in secondary structure between mutant

and wild-type proteins was detected using CD analysis. The wild-type and mutant proteins

underwent similar pH-dependent conformational changes when monitored by intrinsic

tryptophan fluorescence. Urea denaturation of proteins monitored by intrinsic fluorescence

showed no significant differences in the sensitivity of the proteins to this chemical denaturant.

Thermal denaturation monitored by CD and by calorimetry revealed that only the P327R mutant

had a significantly lower midpoint of transition (~ 5 °C) than the wild-type protein, suggesting a

modest decrease in thermal stability. The results show that the HS mutant cdAE1 proteins do not

differ to any great extent in structure from the wild-type protein, suggesting that the HS

mutations may directly affect protein 4.2 binding.

2.2 Introduction

Three cytoplasmic domain mutations associated with HS occur with a normal amount of AE1 at

the red cell membrane. These mutations are E40K (Band 3 Montefiore), G130R (Band 3

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Fukuoka), and P327R (Band 3 Tuscaloosa). These mutations are associated with an 88 %

(homozygous state), 55 % (homozygous state) and 29 % (heterozygous state) decrease in the

amount of protein 4.2 in the red cell, respectively (Inoue et al. 1998, Jarolim et al. 1992a,

Rybicki et al. 1993). Maintaining normal levels of protein 4.2 in the red cell may depend on the

ability of protein 4.2 to assemble with AE1 during biosynthesis since these proteins have been

shown to associate early in erythroblast differentiation (van den Akker et al. 2010a). All three of

these HS mutations result in the incorporation of a positively charged amino acid into a domain

with predominantly negative surface potential. While introduction of a positive charge at these

sites may perturb direct protein 4.2 interactions, these HS mutations may also cause

conformational changes in the protein disrupting the binding site altogether.

The goal of this work was to express and purify the three HS mutant cdAE1 proteins and to

compare their structure and conformational stability to the wild-type cdAE1 protein. The

Memphis I cdAE1 protein was included in the study as an asymptomatic mutant control. The

conformational stability of the Band 3 Tuscaloosa/Memphis I double mutant (P327R/K56E) was

also examined in order to determine the role of the Memphis I background on this HS mutant. I

hypothesize that the three HS cytoplasmic domain mutations in AE1 affect either the folding and

conformational stability of cdAE1, or the direct binding surface of cdAE1. Either may result in

impaired binding of protein 4.2 and lead to its deficiency in the red blood cells of patients with

these mutations.

2.3 Materials and methods

2.3.1 Materials

The following is a list of materials used and their suppliers: pcDNA3 vector (Invitrogen, San

Diego, CA, USA); QuikChange® site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA);

mutagenic primers (ACGT Corp., Toronto, ON, Canada); pETBlue-1 vector and

Tuner(DE3)pLacI Escherichia coli competent cells (Novagen, Madison, WI, USA); growth

media for Escherichia coli (BD, Sparks, MD, USA); chloramphenicol and carbenicillin (Sigma,

St. Louis, MO, USA); isopropyl-β-D-thiogalactopyranoside (IPTG) (Bioshop, Burlington, ON,

Canada); nickel-nitrilotriacetic acid (Ni-NTA) agarose resin (QIAGEN, Germantown, MD,

USA); sequanal grade urea (Pierce, Rockford, IL, USA).

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2.3.2 Plasmid construction and mutagenesis

cdAE1 from Met1 to Ser356 was amplified by PCR from full-length human AE1 located on the

pcDNA3 vector and cloned into the expression vector pETBlue-1, which contains an IPTG-

inducible T7lacO promoter (Giordano et al. 1989). The reverse primer included DNA encoding

six histidine residues which are located at the C-terminus of the protein to provide a

hexahistidine tag for purification. The HS mutants, the K56E variant and the P327R/K56E

double mutant were constructed using the QuikChange® system with complementary mutagenic

primers and the mutations were confirmed by sequencing by the ACGT Corp.

2.3.3 Protein expression and purification

All protein expression was performed in the Escherichia coli strain Tuner(DE3)pLacI. This

strain of E. coli is a derivative of the BL21 strain, but has a mutation in the lac permease gene

which allows uniform entry of IPTG into all cells and reduces basal level protein expression.

pETBlue-1 vectors were expressed in Tuner(DE3)pLacI which contains the gene for T7 RNA

polymerase under the control of the IPTG-inducible lacUV5 promoter (Studier and Moffatt

1986). Cells were grown at 37 °C in Luria Bertani (LB) media containing both carbenicillin (50

µg/ml) and chloramphenicol (34 μg/ml) until the cell density reached A600 of 0.6. Protein

expression was induced with 1 mM IPTG, then cells were grown for an additional 4 h at 37 °C

and cell density reached A600 of 1.3. Cells were harvested by centrifugation (4,400 ×g, 30 min)

and solubilized in 80 ml of lysis buffer per litre of cell culture (lysis buffer: 50 mM sodium

phosphate, 300 mM sodium chloride, 5 mM imidazole, 0.2 % β-mercaptoethanol (βME), 0.2 %

Triton X-100, pH 8.0) containing the following protease inhibitors: 0.70 µg/ml pepstatin, 2.0

µg/ml aprotinin, 4.3 µg/ml leupeptin, and 0.28 µg/ml phenylmethanesulfonyl fluoride (PMSF).

Solubilized cells were allowed to sit on ice for 30 min after addition of lysozyme to 1 mg/ml,

followed by sonication at 40 % duty cycle for 2 min on ice. Purification was carried out by a

batch procedure at 4 °C using 1 ml of Ni-NTA agarose resin (QIAGEN) per 80 ml of cell lysate.

Resin was washed twice with 10 ml of wash buffer (50 mM sodium phosphate, 300 mM sodium

chloride, 20 mM imidazole, 0.2 % βME, pH 8.0). Proteins were eluted three times with 1 ml of

elution buffer (50 mM sodium phosphate, 300 mM sodium chloride, 250 mM imidazole, 0.2 %

βME, pH 8.0). Protein solutions were applied to pre-equilibrated PD-10 gel filtration columns

(Amersham Biosciences) for the purpose of buffer exchange into 10 mM ammonium bicarbonate

and subsequent lyophilization. Proteins were lyophilized overnight and stored at -20 °C. Protein

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purity was determined to be > 95 % by Coomassie staining of samples following SDS-PAGE.

Protein concentrations were determined by the Bio-Rad Bradford total protein assay (Bradford

1976). Protein samples were subjected to mass spectroscopy on an electrospray ionization time-

of-flight (ESI-TOF) instrument for molecular weight analysis to verify the identity of the

proteins. A cysteineless protein where both cysteines of cdAE1 were mutated to alanine

(C201A/C317A) was also submitted for analysis.

2.3.4 Analytical ultracentrifugation

Proteins were freshly purified using Ni-NTA resin and buffer-exchanged into sedimentation

buffer using PD-10 columns without freeze-drying. SE experiments were performed at 20 °C on

an Optima XL-A / XL-I Analytical Ultracentrifuge (Beckman Instruments, Palo Alto, CA) using

an AN50-Ti rotor, quartz windows, and standard six-sector charcoal-filled Epon centerpieces.

Samples were centrifuged at 18,000 × g, 32,000 × g and 50,000 × g for 27 h at each speed to

ensure equilibrium was reached before absorbance measurements were taken. Global analysis of

the data was performed using XL-A / XL-I data analysis software (Origin version 4.1) from

Beckman Instruments. SE experiments were performed on three different concentrations of each

protein (0.32 mg/ml, 0.64 mg/ml and 1.29 mg/ml) in 10 mM sodium phosphate, 50 mM sodium

chloride, pH 7.5.

2.3.5 Circular dichroism

Freeze-dried cdAE1 proteins were dissolved in 10 mM sodium phosphate, 50 mM sodium

fluoride, 1 mM dithiothreitol (DTT), pH 7.0 (Johnson 1990). CD spectra from 190 to 260 nm and

1 nm data pitch were recorded on a Jasco J-810 spectropolarimeter using a final protein

concentration of 0.3 mg/ml cdAE1 in a 1 mm path-length cell at 24 °C. Deconvolution of spectra

was done using the CDPro software package (Sreerama and Woody 2000) for the determination

of secondary structure percentages. For temperature denaturation, samples were heated from 30

°C to 86 °C with a 2 °C data pitch at a scan rate of 2 °C /min and ellipticity was measured at 208

nm. Thermal denaturation data were fit to a standard equation by nonlinear least-squares

regression (using SigmaPlot 2004 version 9.0) assuming a two-state transition for a dimeric

species. Tm is the temperature at the transition midpoint of thermal unfolding. Experiments were

repeated using at least three different preparations of purified protein.

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2.3.6 pH dependence of intrinsic fluorescence

Stock solutions of cdAE1 proteins were made by dissolving proteins in 50 mM sodium

phosphate, 50 mM sodium borate, 70 mM sodium chloride, 1 mM DTT, pH 7.0 (Appell and Low

1981). Stock protein solution was diluted 50 times into the same buffer pre-adjusted to the

desired pH to a final protein concentration of 0.17 mg/ml. Samples were equilibrated for at least

two h at room temperature prior to measurement. In all fluorescence experiments the intrinsic

fluorescence of the proteins was monitored using a Fluorolog FL3-22 fluorescence

spectrophotometer at 24 °C. The excitation wavelength was 290 nm and the fluorescence

emission was measured from 300 to 420 nm for each sample at each pH. Experiments were

repeated using three different preparations of purified protein.

2.3.7 Calorimetry

cdAE1 proteins were dissolved in 10 mM sodium phosphate, 50 mM sodium chloride, pH 7.5 to

a final concentration of 1.3 mg/ml. Heat capacity measurements were obtained on a MicroCal

VP-DSC differential scanning calorimeter. Samples were heated from 25 °C to 90 °C at a rate of

1.5 °C /min. Temperature denaturation data were fit to a two-state transition model using the

Origin 7.0 data analysis software which employs the Marquardt-Levenberg algorithm for least

squares regressions. Experiments were repeated using at least three different preparations of

purified protein.

2.3.8 Urea denaturation measured by intrinsic fluorescence

Stock solutions of cdAE1 proteins were made by dissolving proteins in 50 mM sodium

phosphate, 50 mM sodium borate, 70 mM sodium chloride, 1 mM DTT, pH 7.0. Stock protein

solution was diluted 50 times into the same buffer pre-adjusted to the desired urea concentration

to a final concentration of 0.17 mg/ml. Samples were equilibrated in denaturant at room

temperature for two h prior to measurement. The excitation wavelength was 290 nm and the

fluorescence emission was measured from 300 to 420 nm for each protein at each urea

concentration at 24 °C. Urea denaturation data were fit to a standard equation by nonlinear least-

squares regression (using SigmaPlot 2004 version 9.0) assuming a two-state transition of a

dimeric species. Cm is the urea concentration at the midpoint of the unfolding transition.

Experiments were repeated using six different preparations of purified protein, except for

experiments on the K56E mutant which were repeated using three protein preparations.

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2.3.9 Limited tryptic digestion

Proteins were dissolved in 50 mM sodium phosphate, 50 mM sodium borate, 70 mM sodium

chloride, pH 7.0. Trypsin dissolved in the same buffer was added to each sample to a final

concentration of 6.5 μg/ml (final protein concentration was 0.3 mg/ml). The digestion reaction

was allowed to proceed for five min at room temperature and was stopped by the addition of an

equal volume of 2 × sample buffer containing SDS and βME. Samples were run on SDS-PAGE

and detected by Coomassie blue staining. The relative sizes of the protein bands were determined

by the loading of Protein Molecular Weight Markers (Fermentas, Burlington, ON) on the gel.

2.4 Results

2.4.1 Expression and purification of cdAE1 and cdAE1 HS variants in

Escherichia coli

The region of cdAE1 encompassing amino acids 1-356 of the protein was subcloned from full-

length wild-type AE1 cDNA on the pcDNA3 vector into a pETBlue-1 expression vector and

expressed in E. coli Tuner(DE3)pLacI cells as described in the Methods. These residues extend

from the N-terminus of AE1 to the last residue visible in the crystal structure, followed directly

by a His6 tag. The purification of the His6-tagged wild-type cdAE1 and cdAE1 carrying the

E40K, G130R, and P327R mutations was carried out by Ni-NTA affinity chromatography. This

purification method yielded over 20 mg of protein per litre of cell culture of more than 95 %

purity as determined by SDS-PAGE. The cdAE1 proteins ran as monomers of approximately 41

kDa on SDS-PAGE (data not shown). Mass spectroscopy analysis revealed expected molecular

masses for all proteins with additional molecular masses of either 76 or 152 Da on all of the

proteins (data not shown). These additions were not seen with the cysteineless (C201A/C317A)

mutant. The extra molecular masses were likely due to a βME adduct on one (+76 Da) or both

(+152 Da) of the cysteines.

2.4.2 Analytical ultracentrifugation of wild-type and HS mutant cdAE1

proteins

SE experiments using the analytical ultracentrifuge were carried out to determine whether or not

the HS mutations affected the oligomeric structure of the cdAE1 protein. Purified wild-type

cdAE1 has been shown to exist as a dimer (Appell and Low 1981, Colfen et al. 1996, Wang et

al. 1992a, Zhang et al. 2000a). The predicted sequence molecular weight (MWseq) of the wild-

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type cdAE1 with a His6 tag is 40 866. SE experiments were performed on the wild-type, HS and

K56E mutant cdAE1 proteins using different protein concentrations and rotor speeds, and the

data were fit to a single ideal species model. The analysis gave apparent molecular weights

(MWapp) that were twice that of the MWseq which indicated that the wild-type, HS and K56E

mutant proteins existed as stable dimers in solution. No evidence of concentration dependence of

the molecular weight values was observed over a range from 0.32 – 1.29 mg/ml. The ratios of the

MWapp to the MWseq determined by SE experiments are listed in Table 2.1, and a

representative plot from the SE experiments on wild-type cdAE1 run at 32 000 × g is shown in

Figure 2.1.

2.4.3 Secondary structure analysis of wild-type and HS mutant cdAE1 proteins

CD analysis was carried out to determine whether the HS mutations affect the secondary

structure of the cdAE1 protein. The CD spectrum of the wild-type cdAE1 has been shown to

exhibit a negative extreme at 208 nm and a shoulder at 223 nm typical of an α-helical structure-

containing protein (Appell and Low 1981). Figure 2.2 shows the CD spectra obtained from the

wild-type and HS mutant cdAE1 proteins. The spectra from this study display the same

characteristics as those obtained from native cdAE1, and those of the mutant cdAE1 proteins

overlap with the spectrum of the wild-type protein. Similar spectra were obtained for the

Memphis I mutant protein (data not shown). The helical content of the cdAE1 protein obtained

from the crystal structure was 26 % (Zhang et al. 2000a). The helical content of the wild-type,

HS and K56E mutant cdAE1 proteins obtained by deconvolution of the CD spectra shown in

Table 2.1 are similar to that found in the crystal structure. The HS mutations did not result in any

major change in the secondary structure of cdAE1.

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Figure 2.1: Analytical ultracentrifugation of wild-type cdAE1. Absorbance is plotted as a function of radius, and the residuals from fitting the data to a single

ideal species model are shown. The SE experiment was performed at 20 °C with a rotor speed of

32000 × g for 27 h. The protein concentration was 0.64 mg/ml and the protein was in 10 mM

sodium phosphate, 50 mM sodium chloride, pH 7.5.

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Figure 2.2: CD spectra of wild-type cdAE1 and HS mutants.

Purified proteins were dissolved in 10 mM sodium phosphate, 50 mM sodium fluoride, 1 mM

DTT, pH 7.0 and scanned at 24 °C in a 1 mm cell in a Jasco J-810 spectropolarimeter. The final

concentration of all proteins was 0.3 mg/ml. Spectra are expressed as mean residue ellipticity.

Wild-type cdAE1 (closed circles); E40K (open circles); G130R (closed triangles); P327R (open

triangles).

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2.4.4 Effect of pH on the intrinsic fluorescence of the wild-type and HS mutant

cdAE1 proteins

cdAE1 has been shown to undergo a reversible pH-dependent conformational change that is

characterized by a dramatic increase in the intrinsic tryptophan fluorescence and an increase in

Stokes radius without a change in secondary structure at alkaline pH (Wang et al. 1992a). A

hydrogen bond between W105 of one subunit and D316 of the other subunit, that is present in

the lower pH conformation, is broken at neutral pH (Zhou and Low 2001). At alkaline pH the

conformation of cdAE1 changes extensively, as the peripheral protein binding domain moves

away from the dimerization arm. In the present study, intrinsic tryptophan fluorescence was

measured over a pH range to determine whether or not the HS mutations caused a difference in

the pH-dependent conformational change of the cdAE1 protein. Figure 2.3 shows the intrinsic

fluorescence emission intensity at 347 nm of the wild-type and HS mutant cdAE1 proteins as a

function of pH. The proteins exhibited a similar increase in fluorescence intensity, representing a

dequenching of tryptophans, and an increase in peak wavelength (red-shift) at alkaline pH (data

not shown), indicating that the tryptophans were exposed to a more polar environment. There

was a moderate increase in fluorescence emission intensity between pH 5 and 8, followed by a

more dramatic increase between pH 8 and 10 for all samples. The results indicate that the wild-

type and mutant proteins undergo similar two stage pH-dependent conformational changes.

2.4.5 Thermal denaturation of wild-type and HS mutant cdAE1 proteins by

circular dichroism

The HS mutations did not appear to affect the folded structure or oligomeric state of the cdAE1

protein, but the mutations may affect the thermal stability of this domain. Thermal denaturation

using CD was performed in order to determine if the HS mutants lowered the thermal stability of

the cdAE1 protein. Figure 2.4 shows the results of thermal denaturation as monitored by CD at

208 nm at pH 7.0. The thermal denaturation of all the proteins was irreversible as determined by

rescanning of cooled samples after heating to 86 °C. The data were fit to a two-state transition

model for the purpose of obtaining the midpoints of the thermal denaturation transitions (Tm) so

that they could be compared to one another. Because the transitions were irreversible, these Tms

were relative and were considered to be the apparent Tms of the transitions. The Tm for each

protein is listed in Table 2.1. The wild-type protein had a Tm of 69.0 °C. The P327R cdAE1

protein had a Tm that was 5 °C lower than wild-type.

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Figure 2.3: Intrinsic fluorescence intensity of wild-type cdAE1 and HS mutants as a

function of pH.

Purified proteins were dissolved in 50 mM sodium phosphate, 50 mM sodium borate, 70 mM

sodium chloride, 1 mM DTT, pH 7.0 to a stock concentration of 10 mg/ml. All samples were

filtered through a 0.22 µm syringe filter and 6 μl of each stock protein solution was added to 294

μl (50 × dilution) of the same buffer preadjusted to the desired pH. The final concentration was

0.17 mg/ml for all proteins, and the actual pH was measured in each reaction tube using a pH

meter. The intrinsic fluorescence intensity at 347 nm of wild-type cdAE1 (closed circles), E40K

(open circles), G130R (closed triangles) and P327R (open triangles) is plotted as a function of

pH.

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Table 2.1: Effects of HS mutations on structure and stability of cdAE1 protein

cdAE1

protein

MWapp/

MWseq α-helix (%) Tm (°C) (CD) Tm (°C) (calorimetry) Cm (M)

WT 1.981a 28.5

b ± 3.3 69.0

c ± 2.2 66.2

e ± 1.5 4.79

d ± 0.31

E40K 2.026 28.3 ± 1.7 68.4 b ± 0.3 64.9

b ± 0.3 4.73 ± 0.13

G130R 1.950 27.9 ± 1.5 68.7 b ± 0.4 65.5

b ± 0.3 4.85 ± 0.09

P327R 1.921 28.3 ± 2.6 64.0 b ± 0.4 61.5

b ± 0.2 4.56 ± 0.20

K56E 2.011 26.7 ± 4.4 67.7 d ± 1.5 66.5

f ± 2.0 4.60

b ± 0.15

a Data from nine SE experiments (three different protein concentrations run at three different speeds) were

globally fit to a single ideal species model to obtain the MWapp. b

Values are averages of measurements

obtained from three different preparations of purified protein. c

Values are averages of measurements obtained

from nine different preparations of purified protein. d Values are averages of measurements obtained from six

different preparations of purified protein. e Values are averages of measurements obtained from twelve different

preparations of purified protein. f

Values are averages of measurements obtained from seven different

preparations of purified protein. For CD and calorimetry measurements Tm is the apparent temperature midpoint

of the thermal denaturation transition. Cm is the concentration of urea at the apparent midpoint of the urea-

induced unfolding transition.

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The E40K, G130R and Memphis I (data not shown) cdAE1 proteins did not have significantly

lower melting temperatures. The P327R mutant appears to be less thermally stable than the other

proteins, while the E40K and G130R mutants have similar thermal stabilities to the wild-type

protein.

2.4.6 Thermal denaturation of wild-type and HS mutant cdAE1 proteins by

calorimetry

Thermal denaturation was also performed using differential scanning calorimetry (DSC). The

melting temperature of the wild-type cdAE1 protein measured by DSC has been shown to be pH

dependent and was reported to be 67 °C at pH 7.51 (Appell and Low 1981). Figure 2.5 shows the

results of the baseline-subtracted DSC scans. The thermal denaturation of all the proteins was

irreversible since rescanning cooled samples after heating to 90 °C yielded scans with no

transition. The data were fit to a two-state transition model for the purpose of obtaining

temperature midpoints of transition so that they could be compared to one another. As with the

CD analysis, because the transitions were irreversible, the Tms were relative and were considered

to be the apparent Tms of the transitions. These Tms agree with the temperature corresponding to

the maximum excess heat capacity (Cp), which was used in other studies of proteins with

irreversible thermal denaturation profiles (Idakieva et al. 2005, Nielsen et al. 2003). The Tm

values for each protein at pH 7.5 obtained using DSC are listed in Table 2.1. The wild-type

protein had a Tm of 66.2 °C, which is in agreement with previous studies. The P327R cdAE1

protein had a Tm that was 5 °C lower than wild-type. The E40K, G130R and K56E cdAE1

proteins had similar melting temperatures to wild-type. The P327R/K56E double mutant protein

had a Tm (62.2 °C) that was not significantly different than that of the P327R single mutant (61.5

°C). Although the transition midpoints differ slightly from those obtained from the CD thermal

denaturation experiments, the relative values are similar. The thermal denaturation studies show

that only the P327R mutation results in very modest thermal destabilization of the cdAE1.

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Figure 2.4: Thermal denaturation of wild-type cdAE1 and HS mutants monitored by CD.

Purified wild-type and HS mutant cdAE1 proteins were dissolved in 10 mM sodium phosphate,

50 mM sodium fluoride, 1 mM DTT, pH 7.0 for CD measurements. The final concentration of

the proteins was 0.3 mg/ml. Ellipticity was measured at 208 nm as the temperature was increased

from 30 °C to 86 °C in 2 °C increments. Wild-type cdAE1 (closed circles); E40K (open circles);

G130R (closed triangles); P327R (open triangles).

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Figure 2.5: Thermal denaturation of wild-type cdAE1 and HS mutants by DSC.

The proteins were dissolved in 10 mM sodium phosphate, 50 mM sodium chloride, pH 7.5 at a

final protein concentration of 1.3 mg/ml. The heat capacity was measured as the temperature was

increased from 35 °C to 80 °C and the baseline-subtracted values are presented. No transition

was observed after cooling the samples and reheating. Wild-type cdAE1 (closed circles); E40K

(open circles); G130R (closed triangles); P327R (open triangles).

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2.4.7 Urea denaturation of wild-type and HS mutant cdAE1 proteins

Urea denaturation was also used to study the conformational stability of the cdAE1 proteins.

Urea is a chaotropic agent that causes proteins to denature, and proteins with decreased

conformational stabilities will unfold at lower concentrations of urea (Pace 1986). The

concentration dependence of urea denaturation of the wild-type cdAE1 protein at pH 6.0 has

been shown to cause an increase in intrinsic fluorescence intensity as urea concentration

increased, with the midpoint of the transition at about 5 M urea (Zhou and Low 2001). In the

present study urea denaturation of the wild-type and HS mutant cdAE1 proteins at pH 7.0

resulted in an increase of intrinsic fluorescence intensity as tryptophans became dequenched, and

an increase in peak wavelength as protein unfolding exposed tryptophans to a more polar

environment. Figure 2.6 shows emission intensity spectra for the wild-type cdAE1 at different

urea concentrations and the increase in average emission wavelength as a function of urea

concentration for wild-type and HS mutant proteins. The apparent transition midpoints (Cm) for

each protein are listed in Table 2.1. The Cm values for the mutant proteins were similar to the

wild-type protein and their differences were not statistically significant. Dilution of cdAE1

proteins in 8 M urea into buffer to give a final urea concentration of less than 1 M did not result

in refolding of cdAE1 (data not shown), indicating that the unfolding was irreversible.

2.4.8 Limited tryptic digestion of WT and HS mutant cdAE1 proteins

Limited tryptic digestion of cdAE1 proteins was used to measure the folded structures of the

proteins. Trypsin cleaves after lysine and arginine residues in areas of a folded protein that are

accessible to the protease. Regions of the protein where cleavage sites are buried within the

folded structure of the protein or where the backbone is well-ordered will not be readily cleaved.

Wild-type and HS mutant cdAE1 proteins (0.3 mg/ml) were incubated with trypsin (6.5 µg/ml)

for five min at room temperature and the reaction was stopped by addition of an equal volume of

sample buffer containing SDS and βME. Samples were run on SDS-PAGE and visualized with

Coomassie blue staining as seen in Figure 2.7. Cleavage patterns of the G130R and P327R

mutants were similar to the wild-type pattern. The full-length 41 kDa cdAE1 protein was present

along with the appearance of two smaller bands of approximately 21 kDa and 20 kDa, perhaps

representing the two fragments obtained from cleavage at one site of the protein by trypsin.

Undigested wild-type and HS cdAE1 proteins each appear as a single 41 kDa band.

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The E40K protein gave a different cleavage pattern, with a prominent band of about 36 kDa

and the 21 kDa band. The E40K mutant introduces a new trypsin cleavage site at lysine 40 and

cleavage at this new site would account for the appearance of the 36 kDa band, which is

approximately 40 residues smaller than the full-length protein. A western blot using an antibody

against the N-terminus of cdAE1 confirmed that the 20 kDa band was derived from the N-

terminal region of the protein (data not shown). The 36 kDa band from the E40K digest was not

detected since its first 40 residues had been cleaved off. The 21 kDa band for all of the proteins

was not detected by the N-terminal antibody indicating that this fragment was derived from the

C-terminal region of the protein. Judging from the amino acid sequence of cdAE1, the possible

single site of cleavage that would result in fragment sizes of 20 kDa and 21 kDa is at K174. This

lysine residue is located in an unstructured region of the peripheral protein-binding domain and

is exposed to solvent. The shorter fragment produced from cleavage at this lysine would be from

the N-terminus. K174 is indicated on the topology model of AE1 in Figure 1.6.

The Memphis I mutant protein removes a potential trypsin cleavage site (K56E) in the

protein, but the wild-type and HS mutant proteins examined still have a lysine at position 56. No

fragment corresponding to cleavage at this site was observed for any of the proteins. Trypsin did

not cleave at this position, indicating that either it was not accessible to the protease or that the

backbone is well-ordered in this region. In fact, according to the crystal structure of cdAE1, K56

is located at the N-terminus of the first β-strand of the protein. The protease digestion results

indicate that the HS mutations did not induce gross misfolding of cdAE1 and that the region

around residue 40 is accessible and disordered, while that of residue 56 is not.

2.5 Discussion

HS mutations in the cytoplasmic domain of AE1 were found to cause no major changes in the

structure of the domain. The HS mutants of cdAE1 retained the dimeric structure of the protein

as seen in analytical ultracentrifugation experiments. Even the P327R mutation which is located

at the N-terminus of an α-helix in the dimerization arm did not interfere with the formation of a

dimer by the protein. The mutants also retained the normal secondary structure of the protein as

seen in CD experiments. The mutations may not be expected to cause a major disruption in the

secondary structure since the E40K mutation is located in a structurally unresolved region and

the G130R residue points away from the protein and into the solvent.

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Figure 2.6: Urea denaturation of wild-type and HS mutant cdAE1 proteins.

Purified wild-type and HS mutant cdAE1 proteins were dissolved in 50 mM sodium phosphate,

50 mM sodium borate, 70 mM sodium chloride, 1 mM DTT, pH 7.0 to a stock concentration of

10 mg/ml. All samples were filtered through a 0.22 µm syringe filter and 6 μl of each stock

protein solution was added to 294 μl (50 × dilution) of the same buffer preadjusted to the desired

urea concentration. The final concentration was about 0.17 mg/ml for all proteins. Samples were

equilibrated for 2 h before conducting measurements. The intrinsic fluorescence emission was

measured from 300 - 420 nm (λex, 290 nm) for each protein at each urea concentration.

(A) Intrinsic fluorescence emission spectra of wild-type cdAE1 are plotted for three urea

concentrations: 0 M (black line), 5 M (dark gray line), 8 M (light gray line). (B) The average

emission wavelength of wild-type cdAE1 (closed circles), E40K (open circles), G130R (closed

triangles) and P327R (open triangles) was calculated from each spectrum and plotted as a

function of urea concentration.

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Figure 2.7: Trypsin digestion of wild-type and HS mutant cdAE1 proteins.

Purified wild-type and HS mutant cdAE1 proteins were dissolved in 50 mM sodium phosphate,

50 mM sodium borate, 70 mM sodium chloride, pH 7.0 and incubated with trypsin dissolved in

the same buffer. Digestion proceeded for 5 min at room temperature and the reaction was

stopped by addition of an equal volume of 2 × sample buffer. The final cdAE1 protein and

trypsin concentrations were 0.3 mg/ml and 6.5 μg/ml, respectively. Digested proteins were run

on SDS-PAGE and detected by Coomassie blue staining.

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Arginine at position 327, instead of proline, could allow continuation of the helix that

follows it and no detectable change in the helical content of this mutant was observed. All three

mutants underwent similar pH-dependent conformational changes as the wild-type protein, as

monitored by intrinsic tryptophan fluorescence, indicating that their folded structures are similar.

This pH-dependent behavior is important physiologically since binding of glycolytic enzymes,

deoxyhemoglobin and cytoskeletal proteins to AE1 in the red cell membrane is very sensitive to

pH.

The E40K mutant displayed a different digestion pattern by limited tryptic digestion since it

introduced a new trypsin cleavage site at residue 40. This indicates that the residue at position 40

is in a disordered region accessible to trypsin. The position of the Memphis I mutation, lysine 56,

at the N-terminus of the first β-strand of cdAE1 prevented cleavage at this site. Even though

residues 40 and 56 are only sixteen residues apart in the primary sequence of the protein, their

environments in the tertiary structure of the protein are quite different. Residue E40’s lack of

resolution in the crystal structure also provides evidence of its surface accessibility and disorder

while K56 is one of the first structured residues. Since trypsin was unable to cleave at lysine 56

in the wild-type and HS mutant proteins, this provides evidence for the similarity in their folded

structures.

The P327R mutation caused a modest thermal destabilization of cdAE1 as seen in thermal

denaturation experiments. Interestingly, the thermal stability of the double mutant, P327R/K56E,

was not significantly different from that of the single P327R mutant, and the thermal stability of

the K56E mutant was similar to that of the wild-type protein. This provides evidence for the first

time that the underlying cause for the HS phenotype in the patient with this double mutant is the

P327R mutation, and not K56E.

Conformational destabilization of HS mutant proteins was not seen in the experiments

where urea was used as the denaturant, not even with the P327R mutant. The lack of

conformational destabilization seen with the P327R mutant could be due to different modes of

unfolding by thermal and chemical denaturation. Intrinsic fluorescence spectroscopy, the

technique used to probe for unfolding in the urea experiments, is a measure of the environment

of the four tryptophans in the protein. From these experiments there does not appear to be much

difference between the HS mutant proteins and the wild-type. The results show that protein 4.2

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deficiency in red cells is not the result of a major structural change in cdAE1, but may be the

result of changes at the binding interface due to the introduction of positive charges, which

occurs with each of these HS mutations. The asymptomatic polymorphism K56E also retains the

major structural features of the protein and has similar thermal stability to the wild-type protein.

This is the first study to show that the molecular basis of HS associated with these three HS

mutations is not caused by a structural deformity in the cytoplasmic domain of AE1, which is in

keeping with the normal amount of these mutant proteins at the red cell membrane of these

patients. The phenotype is likely caused by perturbation at the binding interface between cdAE1

and protein 4.2 at the specific sites of mutation. The HS mutation sites are not clustered together

in the folded structure of cdAE1 as seen in the crystal structure, but are located far apart from

each other, which indicates that the cdAE1-protein 4.2 binding interface may be large. The loss

of protein 4.2 binding to the cytoplasmic domain of AE1 may result in degradation or loss of

protein 4.2.

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3 Chapter 3: Protein 4.2 interaction with hereditary

spherocytosis mutants of the cytoplasmic domain of human

anion exchanger 1

A version of this research was originally published in Biochemical Journal. Bustos, S.P. &

Reithmeier, R.A. Protein 4.2 interaction with hereditary spherocytosis mutants of the

cytoplasmic domain of human anion exchanger 1. Biochemical Journal. 2010; 433: 313-322 ©

the Biochemical Society (http://www.biochemj.org/bj/433/bj4330313.htm) (Bustos and

Reithmeier 2010). Jing Li contributed to this work by the generation of the AE1HS mutants and

mdAE1, and in carrying out some replicate experiments of the co-immunoprecipitation and

immunofluorescence assays. Jing Li carried out the Ni-NTA pull-down and subcellular

fractionation experiments. I performed the His6-tagged AE1HS mutant construction, protein 4.2

subcloning and hemagglutinin (HA)-tag engineering, and G2A/C173A protein 4.2 mutant

construction. I designed all of the experiments, analyzed all of the results and conducted replicate

experiments of the co-immunoprecipitation and immunofluorescence assays.

3.1 Abstract

Anion exchanger 1 and protein 4.2 associate in a protein complex bridging the erythrocyte

membrane and cytoskeleton; disruption of the complex results in unstable erythrocytes and HS.

Three HS mutations (E40K, G130R and P327R) in cdAE1 occur with deficiencies of protein 4.2.

The interaction of wild-type AE1, AE1HS mutants, mdAE1, kAE1 and AE1SAO with protein

4.2 was examined in transfected HEK-293 cells. The HS mutants had wild-type expression levels

and plasma membrane localization, and protein 4.2 expression was not dependent on the

presence of AE1. Protein 4.2 was localized throughout the cytoplasm, and co-localized at the

plasma membrane with the HS mutants, mdAE1, and kAE1, but at the ER with AE1SAO. Pull-

down assays revealed diminished levels of protein 4.2 associated with the HS mutants relative to

AE1. mdAE1 did not bind protein 4.2, while kAE1 and AE1SAO bound wild-type amounts of

protein 4.2. A protein 4.2 fatty acylation mutant, G2A/C173A, had decreased plasma membrane

localization compared to wild-type protein 4.2, and co-expression with AE1 enhanced its plasma

membrane localization. Subcellular fractionation showed that the majority of wild-type and

G2A/C173A protein 4.2 was associated with the cytoskeleton of HEK-293 cells. This study

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shows that HS mutations in cdAE1 cause impaired binding of protein 4.2 to AE1, leaving protein

4.2 susceptible to degradation or loss during red cell development.

3.2 Introduction

Since the E40K, G130R and P327R AE1 mutations all affect the levels of protein 4.2 in red

blood cells, I hypothesize that these mutations impair the binding of protein 4.2 to AE1. To test

this hypothesis, the interaction of protein 4.2 with wild-type AE1 and the three cytoplasmic

AE1HS mutants was studied in transfected HEK-293 cells. mdAE1, which is unable to bind

protein 4.2 (Korsgren and Cohen 1986) was included in these studies as a negative control.

As discussed later in the Appendix, the cytoplasmic domain of kAE1 (cdkAE1) is less

thermally stable than erythroid cdAE1 and exists in a more open structure (Pang et al. 2008). As

well, it does not bind ankyrin or glycolytic enzymes (Ding et al. 1994b, Wang et al. 1995b) most

likely due to the absence of the acidic N-terminal tail. We included kAE1 in these studies to see

what effect the missing tail and altered structure would have on protein 4.2 binding. Also, protein

4.2 is expressed in kidney cells (Friedrichs et al. 1989) where it may perform a similar function

as in erythrocytes. We also included AE1SAO in our studies to see if this mutant’s increased

cytoskeletal attachment (Liu et al. 1995, Liu et al. 1990, Sarabia et al. 1993) translated into an

increased interaction with protein 4.2.

The localization and interaction of protein 4.2 with these AE1 proteins was examined in

HEK-293 cells, as was the role of fatty acid modifications on protein 4.2 localization. Protein

4.2 had a broad distribution in transfected cells, was predominantly associated with the

cytoskeletal fraction, and co-localized with AE1 at the plasma membrane. The AE1HS mutants,

but not kAE1 or AE1SAO, had impaired protein 4.2 binding; this weakened interaction may

account for the loss of protein 4.2 during red cell development.

3.3 Materials and methods

3.3.1 Materials

The following is a list of materials used (Suppliers): pcDNA3 vector (Invitrogen); mutagenic

primers (ACGT Corp.); QuikChangeTM

site-directed mutagenesis kit (Stratagene); HEK-293

cells (ATCC); Dulbecco's modified Eagle's medium (DMEM), calf serum, penicillin,

streptomycin (Gibco BRL); LipofectamineTM

2000 (Invitrogen); C12E8 (Nikko Chemical Co.);

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Protein G-Sepharose (Amersham Biosciences); poly-L-lysine (Sigma-Aldrich); Ni-NTA agarose

resin (QIAGEN); mouse anti-AE1 (BRIC 6) antibody, which recognizes an extracellular epitope

at the C-terminus of AE1 (Bristol Institute for Transfusion Sciences); mouse anti-AE1 antibody,

which recognizes an intracellular epitope of AE1 (a gift from Dr Michael L. Jennings, University

of Arkansas for Medical Sciences, Little Rock, AR, USA); rabbit anti-CtAE1 antibody raised

against the last 16 amino acids of AE1 (SynPep Corporation); mouse anti-HA antibody

(Covance); rat anti-HA antibody (Roche); rabbit anti-calnexin (CNX) antibody (Stressgen

Biotech); lectin peanut agglutinin (PNA) Alexa Fluor® 488 conjugate (Molecular Probes);

mouse anti-actin antibody (Chemicon); mouse anti-GAPDH antibody (Millipore); goat

peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG (New England Biolabs); Cy3-

conjugated goat anti-mouse, anti-rat and anti-rabbit antibodies (Jackson ImmunoResearch);

Alexa Fluor® 488-conjugated goat anti-mouse and anti-rat antibodies (Molecular Probes);

Chemiluminescence Kit (Roche).

3.3.2 Site-directed mutagenesis

The coding sequence for wild-type human AE1 was inserted into the XhoI and BamHI sites of

the pcDNA3 vector. The construction of AE1SAO and kAE1 are described in Cheung and

Reithmeier (2005) and Quilty et al. (2002), respectively. mdAE1 was constructed by PCR on the

full-length AE1 protein with a methionine engineered at the start of the DNA sequence coding

for amino acids Asp369-Val911, followed by subcloning into the pcDNA3 vector. Asp369 is the

first perfectly conserved residue in the human AE1 family (AE1, AE2 and AE3) and defines the

beginning of the membrane domain. The AE1HS mutants were created using the QuikChangeTM

mutagenesis kit using complementary mutagenic primers, with wild-type AE1 as template. The

coding sequence for wild-type human protein 4.2, Type II, was subcloned from the pGEM-

7Zf(+/-) vector into the EcoRI site of the pcDNA3 vector. The C-terminal HA tag on protein 4.2

was created by PCR, inserting the tag after position Ala691. The G2A/C173A fatty acylation

mutant of protein 4.2 was created using the QuikChangeTM

mutagenesis kit using complementary

mutagenic primers, with wild-type HA-tagged protein 4.2 as template. HA-tagged protein 4.2

and the HA-tagged G2A/C173A mutant were used in all experiments, but were referred to as

protein 4.2 and G2A/C173A in the text, respectively. Sequencing of constructs was performed by

ACGT Corp.

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3.3.3 Transient transfection and expression of AE1 and protein 4.2 in HEK-

293 cells

HEK-293 cells were grown in DMEM supplemented with 10 % (v/v) calf serum, 0.5 %

penicillin and 0.5 % streptomycin under 5 % CO2 at 37 °C as described (Popov et al. 1999).

Cells were transfected by the Lipofectamine method (Sells et al. 1995) with 2 μg of plasmid

DNA per well of a six-well plate.

3.3.4 SDS-PAGE and immunoblotting

Proteins were resolved by SDS-PAGE (8 % or 10 % gels) (Laemmli 1970) and transferred to

nitrocellulose membrane (Towbin et al. 1979). AE1 was detected with a mouse monoclonal anti-

AE1 (Jennings) antibody (Jennings et al. 1986). HA-tagged protein 4.2 was detected using

mouse monoclonal anti-HA antibody. Actin was detected using mouse anti-actin antibody and

GAPDH was detected using mouse anti-GAPDH antibody. Goat peroxidase-conjugated anti-

mouse IgG was then added followed by detection by chemiluminescence and film exposure or by

a VersaDoc Imaging System Model 5000. Band intensities of immunoblots in the linear range of

intensity were determined using the ImageJ software (version 1.41o).

3.3.5 Immunofluorescence and confocal microscopy

HEK-293 cells transfected with pcDNA3 plasmids were grown on glass cover slips. In some

cases, cover slips were coated with poly-L-lysine, but this made no difference in the growth or

adherence of the cells. Cells were fixed with 3.8 % (w/v) paraformaldehyde for 15 min and

washed once with 100 mM glycine. Cells were either non-permeabilized or permeabilized and

incubated with antibodies. Non-permeabilized cells were blocked with 0.2 % (w/v) bovine serum

albumin (BSA) for 30 min, followed by incubation with 1:100 diluted mouse anti-AE1 (BRIC 6)

or 1:100 diluted PNA Alexa Fluor® 488 conjugate in 0.2 % BSA for 30 min. These cells were

then permeabilized with 0.2 % (v/v) Triton X-100 for 5 min and blocked with 0.2 % (w/v) BSA

for 30 min. Next, 1:250 diluted rat anti-HA antibody was added in 0.2 % BSA for 30 min. Cells

that were permeabilized at the beginning were permeabilized with 0.2 % (v/v) Triton X-100 for 5

min and blocked with 0.2 % (w/v) BSA for 30 min. These cells were then incubated with 1:500

diluted mouse anti-AE1 (Jennings) antibody, 1:100 diluted mouse anti-AE1 (BRIC 6) antibody,

1:250 diluted rat anti-HA antibody, or 1:250 diluted rabbit anti-CNX antibody in 0.2 % BSA for

30 min. Following several washes, samples were incubated with 1:1000 dilution of Alexa

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Fluor® 488-conjugated goat anti-mouse antibody, Alexa Fluor® 488-conjugated goat anti-rat

antibody, Cy3-conjugated goat anti-mouse antibody, Cy3-conjugated donkey anti-rat antibody,

or Cy3-conjugated donkey anti-rabbit antibody for 30 min. A Zeiss laser confocal microscope

LSM 510 was used to observe the samples.

3.3.6 Ni-NTA pull-down

HEK-293 cells transiently transfected with His6-tagged AE1 and HA-tagged protein 4.2

constructs, were harvested with lysis buffer (1 % C12E8, 300 mM NaCl, and 10 mM imidazole

with protease inhibitors in PBS). Cell lysates were centrifuged at 14 000 g for 30 min at 4 °C to

remove insoluble material. The supernatants were added to 50 μl of a 50 % slurry of Ni-NTA

agarose in binding buffer (0.1 % C12E8, 300 mM NaCl and 10 mM imidazole with protease

inhibitors in PBS) and incubated for 2 h at 4 °C. Resin was washed with 0.3 ml of wash buffer

(0.2 % C12E8, 300 mM NaCl and 30 mM imidazole with protease inhibitors in PBS) three times.

Bound proteins were eluted with elution buffer (0.5 % C12E8, 300 mM NaCl and 500 mM

imidazole in PBS) and solubilized in 2 × SDS sample buffer. Samples were analyzed by SDS-

PAGE (8 % gels) and immunoblotting was performed as described above. Band intensities were

determined by ImageJ 1.41o software. The amount of protein 4.2 associated with the amount of

AE1 eluted from the resin was calculated from immunoblots from eight separate transfection

experiments and normalized to the total amount of protein 4.2 expressed in the cells. Each value

was reported as relative to that of AE1, which was set to 100 %, to give a value of protein 4.2

relative binding. Results for protein 4.2 binding are given as means ± S.D. Mean values were

considered to be significantly different (p < 0.05) when the Student’s t test was used.

3.3.7 Co-immunoprecipitation

HEK-293 cells transiently transfected with AE1 and HA-tagged protein 4.2 constructs were

harvested with lysis buffer (1 % C12E8 with protease inhibitors in PBS). Cell lysates were

centrifuged at 14 000 × g for 30 min at 4 °C to remove insoluble material. AE1 was

immunoprecipitated from supernatants with 4 μl of rabbit anti-CtAE1 antibody followed by 100

μl of Protein G-Sepharose. Proteins were eluted with 25 μl 0.1 M glycine, pH 2.5, on ice for 20

min. Two μl of 1 M Tris, pH 9.0, was added to the eluate and proteins were solubilized in 2 ×

SDS sample buffer. Samples were analyzed by SDS-PAGE (8 % gels) and immunoblotting was

performed as described above. Band intensities were determined by ImageJ software (version

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1.41o) from various blot exposures ensuring that the band intensities were in the linear range.

Linear dilutions of protein were included on the blots to ensure linearity. The amount of protein

4.2 associated with the amount of AE1 eluted from the resin was calculated from immunoblots

from eight separate transfection experiments and normalized to the total amount of protein 4.2

expressed in the cells. Each value was reported as relative to that of AE1, which was set to 100

%, to give a value of protein 4.2 relative binding. Results for protein 4.2 binding are given as

means ± S.D. Mean values were considered to be significantly different (p < 0.05) when the

Student’s t test was used.

3.3.8 Subcellular fractionation of HEK-293 cells expressing wild-type or

G2A/C173A protein 4.2

HEK-293 cells transiently transfected with HA-tagged wild-type protein 4.2 or the G2A/C173A

protein 4.2 mutant were suspended in PBS with protease inhibitors, followed by sonication at 50

% duty cycle for 20 pulses on ice to lyse cells. Cell lysates were centrifuged at 1500 × g for 10

min at 4 °C to remove cell debris and unbroken cells. The supernatant, containing the membrane,

cytoskeletal and cytoplasmic fractions (labeled the Total fraction) was collected and centrifuged

at 100 000 × g for 1 h at 4 °C. The resulting supernatant contained the soluble cytoplasmic

fraction and was labeled S1. The pellet was washed with 1 ml of PBS with protease inhibitors

and centrifuged at 100 000 × g for 1 h at 4 °C and the wash fraction labeled Sw. The pellet,

containing the membrane and cytoskeletal fractions, was resuspended in lysis buffer (1 % C12E8

with protease inhibitors in PBS) and was labeled P1. Samples were slowly rotated for 1 h at 4 °C

to solubilize the membrane proteins in the detergent. Samples were then centrifuged at 100 000

× g for 1 h at 4 °C. The resulting supernatant contained detergent-soluble membrane protein and

was labeled S2. The pellet contained the cytoskeletal fraction and was labeled P2. Samples were

solubilized in 2 × SDS sample buffer and analyzed by SDS-PAGE (10 % gels). Immunoblotting

was performed as described above and band intensities were determined by ImageJ software

(version 1.41o). Detection of GAPDH was used as a marker for soluble cytoplasmic proteins.

Detection of actin was used as marker for soluble (G-actin) and cytoskeletal (F-actin) proteins.

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3.4 Results

3.4.1 Expression of protein 4.2 and AE1 proteins in transfected HEK-293 cells

Protein 4.2 was expressed alone or co-expressed with AE1, the HS mutants (E40K, G130R,

P327R), as well as mdAE1, kAE1 and AE1SAO in transiently transfected HEK-293 cells. Figure

3.1 shows a representative immunoblot of total cell extracts. AE1 and AE1HS proteins were

detected as approximately 95 kDa bands (A: lanes 2, 4, 5, 6). No immunoreactive band was seen

in the empty vector-transfected control lane (A: lane 1). All three AE1HS proteins had similar

expression levels compared to the wild-type protein. The immunodetection of mdAE1 (A: lane

3) was variable and often lower than AE1 due to the presence of multiple bands. Expression

(data not shown) of kAE1 was comparable to AE1, while AE1SAO was lower as reported

previously (Cheung et al. 2005a). HA-tagged protein 4.2 expressed in HEK-293 cells in the

absence or presence of AE1 proteins was readily detected as an approximately 72 kDa

immunoreactive band (B: lanes 1-6). No HA-immunoreactive band was seen in the non-

transfected control cells (data not shown). In multiple transfection experiments, there was no

statistically significant difference in the amount of protein 4.2 expressed in the absence or

presence of AE1, AE1HS mutants, mdAE1, kAE1 or AE1SAO. These results show that the

AE1HS mutants had similar stabilities compared to AE1 and that co-expression with AE1 was

not necessary for the expression of protein 4.2 in transfected HEK-293 cells.

3.4.2 Localization of AE1 proteins in HEK-293 cells

The localization of wild-type and mutant AE1 proteins expressed in HEK-293 cells was

determined by immunofluorescence and confocal microscopy. The first column in Figure 3.2

shows immunofluorescence of AE1 proteins in non-permeabilized cells. AE1 was readily

detected at the cell surface indicating that it had trafficked to the plasma membrane, as seen in

previous studies (Tang et al. 1998). The three AE1HS mutants were also detected at the cell

surface at similar intensities, indicating their ability to traffic to the plasma membrane. The

mdAE1 lacking the entire cytoplasmic domain was also detected at the cell surface, indicating

that the TM domain alone is sufficient for plasma membrane localization. The kAE1 isoform

was also able to traffic to the plasma membrane. In contrast, AE1SAO was not detected at the

cell surface, indicating its inability to traffic from the ER to the plasma membrane in transfected

HEK-293 cells, as was previously reported (Cheung et al. 2005a).

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A

1 2 3 4 5 6

M

empty

vector

AE1

md

AE1

E40K

G130R

P327R

B

Protein 4.2 expressed with: 1 2 3 4 5 6

M

empty

vector

AE1

md

AE1

E40K

G130R

P327R

Figure 3.1: Expression of AE1 proteins and protein 4.2 in HEK-293 cells.

Immunoblot analysis was performed on whole-cell detergent extracts from HEK-293 cells

expressing AE1 proteins and protein 4.2. A mouse monoclonal anti-AE1 antibody was used to

detect AE1 proteins (A) and a mouse monoclonal anti-HA antibody was used to detect HA-

tagged protein 4.2 (B). Incubation with peroxidase-conjugated anti-mouse IgG and detection by

chemiluminescence was then performed (see Methods).

total AE1

total mdAE1

total p4.2

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The second column of Figure 3.2 shows immunofluorescence of AE1 proteins in cells that

have been subsequently permeabilized. In these cells AE1, AE1HS, mdAE1 and kAE1 proteins

showed strong staining at the plasma membrane, indicating that most of the AE1 is at the cell

surface. This was seen by the yellow staining pattern in the merged images showing the strong

overlap of fluorescent signals from intact and permeabilized cells. In contrast, AE1SAO was not

detected at the cell surface, but only in permeabilized cells due to its ER localization (Cheung et

al. 2005a).

3.4.3 Co-localization of protein 4.2 and AE1 in HEK-293 cells

HA-tagged protein 4.2 was expressed alone or with AE1, AE1HS, mdAE1, kAE1 and

AE1SAO proteins, and visualized by immunofluorescence and confocal microscopy to

determine its intracellular localization in HEK cells. The first row of Figure 3.3 shows

immunofluorescence of protein 4.2 expressed alone in permeabilized cells and CNX, an ER-

resident protein. Protein 4.2 (green) displayed a wide distribution throughout the cell and also co-

localized with CNX (blue) as seen by the cyan colour in the merged image. This suggests that

some protein 4.2 localizes at the ER. In the first column of the remaining rows, protein 4.2

displayed a wide cellular distribution when expressed with all AE1 proteins, similar to when

expressed alone. The second column shows the immunofluorescence of AE1 proteins in

permeabilized cells. The proteins were predominantly localized at the cell surface with the

exception of AE1SAO, which was intracellular. In the merged images in the third column, the

yellow colour indicates that a fraction of protein 4.2 co-localized to the plasma membrane with

AE1, AE1HS, mdAE1 and kAE1 proteins. Its co-localization with mdAE1 is interesting since

mdAE1 lacks the cytoplasmic domain, which is needed for protein 4.2 interaction (Korsgren and

Cohen 1986). Protein 4.2 co-localized with AE1SAO, indicating its partial localization at the ER

similar to its co-localization with CNX above. The proteins do not co-localize completely, as

seen by distinct green and red staining in the merged image, indicating localization of protein 4.2

in other cellular compartments. Thus, protein 4.2 has a wide intracellular distribution in

transfected HEK-293 cells localizing to the ER and other intracellular compartments, as well as

to the plasma membrane.

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non-perm. perm.

AE1 AE1 merge

Figure 3.2: Immunofluorescence

images of wild-type and mutant AE1 in

HEK-293 cells.

Non-permeabilized HEK-293 cells

expressing wild-type or mutant AE1

proteins were incubated with mouse anti-

AE1 antibody (BRIC 6) against an

external epitope, followed by incubation

with Alexa Fluor® 488-conjugated goat

anti-mouse antibody to detect cell surface

AE1 (green). Cells were then washed,

permeabilized and incubated with the

same primary antibody followed by

incubation with Cy3-conjugated goat anti-

mouse antibody to detect total AE1 (red).

Confocal microscopy was then performed.

An enlarged region of the cell is shown in

the inset for each image. In the merged

image, yellow indicates the co-localization

of cell surface and total AE1. non-perm.,

non-permeabilized; perm., permeabilized.

AE1

E40K

G130R

P327R

mdAE1

kAE1

AE1SAO

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perm. perm.

protein 4.2 CNX merge

protein 4.2 AE1 merge

Figure 3.3: Immunofluorescence images of

protein 4.2 and AE1 proteins in HEK-293

cells.

Permeabilized cells expressing HA-tagged

protein 4.2 alone or co-expressed with wild-

type or mutant AE1 proteins were incubated

with rat anti-HA and rabbit anti-CNX or mouse

monoclonal anti-AE1 (Jennings) antibodies.

Incubation of samples with fluorescently-

labeled secondary antibodies and confocal

microscopy was performed (see Methods). In

the merged image of the top row, cyan

indicates co-localization of protein 4.2 (green)

and CNX (blue). In the remaining merged

images, yellow indicates co-localization of

protein 4.2 (green) and AE1 (red). perm.,

permeabilized.

p4.2

E40K

G130R

P327R

AE1

p4.2

p4.2

p4.2

p4.2

p4.2

kAE1

AE1SAO

p4.2 mdAE1

p4.2

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3.4.4 Interaction of protein 4.2 with AE1 proteins in HEK-293 cells

To examine the interaction of protein 4.2 with the AE1 proteins we conducted Ni-NTA pull-

down assays on HEK-293 cells transiently expressing His6-tagged AE1 proteins and HA-tagged

protein 4.2. Figure 3.4 shows representative immunoblots of protein 4.2 associated with AE1

(top panel), AE1 pulled down (middle panel), and total protein 4.2 in the HEK-293 lysate

(bottom panel). The amount of protein 4.2 bound to AE1 proteins was calculated as described in

the Methods. The control experiment with protein 4.2 expressed in the absence of AE1 (lane 1)

showed background amounts of bound protein 4.2. The AE1HS mutants consistently bound less

protein 4.2 at levels of 54 ± 14 % (E40K), 43 ± 14 % (G130R), and 65 ± 29 % (P327R), n = 8,

relative to wild-type AE1. Taking into account the dilution factor of the total protein 4.2 fraction

compared to the bound protein 4.2 fraction, it was determined from their band densities that

wild-type AE1 bound approximately 9 % of the total cellular protein 4.2.

As expected, the mdAE1 was unable to pull down protein 4.2 above background levels (lane

6). The kAE1 and AE1SAO proteins bound similar amounts of protein 4.2 at levels of 110 ± 21

% and 99 ± 33 %, n = 8, relative to wild-type AE1, respectively. These small differences were

not statistically significant indicating that the loss of the first 65 amino acids for kAE1 and

deletion of nine amino acids at the first TM in AE1SAO do not diminish protein 4.2 binding.

To confirm the protein 4.2 binding impairment by the AE1HS mutants seen in the Ni-NTA

pull-down assays, we conducted co-immunoprecipitation (co-ip) experiments on HEK-293 cells

transiently expressing AE1HS proteins and protein 4.2. Figure 3.5A shows representative

immunoblots of protein 4.2 associated with AE1 (top panel), AE1 bound to the resin (middle

panel), and total protein 4.2 in the HEK-293 lysate (bottom panel). The amount of protein 4.2

bound to AE1HS proteins relative to wild-type AE1 was calculated as described in the Methods.

The control experiment with protein 4.2 expressed in the absence of AE1 (lane 1) showed no

bound protein 4.2. The AE1HS proteins (lanes 3-5) consistently co-purified less protein 4.2

relative to AE1 (lane 2). In Figure 3.5B we show the results for AE1 (lane 1) and mdAE1 (lane

2) where the amount of immunoreactive mdAE1 expressed was comparable to AE1. As seen in

lane 2, there was no bound protein 4.2 in agreement with the Ni-NTA results.

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Protein 4.2 expressed with: 1 2 3 4 5 6 7 8

M

empty

vector

AE1

E40K

G130R

P327R

mdAE1

kAE1

AE1SAO

Figure 3.4: Ni-NTA pull-down of wild-type and mutant His6-tagged AE1 with protein 4.2 in

HEK-293 cells.

His6-tagged AE1 in whole-cell extracts was prepared with the detergent C12E8 and pulled-down

using Ni-NTA agarose. Total and bound fractions were resolved by SDS-PAGE and

immunoblots were probed with mouse anti-AE1 (Jennings) antibody to detect AE1 and mouse

anti-HA antibody to detect HA-tagged protein 4.2 (see Methods). Immunoblot band intensities

were measured from eight independent experiments. The amount of protein 4.2 bound to the

P327R HS mutant appears high in this blot. However, after correcting for bound AE1 and total

protein 4.2, the amount of protein 4.2 bound to P327R HS mutant was less than that bound to

AE1.

bound p4.2

bound AE1

bound mdAE1

total p4.2

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A

Protein 4.2 expressed with: 1 2 3 4 5

M

empty

vector

AE1

E40K

G130R

P327R

B

Protein 4.2 expressed with: 1 2

M

AE1

md

AE1

Figure 3.5: Co-immunoprecipitation (co-

ip) of wild-type and mutant AE1 with

protein 4.2 in HEK-293 cells.

(A) AE1 in whole-cell extracts prepared

with the detergent C12E8 was

immunoprecipitated with rabbit anti-CtAE1

antibody followed by Protein G Sepharose

binding. Total and bound fractions were

resolved by SDS-PAGE and immunoblots

probed with mouse anti-AE1 (Jennings)

antibody to detect AE1 and mouse anti-HA

antibody to detect any co-purifying HA-

tagged protein 4.2 (see Methods).

Immunoblot band intensities were measured

from eight independent experiments. The

mdAE1 lanes were excluded from these

blots due to variable and low expression

level, and the remaining lanes on the same

blots were spliced together for clarity

(splice points are denoted by vertical black

lines).

(B) Immunoblots following co-ip of AE1

and mdAE1 and associated protein 4.2 from

an experiment with comparable amounts of

bound AE1 and mdAE1 proteins.

bound AE1

bound mdAE1

bound p4.2

bound AE1

total p4.2

bound p4.2

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Also in agreement with the Ni-NTA pull-down results, the three AE1HS mutants consistently

bound less protein 4.2 at levels of 75 ± 16 % (E40K), 68 ± 16 % (G130R), and 79 ± 17 %

(P327R), n = 8, relative to wild-type AE1. Clearly, protein 4.2 could still interact with all three

AE1HS mutants, however at a statistically significant lower level compared to wild-type AE1.

Thus, the HS mutations in the cdAE1 result in impaired binding of protein 4.2 in HEK-293 cells.

3.4.5 Co-localization of wild-type and G2A/C173A protein 4.2 and AE1 in

HEK-293 cells

The ability of protein 4.2 to co-localize with mdAE1 at the plasma membrane of HEK-293

cells despite a lack of interaction was puzzling. Protein 4.2’s requirement of Gly2 myristoylation

for plasma membrane localization in Sf9 cells led us to believe its acylation state may allow for

similar localization in HEK-293 cells in the absence of cdAE1. To confirm this, we constructed

a double mutant removing sites of myristoylation and palmitoylation (G2A/C173A) and

observed its co-localization with AE1 proteins. Figure 3.6 shows the immunofluorescence of

HA-tagged protein 4.2 and the G2A/C173A (GC) mutant expressed with AE1, mdAE1 and

AE1SAO. In the first row, wild-type protein 4.2 is widely distributed throughout the cell and co-

localizes with AE1 at the plasma membrane, as seen by the yellow colour in the merged image.

In the second row, the GC mutant has a similar cellular distribution and co-localizes to the

plasma membrane with AE1. In the third row, the co-localization of protein 4.2 and mdAE1 is

again seen by the yellow colour at the plasma membrane. However, in the fourth row when the

GC mutant is expressed with mdAE1 there is very little yellow staining at the plasma membrane,

indicating poor plasma membrane localization. This suggests that the fatty acid moieties promote

protein 4.2 targeting to the plasma membrane. This also suggests that the presence of wild-type

AE1 allows the GC mutant to localize to the plasma membrane as seen in the second row,

consistent with an interaction between these proteins. The fifth row shows, as before, that wild-

type protein 4.2 also co-localizes with AE1SAO at the ER. In the sixth row, the GC mutant is

also seen to co-localize with AE1SAO at the ER. Co-localization of protein 4.2 and the GC

mutant with AE1SAO is not complete as seen by distinct green and red staining in the merged

image. This indicates that both wild-type and GC protein 4.2 are associated with other

intracellular compartments in addition to the ER.

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perm. non-perm.

protein 4.2 AE1 merge

perm. perm.

protein 4.2 AE1 merge

Figure 3.6: Immunofluorescence

images of wild-type and

G2A/C173A protein 4.2 and AE1

proteins in HEK-293 cells.

Non-permeabilized cells expressing

wild-type or mutant protein 4.2 and

wild-type or mdAE1 proteins (first

four rows) were incubated with

mouse monoclonal anti-AE1

antibody (BRIC 6) against an

external epitope. Cells were then

permeabilized and incubated with

rat anti-HA antibody. In the last two

rows, permeabilized cells

expressing wild-type or mutant

protein 4.2 and AE1SAO were

incubated with mouse anti-AE1 and

rat anti-HA antibodies. Incubation

of samples with fluorescently-

labeled secondary antibodies and

confocal microscopy was

performed (see Methods). In the

merged images, yellow indicates

co-localization of protein 4.2 and

AE1. perm., permeabilized; non-

perm., non-permeabilized.

p4.2

GC

GC

AE1

AE1SAO

AE1SAO

p4.2 mdAE1

mdAE1 GC

p4.2 AE1

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3.4.6 Co-localization of wild-type and G2A/C173A protein 4.2 and cell surface

glycans in the absence or presence of AE1 in HEK-293 cells

In order to further support the idea that the GC mutant displays impaired plasma membrane

targeting, HA-tagged protein 4.2 and the GC mutant were expressed either alone or with AE1,

and their localization to the plasma membrane using PNA binding was determined. PNA is a

lectin that binds to the disaccharide galactose β1,3 N-acetylgalactosamine found in

oligosaccharides of glycoproteins (Slupsky et al. 1993), serving as a marker of the outer surface

of the plasma membrane. In the first row of Figure 3.7, protein 4.2 expressed alone is widely

distributed within the cell (green) and the red stain, representing bound PNA, marks the outside

surface of the cell. Even though PNA is on the outside of the bilayer and protein 4.2 on the inner

side, we see an overlap of fluorescence seen by the yellow colour in the merged image since the

confocal microscope cannot resolve the thickness of the membrane. A distinctive green-yellow-

red pattern (going from the inside of the cell outward) is seen in the merged image, indicating

that protein 4.2 associates with the plasma membrane in the absence of AE1. The same pattern is

seen in the second row when protein 4.2 is expressed with AE1. In the third row when the

acylation mutant is expressed alone there is no yellow colour in the merged image, indicating its

inability to associate with the plasma membrane. In the fourth row when the acylation mutant is

co-expressed with AE1 the yellow colour is seen in the merged image, indicating that AE1 can

localize the acylation mutant of protein 4.2 to the plasma membrane.

3.4.7 Subcellular fractionation of HEK-293 cells expressing wild-type or

G2A/C173A protein 4.2

In order to confirm that impaired plasma membrane localization was not indirectly caused by

impaired cytoskeletal attachment by the double mutant we performed subcellular fractionation of

HEK-293 cells expressing HA-tagged wild-type and G2A/C173A mutant protein 4.2. The bulk

of wild-type and GC mutant protein 4.2 was associated with the detergent-insoluble

cytoskeleton. The top two panels in Figure 3.8 are representative immunoblots showing wild-

type and GC protein 4.2 in various fractions. After centrifugation at 100 000 × g the majority of

protein 4.2 (73 ± 3 %, n = 4) was associated with the membrane/cytoskeleton fraction (P1)

versus the cytoplasmic fraction (S1). Similar results (71 ± 6 %, n = 4) were seen with the

acylation mutant.

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89

perm. non-perm.

protein 4.2 PNA merge

Figure 3.7: Immunofluorescence images of cell surface glycans using PNA and wild-type

and G2A/C173A protein 4.2 expressed in the absence or presence of AE1 in HEK-293 cells.

Non-permeabilized HEK-293 cells expressing wild-type or mutant protein 4.2 proteins in the

absence or presence of AE1 were incubated with PNA Alexa Fluor® 488 conjugate to detect the

outer membrane. Cells were then permeabilized and incubated with rat anti-HA antibody to

detect HA-tagged protein 4.2 or G2A/C173A. Incubation of samples with fluorescently-labeled

anti-rat secondary antibody and confocal microscopy was performed as described in the

Methods. In the merged image, yellow indicates the co-localization of protein 4.2 (intracellular)

and PNA (extracellular) at the level of the plasma membrane. perm., permeabilized; non-perm.,

non-permeabilized.

PNA

GC+AE1

p4.2+AE1

PNA

p4.2 alone PNA

GC alone PNA

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Detergent extraction of the membrane/cytoskeletal fraction revealed that most (87 %) of the

membrane-associated protein 4.2 was in the cytoskeletal fraction (P2) with a small amount in the

membrane fraction (S2). Similar results (92 %) were seen with the acylation mutant, although the

amount solubilized by detergent was decreased relative to wild-type protein 4.2 (8 % versus 13

%). These observations support a previous report (Risinger et al. 1996) where myristoylated and

non-myristoylated (G2A) protein 4.2 expressed in Sf9 cells were both associated with the

particulate fraction. These results indicate that fatty acylation does not affect protein 4.2

cytoskeleton association.

The bottom two panels of Figure 3.8 show the detection of actin and GAPDH in the various

fractions of HEK-293 cells expressing wild-type or GC mutant protein 4.2. In HEK-293 cells

most of the actin (~ 75 %) is in the soluble versus the filamentous form (Oprea et al. 2008). In

this study, the majority of the actin was seen in the cytoplasmic fraction (S1) as opposed to the

membrane/cytoskeletal fraction (P1). Of the amount present in the membrane/cytoskeletal

fraction, most of it was in the cytoskeletal fraction (P2). Virtually all of the GAPDH, a soluble

protein, was detected in the cytosol, with a negligible amount detected in the

membrane/cytoskeleton fraction. These results support our assignment of cellular compartments

to the various fractions, allowing us to localize the majority of wild-type and GC protein 4.2 to

the cytoskeletal compartment with confidence.

3.5 Discussion

In the current study, I was able to express full-length HA-tagged protein 4.2 with AE1 proteins in

HEK-293 cells. The AE1HS proteins were expressed at similar levels to AE1 and could traffic to

the plasma membrane of the cells. These findings are consistent with the observation that the

levels of these HS mutants in patient red cells are normal (Inoue et al. 1998, Jarolim et al. 1992a,

Rybicki et al. 1993). I also demonstrated that protein 4.2 was expressed at similar levels in HEK-

293 cells in the absence or presence of AE1 proteins. Thus, protein 4.2 association with AE1 is

not required for its expression in HEK cells.

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1 2 3 4 5 6 M Total S1 Sw P1 S2 P2

Figure 3.8: Subcellular fractionation of HEK-293 cells expressing wild-type or G2A/C173A

protein 4.2.

Following subcellular fractionation of HEK-293 cells expressing HA-tagged wild-type and GC

mutant protein 4.2, fractions were run on SDS-PAGE and transferred to nitrocellulose. Blots

were probed with mouse anti-HA antibody, mouse anti-actin antibody and mouse anti-GAPDH

antibody to detect protein 4.2, actin and GAPDH, respectively (see Methods). Immunoblot band

intensities were measured from four independent experiments. S1, cytoplasmic fraction; Sw,

wash fraction; P1, membrane/cytoskeletal fraction; S2, detergent-solubilized membrane fraction;

P2, cytoskeletal fraction.

WT p4.2

G2A/C173A p4.2

Actin with WT p4.2

GAPDH with WT p4.2

Actin with GC p4.2

GAPDH with GC p4.2

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When co-expressed in HEK-293 cells, protein 4.2 and AE1 co-localized at the plasma

membrane. Similar observations were made with the AE1HS mutants and kAE1, but also with

mdAE1, which does not interact with protein 4.2. Furthermore, protein 4.2 could target to the

plasma membrane in the absence of AE1, as seen by its co-localization with PNA. This result

agrees with previous reports of protein 4.2 localization at the plasma membrane of Xenopus

oocytes (Toye et al. 2005) and Sf9 cells (Risinger et al. 1996) in the absence of AE1. We also

found protein 4.2 widely distributed among intracellular compartments, including the ER, as

seen by its co-localization with AE1SAO and CNX. These results show that protein 4.2

associates with various membranes within the cell in the absence of AE1, including the plasma

membrane, the ER membrane, and possibly other intracellular membranes in transfected HEK-

293 cells.

Ni-NTA pull-down experiments revealed that protein 4.2 can associate with AE1 proteins in

whole-cell detergent extracts. Protein 4.2 co-purified with AE1, AE1HS, kAE1 and AE1SAO,

but not mdAE1. Protein 4.2 binding to AE1HS mutants was diminished relative to AE1,

indicating an impaired interaction. Co-immunoprecipitation assays supported the Ni-NTA pull-

down results. We were not expecting binding of protein 4.2 to AE1HS mutants to be completely

abolished since there is only a partial deficiency of protein 4.2 in HS patient red cells.

Introduction of a positive charge by the E40K, G130R or P327R mutants may serve to disrupt

the acidic protein 4.2 binding surface on cdAE1. As shown in the crystal structure of cdAE1

(Figure 1.8), these mutations are not localized to a “hot spot”, but are widely distributed on one

surface of the protein. Residues 40 and 130 on one monomer occur on the same side of the

domain as residue 327 from the other monomer, perhaps requiring dimer formation to create the

binding surface for protein 4.2. The cdAE1-binding regions shown in the protein 4.2 homology

model (Figure 1.8) are predicted to form a binding surface that could be large enough to interact

with all three AE1HS mutation sites. It is not surprising then that alterations of residues at these

distant sites in cdAE1 affect protein 4.2 binding.

The binding of protein 4.2 to kAE1 as measured in Ni-NTA pull-downs was not significantly

different from that of AE1. This result may seem puzzling when we consider that the kidney

isoform is missing the first 65 residues of the N-terminus, while the E40K HS mutant which has

a single point mutation in this region shows impaired binding to protein 4.2. However, this

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93

mutation replaces an acidic with a basic residue. This introduction of a positive charge into a

potential binding spot may compromise protein 4.2 binding in a way that absence of the region

does not. As was mentioned earlier, it is believed that the Arg-Arg motif of protein 4.2 (residues

34 and 35) is necessary for cdAE1 interaction and that this motif has an electrostatic interaction

with an acidic region of cdAE1, possibly at the N-terminus. If this acidic region were to become

more basic, the electrostatic repulsion could result in the binding impairment we saw with E40K.

The cytoplasmic domain of kAE1 has been shown to be a more open structure than AE1 (Pang et

al. 2008), as discussed in the Appendix, and it is not known how this would affect protein

binding. It is possible that this open structure compensates for the missing central β-strand. This

kAE1 structural difference and its possible effect on protein 4.2 binding would have to be

explored using structural techniques, such as nuclear magnetic resonance (NMR). Wild-type

levels of AE1SAO binding to protein 4.2 were not surprising as the cytoplasmic domain of this

mutant is unchanged, deficiencies of protein 4.2 in AE1SAO red cells have not been reported,

and AE1SAO exhibits increased binding to the cytoskeleton in red blood cells (Liu et al. 1995).

The co-localization of protein 4.2 with mdAE1 at the plasma membrane, despite a lack of

interaction, turned our attention to the possible role of fatty acid modifications in plasma

membrane localization. We found that G2A/C173A protein 4.2 had diminished plasma

membrane localization when expressed alone or with mdAE1. This suggests that protein 4.2 is

able to associate with the plasma membrane via its lipid anchors, as suggested in previous reports

(Das et al. 1994, Risinger et al. 1996). This is at least the case in HEK-293 cells where AE1 is

not needed for protein 4.2 expression or plasma membrane localization. Subcellular

fractionation studies showed that the majority of wild-type and GC protein 4.2 expressed in

HEK-293 cells was associated with the cytoskeleton. This demonstrates that the GC mutation

does not affect cytoskeletal binding. A portion (< 30 %) of wild-type and GC protein 4.2 was

seen in the cytoplasmic fraction, which agrees with the immunofluorescence localization results

where the protein was broadly localized throughout the cell.

The current study shows that AE1 is not required for the expression and plasma membrane

localization of protein 4.2 in HEK-293 cells. As well, we have shown that the three AE1HS

mutants, E40K, G130R and P327R, have impaired binding to protein 4.2. How this impaired

binding translates into protein 4.2 deficiency in erythrocytes is yet to be determined. Protein 4.2

and AE1 have been found to appear simultaneously during erythropoiesis, and interact with each

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other as soon as they are expressed (van den Akker et al. 2010a). Indeed, protein 4.2 may

associate with AE1 at the ER during their biosynthesis and they may traffic together to the cell

surface. The fate of protein 4.2 that gets expressed but is unable to fully associate with AE1HS

mutants during differentiation is not known. Protein association with the cytoskeleton can

determine protein sorting during enucleation of the differentiating red cell (Lee et al. 2004),

where an increased cytoskeletal association causes retention in the reticulocyte. It is possible that

the portion of protein 4.2 unable to bind to AE1HS never makes it to the membrane, or does not

localize to a region of the membrane where protein 4.2 can bind to the cytoskeleton. This would

prevent its retention in the reticulocyte during enucleation and explain its deficiency in

erythrocytes of patients with these three cytoplasmic AE1HS mutations. Studies of protein 4.2

sorting during enucleation of red cell precursors from healthy individuals and HS patients, or

relevant HS mouse models, would help to determine the mechanism of protein 4.2 loss in

hereditary spherocytosis.

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4 Discussion and future directions

In this thesis, I have made two significant findings. The first finding is that three HS mutations,

E40K, G130R and P327R, in cdAE1 do not cause gross conformational changes in the structure

of the domain. However, the P327R mutant displayed a slight thermal destabilization with a Tm

that was 5 ºC lower than that of wild-type cdAE1, as determined by calorimetry and CD thermal

melts. The second finding is that these mutations in full-length AE1 cause impaired binding to

protein 4.2 as determined in HEK-293 cells. In addition to these results, I have created several

other variants of AE1 and protein 4.2 to be used as reagents in future studies. Table 4.1 lists all

the AE1 and protein 4.2 mutant DNA constructs that I have designed and made, their research

purpose and the results of their expression and characterization, if applicable. Following the table

is a more detailed analysis of the major results of this thesis and discussion of future research,

where I will refer back to the table when discussing possible uses for these constructs.

Table 4.1: Mutant protein DNA constructs

Mutation/

Common

Name

Protein/

Vector/

Expression System

Predicted

Product

Comments / Research Purpose / Results

E90K

Cape Town

cdAE1 + His6 tag

pETBlue1

E. coli

HS mutant - causes HS in trans with Prague III

P147S

Mondego

cdAE1 + His6 tag

pETBlue1

E. coli

HS mutant - causes HS in cis with E40K and in trans with Coimbra

- constructed by Allison Pang under my supervision

W75F cdAE1 + His6 tag

pETBlue1

E. coli

Trp mutant - to study specific sites of folding by intrinsic fluorescence

W81F cdAE1 + His6 tag

pETBlue1

E. coli

Trp mutant - to study specific sites of folding by intrinsic fluorescence

W94F cdAE1 + His6 tag

pETBlue1

E. coli

Trp mutant - to study specific sites of folding by intrinsic fluorescence

W105F cdAE1 + His6 tag

pETBlue1

E. coli

Trp mutant - to study specific sites of folding by intrinsic fluorescence

E40A cdAE1 + His6 tag

pETBlue1

E. coli

Ala mutant - to test site of HS mutation using Ala

- wild-type CD spectrum

G130A cdAE1 + His6 tag

pETBlue1

E. coli

Ala mutant - to test site of HS mutation using Ala

- wild-type CD spectrum

P327A cdAE1 + His6 tag

pETBlue1

E .coli

Ala mutant - to test site of HS mutation using Ala

- wild-type CD spectrum

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Table 4.1: Mutant protein DNA constructs (continued)

Mutation/

Common

Name

Protein/

Vector/

Expression System

Predicted

Product

Comments / Research Purpose / Results

C201A cdAE1 + His6 tag

pETBlue1

E. coli

single-Cys - to make the Cys-less mutant

- can use for Cys-scanning methods

C317A cdAE1 + His6 tag

pETBlue1

E. coli

single-Cys - to make the Cys-less mutant

- can use for Cys-scanning methods

C201/317A cdAE1 + His6 tag

pETBlue1

E. coli

Cys-less - same CD spectrum as WT, maybe slightly higher thermal

stability by calorimetry

- allows use of Cys-scanning methods

- helped determine extra mass on cdAE1 was β-

mercaptoethanol adduct by mass spectrometry

kC201A cdAE1 + His6 tag

pETBlue1

E. coli

kidney

single-Cys

- to make the Cys-less mutant

- can use for Cys-scanning methods

kC317A cdAE1 + His6 tag

pETBlue1

E. coli

kidney

single-Cys

- to make the Cys-less mutant

- can use for Cys-scanning methods

L319K cdAE1 + His6 tag

pETBlue1

E. coli

monomer - puts positive charge in hydrophobic pocket and β-strand

of dimer interface to disrupt dimer

- mixture of monomers and dimers by SE

L319E cdAE1 + His6 tag

pETBlue1

E. coli

monomer - puts negative charge in hydrophobic pocket and β-strand

of dimer interface to disrupt dimer

- not pure enough for SE analysis

L321K cdAE1 + His6 tag

pETBlue1

E. coli

monomer - puts positive charge in hydrophobic pocket and β-strand

of dimer interface to disrupt dimer

- not pure enough for SE analysis

L321E cdAE1 + His6 tag

pETBlue1

E. coli

monomer - puts negative charge in hydrophobic pocket and β-strand

of dimer interface to disrupt dimer

- not pure enough for SE analysis

L319K/L321K cdAE1 + His6 tag

pETBlue1

E. coli

monomer - puts two positive charges in hydrophobic pocket and β-

strand of dimer interface to disrupt dimer

- dimer by SE

L319E/L321E cdAE1 + His6 tag

pETBlue1

E. coli

monomer - puts two negative charges in hydrophobic pocket and β-

strand of dimer interface to disrupt dimer

- monomer by SE

Δh10 cdAE1 + His6 tag

pETBlue1

E. coli

monomer - missing helix 10 of dimerization arm

- mixture of monomers and dimers

Δh10,s11 cdAE1 + His6 tag

pETBlue1

E. coli

monomer - missing helix 10 and strand 11

- no expression

Δh9,10,s11 cdAE1 + His6 tag

pETBlue1

E. coli

monomer - missing helix 9 and 10, and strand 11

- low expression, poor purification, poor solubility

Δh8, 9,10,s11 cdAE1 + His6 tag

pETBlue1

E. coli

monomer - missing helix 8, 9, and 10 and strand 11

- low expression, poor purification, poor solubility

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Table 4.1: Mutant protein DNA constructs (continued)

Mutation/

Common

Name

Protein/

Vector/

Expression System

Predicted

Product

Comments / Research Purpose / Results

E40K

Montefiore

AE1-His6

pcDNA3

HEK-293 cells

HS mutant

(AE1HS)

- used in Ni-NTA pull-down assays

- good expression

G130R

Fukuoka

AE1-His6

pcDNA3

HEK-293 cells

HS mutant

(AE1HS)

- used in Ni-NTA pull-down assays

- good expression

P327R

Tuscaloosa

AE1-His6

pcDNA3

HEK-293 cells

HS mutant

(AE1HS)

- used in Ni-NTA pull-down assays

- good expression

Wild-type protein 4.2-GST

pGEX5-1

E. coli

full-length

wild-type

- for in vitro binding studies with cdAE1

- no expression

1-238 aa protein 4.2-GST

pGEX5-1

E .coli

23kDa N-

terminus

- for in vitro binding studies with cdAE1

- poor expression and purification

1-144 aa protein 4.2-GST

pGEX5-1

E. coli

cdAE1-

binding site

- for in vitro binding studies with cdAE1

- contains Arg34-Arg35 binding motif

- poor expression and purification

145-238 aa protein 4.2-GST

pGEX5-1

E. coli

cdAE1-

binding site

- for in vitro binding studies with cdAE1

- contains palmitoylatable Cys173

- good expression, fair purification with degradation

145-203 aa protein 4.2-GST

pGEX5-1

E. coli

β-hairpin - for in vitro binding studies with cdAE1

- contains palmitoylatable Cys173

- good expression and purification

Wild-type protein 4.2-HA

pcDNA3

HEK-293 cells

full-length

wild-type

- for immunofluorescent localization and pull-down assays

- good expression and plasma membrane localization

- good binding to AE1, impaired binding to AE1HS

C173A protein 4.2-HA

pcDNA3

HEK-293 cells

acylation

mutant

- for immunofluorescence localization: trafficking effects

of palmitoylation

G2A/C173A protein 4.2-HA

pcDNA3

HEK-293 cells

double

acylation

(GC) mutant

- for immunofluorescence localization: trafficking effects

of palmitoylation and myristoylation

- good expression and poor plasma membrane localization

Arg34-Arg35

Glu34-Glu35

protein 4.2-HA

pcDNA3

HEK-293 cells

binding

rescue

mutant

- for pull-down assays to measure interaction with AE1

and E40K: possible interaction rescue mutant

- good expression and preliminary co-ip assay showing

rescue of binding to E40K AE1

E40K

Montefiore

AE1-myc

pFB-Neo

K562 cells

HS mutant

(AE1HS)

- used in immunofluorescent localization

- good expression and localization

- unable to make stable transfectants

G130R

Fukuoka

AE1-myc

pFB-Neo

K562 cells

HS mutant

(AE1HS)

- used in immunofluorescent localization

- good expression and localization

- unable to make stable transfectants

P327R

Tuscaloosa

AE1-myc

pFB-Neo

K562 cells

HS mutant

(AE1HS)

- used in immunofluorescent localization

- good expression and localization

- unable to make stable transfectants

Wild-type protein 4.2-HA

pFB-Neo

K562 cells

full-length

wild-type

- used in immunofluorescent localization

- variable expression and inconsistent localization

- unable to make stable transfectants

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4.1 Structure and conformational stability of the cytoplasmic domain

of AE1

The E40K, G130R and P327R mutations in cdAE1 are associated with HS and a lower amount

of protein 4.2 in the red cell, while maintaining a normal amount of AE1 at the plasma

membrane. I hypothesized that these mutations would cause either a change in the structure of

the domain or a change in the interaction surface, thereby preventing proper binding of protein

4.2. I found that these mutations do not cause gross changes in the structure, folding or

conformational stability of the domain. The secondary structures of these mutant proteins were

similar to wild-type as determined by CD. The mutant proteins retained the dimeric structure of

the wild-type protein, as determined by SE experiments using the analytical ultracentrifuge. The

P327R mutant was expected to affect the dimeric state since this mutation is located at the N-

terminus of an α-helix in the dimerization domain, but this and another study (Zhou et al. 2007)

confirmed that it remained a dimer. This mutation, however, caused a slight thermal

destabilization of the domain with a Tm that was 5 ºC lower than for wild-type cdAE1.

The mutant proteins underwent similar pH-dependent conformational changes when

monitored by intrinsic tryptophan fluorescence. In this experiment, the fluorescence intensity

increased in a similar manner for all the proteins as pH was raised between 5 and 10,

representing a dequenching of tryptophan residues. Tryptophan residues located within the

interior of globular proteins are most likely hydrogen-bonded to other groups (Voet and Voet

2004). The cdAE1 protein has four tryptophans per subunit: W75, W81, W94 and W105 (see

Figure A1). In fact, a hydrogen bond between W105 of one subunit and D316 of the other

subunit that is present in the lower pH conformation is broken in a higher pH conformation of

cdAE1 as the peripheral protein binding domain moves away from the dimerization arm (Zhou

and Low 2001). Nearby groups can quench tryptophan fluorescence, hence the increase in

fluorescence intensity as the protein begins to open up with breaking of hydrogen bonds caused

by increasing pH. There was also a similar increase in peak wavelength (red-shift) at alkaline

pH, indicating that the tryptophans were exposed to a more polar environment. These results

indicate that the proteins have similarly folded structures. Urea denaturation of proteins

monitored by intrinsic fluorescence showed no significant differences in the sensitivity of the

proteins to this chemical denaturant, again indicating the proteins had similarly folded structures.

I have constructed cdAE1 variants where each of the four tryptophan residues in cdAE1 has been

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mutated to phenylalanine: W75F, W81F, W94F and W105F. These cdAE1 Trp mutants, listed in

Table 4.1, can be used to study their role in stabilizing the folded structure of cdAE1, and further

pinpoint specific regions of the protein affected by various denaturing conditions. As well, these

mutants could potentially be used as templates to make cdAE1 variants with single tryptophan

residues. Replacement of tryptophan with phenylalanine is a conservative substitution in terms of

retaining hydrophobicity, but its hydrogen-bonding capability will be removed which may have

an impact on protein folding.

The thermal stabilities of the mutant proteins were measured by CD during thermal melts

and by DSC. By both methods the E40K and G130R mutants were found to have similar

stabilities to the wild-type cdAE1. The P327R mutant had an apparent midpoint transition that

was 5 ºC lower than wild-type indicating a slight thermal destabilization of the domain. Another

research group using SDSL in combination with EPR and DEER spectroscopies found that this

mutant had no global effect on the structure, but did affect the packing of the helix in the

dimerization arm where it resides (Zhou et al. 2007). The P327 residue from one subunit occurs

on the same side of the dimer as E40 and G130 from the other subunit. This made the creation of

a monomer mutant of cdAE1 desirable, both for structural purposes and for binding assays. It is

likely that formation of the dimer is necessary for protein 4.2 binding since the three sites of HS

mutation may form part of a large binding surface.

The dimer interface is composed of a β-sheet formed by one β-strand from each monomer.

As well, nine leucine residues contributed from both subunits interact in a hydrophobic pocket.

Two of these leucine residues, Leu319 and Leu321, also reside on the β-strands used to form the

β-sheet. Given the dual role of these leucine residues, I decided they were good candidates to

mutate for dimer disruption. As listed in Table 4.1, I mutated one or both of Leu319 and Leu321

to lysine or glutamate in hopes that introduction of one or two charged residues into the

hydrophobic pocket would disrupt the hydrophobic interactions and weaken the bonding

between β-strands. I also made more extreme mutations where I deleted successive helices and

strands from the C-terminus of cdAE1, which includes the dimerization arm. Deletion of the

final helix, but retention of the β-strand, resulted in formation of a mixture of dimers and

monomers as determined by SE experiments using the analytical ultracentrifuge. Deletion of the

β-strand in the other three deletion mutants resulted in low to no protein expression. Of the single

point mutants, only L319K yielded pure enough protein for analysis, but resulted in a dimer-

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monomer mix. The L319/321K double mutant resulted in dimer formation, but the L319/321E

double mutant resulted in a monomer and was further characterized. As determined by

calorimetry, it was not surprising that the melting temperature was lower for the L319/321E

monomer than for wild-type cdAE1 since dimer dissociation was not a part of the thermal

unfolding pathway. This monomer could be used in future binding studies with protein 4.2 to

determine whether dimer formation is in fact necessary for conformational stability and protein

interactions.

The cdAE1 mutant A285D (Band 3 Boston) represents a point mutation in the cytoplasmic

domain resulting in defective protein expression leading to HS (Jarolim et al. 1996). The mutant

mRNA is present in normal amounts but AE1 protein level is decreased suggesting the mutation

results in a protein product that gets degraded. Deficiency of AE1 at the cell surface removes

points of contact between the membrane and cytoskeleton, leading to HS. It would certainly be

worthwhile to study AE1 A285D in transfected HEK-293 cells to compare its expression level

and protein half-life to those of wild-type AE1. However, in cdAE1 with the three HS mutations

E40K, G130R and P327R, protein degradation of AE1 is not the cause of HS since normal

amounts of AE1 are detected at the red cell membrane and almost all proteins known to associate

with cdAE1 are seen in normal amounts in patient red cells. The only deficient protein in the

whole AE1 complex is protein 4.2. This led to the hypothesis that these three HS mutations,

through conformational changes or interaction surface changes, cause impaired binding of

protein 4.2 to cdAE1 resulting in protein 4.2 deficiency. This was the basis for the next part of

the present study.

4.2 Protein 4.2 interaction with HS mutants of cdAE1

Full-length protein 4.2 HA-tagged at its C-terminus was successfully expressed in the HEK-293

human cell line. Previous unsuccessful attempts prompted researchers to instead express only the

23 kDa N-terminal region with a FLAG tag (Mandal et al. 2003). The expression of full-length

protein 4.2 in this thesis was possible in the presence or absence of AE1, indicating that AE1 was

not needed for protein 4.2 expression in these cells, as it may be in red blood cells. Protein 4.2

was found throughout the HEK-293 cells, including the cytoplasm, ER and plasma membrane. It

was predominantly associated with the cytoskeletal fraction. It was able to reach the plasma

membrane in the absence of AE1, showing its lack of dependence on AE1 for membrane

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localization. When co-expressed, protein 4.2 co-localized with AE1HS mutants at the plasma

membrane, as well as with kAE1 and mdAE1. Since protein 4.2 is unable to interact with the

membrane domain (Korsgren and Cohen 1986) this co-localization does not suggest any

interaction between protein 4.2 and AE1, but simply indicates that protein 4.2 is able to localize

to the plasma membrane. Protein 4.2 co-localized with AE1SAO at the ER indicating its ability

to localize to that internal membrane. Again, interaction cannot be inferred from this result since

protein 4.2 also co-localized with CNX at the ER in the absence of AE1SAO. The broad

distribution of protein 4.2 in HEK-293 cells may be a consequence of over-expression of this

protein.

I showed that protein 4.2 was able to interact with AE1 using co-ip and Ni-NTA pull-down

assays. This interaction has been shown in the past between red cell-purified protein 4.2 and red

cell IOVs (Korsgren and Cohen 1986) as well as with chymotrypsin-released cdAE1 from red

cells (Korsgren and Cohen 1988). The interaction has also been shown in pull-downs from

Xenopus oocytes (Toye et al. 2005), and now I have demonstrated it in human HEK-293 cells.

This expression system allows for an efficient and economical analysis of protein 4.2 binding

with AE1 and its mutants in a human cell line. AE1HS proteins were also able to bind protein

4.2, but the binding was impaired. The binding was not abolished by these mutants, and was not

expected to be, since even in patient red cells there is some protein 4.2 present. It is possible that

the binding interface between protein 4.2 and cdAE1 is so large that one point mutation will not

cause complete binding impairment since other parts of the binding surface remain intact. The

introduction of a positive charge by each of the three HS mutations may cause electrostatic

repulsion, enough to repel the region of protein 4.2 that binds there.

Surprisingly, kAE1 was able to bind similar amounts of protein 4.2 as erythroid AE1. The

kidney isoform is missing the first 65 residues, which includes the site of the E40K mutation.

Since a positively-charged Arg-Arg motif in protein 4.2 has been found to be essential for

interaction with cdAE1 (Rybicki et al. 1995), it may be that introduction of a positive charge into

a very acidic binding region of cdAE1 compromises the binding in a way that absence of this

region does not. The kidney isoform, with its more open structure (Pang et al. 2008), may

compensate structurally for the missing central β-strand in such as way as to accommodate the

binding of protein 4.2. Wild-type levels of AE1SAO binding to protein 4.2 were not surprising as

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the cytoplasmic domain of this mutant is unchanged and deficiencies of protein 4.2 in AE1SAO

red cells have not been reported.

The fatty acylation mutant of protein 4.2, G2A/C173A (GC), showed reduced plasma

membrane localization in the absence of AE1 and in the presence of mdAE1. When co-expressed

with wild-type AE1, it localized to the plasma membrane indicating a protein interaction. Both

wild-type and GC protein 4.2 were found to be mostly associated with the cytoskeleton of HEK-

293 cells. This would put protein 4.2 in a good position for binding to plasma membrane

proteins, such as AE1. The fatty acyl moieties on protein 4.2 would also help it interact with the

plasma membrane. With GC protein 4.2 lacking these fatty acid modifications, even though it is

present in the same amounts as wild-type protein 4.2 at the cytoskeleton, it is unable to interact

with the plasma membrane on its own. It is only when co-expressed with AE1 that it becomes

localized to the plasma membrane, probably through protein interaction.

Future studies would focus on characterizing the interaction between cdAE1 and protein 4.2

in vitro by using purified full-length protein 4.2 and purified cdAE1 proteins. In the absence of

pure protein 4.2, a detergent-free cell lysate containing the GC mutant form of protein 4.2 could

be used in semi-quantitative binding experiments with pure cdAE1. Lack of detergent may allow

for more native interactions to occur between protein 4.2 and cdAE1 since these proteins interact

in the cytoplasm, not the membrane. I have grown HEK-293 cells expressing protein 4.2 and the

GC mutant and lysed them using detergent, and by needle aspiration followed by sonication,

respectively. The latter method yielded more protein in the soluble versus particulate fraction.

His6-tagged cdAE1 and protein 4.2-expressing HEK-293 lysate could be incubated together

followed by pull-down on Ni-NTA resin. Alternatively, the His6-tagged cdAE1 could be first

immobilized on Ni-NTA resin, to mimic the situation in red cells where cdAE1 is immobilized at

a high density at the plasma membrane as part of AE1. Then HEK-293 lysate expressing protein

4.2 would be added for binding to cdAE1. The advantage of using HEK-293 cells for protein

expression is that full-length, and presumably properly folded, protein 4.2 can be obtained, as

opposed to expression in E. coli or cell-free translation. The disadvantage of this method is that

by using a cell lysate, many other proteins are present and we cannot say with certainty that an

interaction reflects direct binding between proteins. Ni-coated microplates could also be used for

this purpose allowing measurement of protein 4.2 binding to several cdAE1 variants and

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concentrations at one time. However, the same advantages and disadvantage apply to this

method as with the Ni-NTA pull-down.

A variation on this pull-down assay would be to use a rabbit reticulocyte cell-free system to

express protein 4.2 using [35

S]-methionine for detection. This would provide radiolabeled protein

4.2 to be used in pull-down assays. This system would allow for protein 4.2 to be enriched in the

lysate, although it would contain a large amount of hemoglobin which also binds to cdAE1. An

advantage would be that radiographic detection would be much more sensitive and direct, since

[35

S]-Met would be incorporated into protein 4.2, thereby removing the primary and secondary

antibody steps involved in immunodetection. I have expressed full-length protein 4.2 in this

system and preliminary results have shown its ability to bind cdAE1 and HS mutants above

background. However, the yield for this expression system is low and obtaining higher yields

would be very costly.

There has been much difficulty in expressing and purifying full-length protein 4.2 in

bacteria by myself and others (Bhattacharyya et al. 1999, Korsgren et al. 2010). For this reason I

have created several protein 4.2 GST-fusion proteins in the hope that at least the cdAE1-binding

regions could be expressed in E. coli and purified using glutathione resin for use in binding

assays (see Table 4.1). The boundaries of these fusion proteins were determined based on natural

protease cleavage sites (Bhattacharyya et al. 1999), regions known to bind cdAE1

(Bhattacharyya et al. 1999, Rybicki et al. 1995) and predicted surface-exposed residues based on

the protein 4.2 homology model (Figure 1.8). I designed protein 4.2 GST fusion proteins to

express full-length protein 4.2, the 23 kDa N-terminal domain (residues 1-238), the cdAE1-

binding domain encompassing residues 1-144, the cdAE1-binding region encompassing residues

145-238 and the region predicted to form a β-hairpin (residues 145-203). Following vector

transformation into E. coli, only the shortest fusion proteins containing the predicted β-hairpin

(Figure 1.8) encompassing residues 145-238 and 145-203 were successfully expressed and

purified. Since this hairpin region appears to be important for cdAE1 binding (Bhattacharyya et

al. 1999) these GST-fusion proteins could be used in binding assays with wild-type and HS

cdAE1 to determine the hairpin-binding region on cdAE1. These fusion proteins could be used in

Ni-NTA pull-down assays where His6-tagged cdAE1 proteins are first immobilized on resin

followed by fusion protein incubation. The reciprocal experiment could also be performed where

GST-fusion proteins are first immobilized on glutathione resin followed by cdAE1 incubation. If

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one of the sites of HS mutation were a region of interaction for any particular protein 4.2

fragment, this would become obvious in the binding studies. The hairpin fragment could also be

used in competition binding assays with full-length protein 4.2.

These fusion proteins could also be used in far western, or blot overlay, assays. In this case

fusion proteins would be run on SDS-PAGE and transferred to nitrocellulose. Wild-type and HS

mutant cdAE1 proteins would then be added to separate protein 4.2-GST blots, followed by a

western blot procedure using anti-cdAE1 antibodies to detect and compare binding. I have

performed the reciprocal version of this experiment where cdAE1 proteins are run on the gel and

transferred to the blot, followed by incubation with β-hairpin-GST fusion protein. Results were

inconclusive, partly owing to the fact that cdAE1 probably needs to dimerize for protein 4.2

binding, but SDS-PAGE is denaturing and most likely does not allow proper refolding of the

cdAE1 domain. The same problem could occur when the protein 4.2 GST fusion proteins are run

on the SDS-PAGE gel.

It may be preferable to use an expression system that allows protein 4.2 to be myristoylated

and palmitoylated, as it is in nature, since these modifications may have an effect on cdAE1

binding. Because of the need for these co- and post-translational modifications and protein

aggregation problems, bacterial systems are not ideal. Using purification tags, such as a His6 tag

or GST tag, would allow purification of protein 4.2 from HEK-293 cells on Ni-NTA or

glutathione columns, respectively. Alternatively, Protein G-Sepharose and an anti-protein 4.2

antibody could be used to purify protein 4.2, or an anti-HA antibody to purify HA-tagged protein

4.2. But this would be more costly, and protein 4.2 would still have to be separated from the

antibody used to purify it. In any case, the expression of protein 4.2 in HEK-293 cells would

have to be scaled up quite a bit to achieve high enough yields for structural and in vitro binding

studies.

I have also expressed the AE1HS mutants and protein 4.2 in K562 cells, an erythroleukemia

cell line. The advantage of these cells is that they are from the erythroid lineage and may contain

many of the same proteins in erythroid precursors that make for a more native environment for

AE1 and protein 4.2. I had hoped that by using a viral transfection method with these cells,

including viral expression vector pFB-Neo (see Table 4.1), I could create stable transfectants, but

this proved to be quite difficult. As well, while expression of AE1 proteins and their plasma

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membrane localization were consistent, this was not the case with protein 4.2. These cells gave

variable protein 4.2 expression and localization, where protein 4.2 was sometimes seen at the

plasma membrane and other times throughout the cell. Successful expression was heavily

dependent on the health and age of the cells and their use became increasingly cumbersome. For

this reason, HEK-293 cells were used for interaction and localization studies and gave more

reliable and reproducible results.

4.2.1 Quantitative in vitro binding analysis

Binding of purified protein 4.2 to HS mutants of cdAE1 could be performed, as well as to other

cdAE1 variants where selected residues have been mutated to determine specific binding

regions. By using quantitative methods such as isothermal calorimetry (ITC), surface plasmon

resonance (Biacore) or analytical ultracentrifugation, we could assign importance to each

mutation site depending on its ability to disrupt the protein interaction. The major advantage of

these quantitative methods is that binding affinities can be determined for each interaction.

Another advantage is that direct interactions between two proteins can be measured. I would be

able to compare direct binding between cdAE1 and the HS mutants. I have also created alanine

mutants in cdAE1 where alanine has replaced each of the HS mutations: E40A, G130A and

P327A (Table 4.1). These mutant proteins have similar CD spectra to wild-type cdAE1. Using

these Ala mutants in binding assays with protein 4.2 will help determine whether the binding

defect is caused by the introduction of a positive charge in cdAE1 or because of the absence of

specific residues, namely glutamate, glycine and proline. Perhaps these residues have specific

roles in the binding that can only be fulfilled by the native residue or those with similar

properties. These Ala mutations can also be made in full-length AE1 for expression in HEK-293

cells and for use in co-ip assays. The monomer mutant of cdAE1 that I have constructed could

also be used in binding assays to determine whether dimer formation is necessary for protein 4.2

binding. Double and triple HS mutations could also be made in cdAE1, for example, the double

mutant E40K/G130R and the triple mutant E40K/G130R/P327R. An additive effect by these

mutants would be seen as a more pronounced protein 4.2 binding impairment than was seen for

any of the single HS mutants. This would strengthen the idea that these three HS mutation sites

contribute to a large protein 4.2-binding surface.

Mutations would also be made in protein 4.2 in order to characterize important residues

involved in the interaction. I have already created a protein 4.2 mutant where the important

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Arg34-Arg35 motif has been mutated to Glu34-Glu35 (EE protein 4.2) and I have done

preliminary co-ip experiments in HEK-293 cells with wild-type and E40K AE1. If the Arg-Arg

motif does indeed interact with E40 in cdAE1 as proposed (Rybicki et al. 1995), and the lysine at

position 40 does electrostatically repel the Arg-Arg motif, then it is reasonable to expect the EE

mutant to restore E40K binding to wild-type levels. In this case, the salt bridge would be

restored. In fact, in my preliminary co-ip experiments I saw that EE protein 4.2 was able to bind

E40K AE1 to a similar level as wild-type protein 4.2 binding to wild-type AE1. More trials

would have to be done to confirm these results. As well, purification of the EE protein 4.2

mutant would enable more quantitative in vitro binding studies with wild-type and E40K cdAE1.

It would be interesting to study the AE1-protein 4.2-ankyrin complex, since protein 4.2

binds to both cdAE1 and ankyrin (Korsgren and Cohen 1988) and strengthens the AE1-ankyrin

interaction (van den Akker et al. 2010a). Even though these three proteins exist in a complex, a

fraction of AE1 extracted from red cells is associated only with protein 4.2 (Yu and Steck 1975)

showing that these two proteins can interact on their own without ankyrin. Study of a three-

protein complex is much more complicated than a two-protein complex, hence our focus at this

point only on the AE1-protein 4.2 interaction. In the future, addition of ankyrin into the mix will

help to further characterize the interaction of these three proteins and the effect of AE1HS

mutations. Ankyrin is, however, a very large cytoskeletal protein consisting of 1880 residues,

with many binding partners (Lux et al. 1990). The N-terminal region of ankyrin is the

membrane-binding domain and contains 24 ankyrin repeats. The D34 region contains repeats 13-

24 (residues 402-827) and is known to bind to cdAE1 and protein 4.2 (Su et al. 2006). The D34

domain is easily expressed in E. coli and purified using affinity tags engineered into the

recombinant protein. The use of purified cdAE1, protein 4.2 and D34 ankyrin would make

possible the characterization of this three-protein complex by quantitative binding studies.

4.2.2 Structure determination

Determining the binding interface between cdAE1 and protein 4.2 by introducing point

mutations and measuring protein affinities would help in the identification of important residues

involved in binding, and the effect of various natural mutations. However, a structure of the

cdAE1-protein 4.2 complex would be far more informative. A crystal structure of cdAE1 at low

pH has been solved (Zhang et al. 2000a), but no structure of protein 4.2 exists as of yet, let alone

in complex with cdAE1. The problem of protein 4.2 structure determination lies with expression

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and purification of high amounts of protein. It may be that expression of protein 4.2

simultaneously with cdAE1 would help stabilize it, allowing for purification of the complex.

Expressing the proteins together may also negate the need for detergents to solubilize protein 4.2.

Since protein 4.2 and AE1 appear together and interact as soon as they are expressed in red blood

cell precursors (van den Akker et al. 2010a) this may provide a more suitable environment for

the production of a complex. In this case the proteins would be purified together. Since a

complex is what we seek for structure determination, this may be the ideal situation. cdAE1

could only be crystallized at low pH and this structure may not completely represent the native

form. However, structural features determined by SDSL in combination with EPR and DEER

spectroscopies at neutral pH were in agreement with those in the crystal structure (Zhou et al.

2005b). Co-expression and co-purification of cdAE1 with protein 4.2 may also allow structure

determination of both proteins at neutral pH if it is found that the proteins stabilize each other in

solution. Another way to obtain high levels of a cdAE1-protein 4.2 complex would be to

trypsinize inside-out red cell ghosts which would release cdAE1 from the membrane along with

interacting protein 4.2. Even if only 10 % of AE1 is associated with protein 4.2 at the membrane

(Yu and Steck 1975) that is still a large number of cdAE1-protein 4.2 complexes that can be

collected, since there are about 1.2 million AE1 molecules per red cell. Removal of protein 4.2

from IOVs required harsh alkaline conditions (Steck and Yu 1973) which are needed to disrupt

the cdAE1-protein 4.2 interaction. Such harsh conditions may not be required if protein 4.2 is to

be isolated in a complex with cdAE1, since the latter may be proteolytically cleaved from the

membrane. From there, the complex may be purified using traditional protein purification

techniques, such as anion exchange chromatography and gel filtration chromatography.

Determining the structure of the monomer mutant of cdAE1 would be necessary to

determine whether the monomer is folded in the same way as the individual subunits within the

dimer, or becomes misfolded when expressed alone. Binding experiments could proceed with

confidence if it is discovered that the monomer truly represents a single subunit from the dimer

that is properly folded. Following purification of large amounts of monomer cdAE1,

crystallization screens would be performed to determine the best conditions for crystal

formation. Alternatively, NMR could be used for structure determination of the cdAE1 monomer

as well as wild-type cdAE1 at neutral pH. A structure for the kidney isoform of cdAE1 would

also be desirable, as well as that of the variant missing the N-terminal 54 residues from cdAE1

(cdΔ54AE1). I have obtained preliminary 2D NMR spectra for all of these proteins, some at

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varied pH, temperature, time and magnetic field strength. Thus far, the monomer cdAE1 gives

the best spectra with the most resolved peaks, while cdAE1, cdkAE1 and cdΔ54AE1 appear to

be aggregated. The poor spectra may be due to the size of the proteins since cdAE1 forms a

dimer of 43 kDa subunits. Conditions would have to be optimized for all constructs in order to

obtain spectra that can be reasonably compared.

4.2.3 Interaction of protein 4.2 and AE1 during red cell development

Great interest lies in studying the dynamic interactions that occur during red cell development.

Protein 4.2 and AE1 have been found to appear simultaneously during early erythropoiesis, and

interact with each other as soon as they are expressed (van den Akker et al. 2010a) as determined

from differentiating erythroblasts. CD34+ cells are early hematopoietic progenitors that can be

stimulated to differentiate into erythroblasts, and later into erythrocytes. CD34+ cells mainly

reside in adult bone marrow, but can also be isolated from peripheral blood (Lataillade et al.

2005). The use of CD34+ cells from peripheral blood of healthy donors and AE1HS patients

would allow the study of protein 4.2 interactions with AE1 and AE1HS from the moment they

are expressed and interact, and onward in the differentiation process. Co-immunoprecipitation

assays using anti-AE1 or anti-protein 4.2 antibodies could be employed at various stages of

differentiation. We would be able to study the trafficking of the AE1-protein 4.2 complex as it

moves through the ER, Golgi complex, and finally to the plasma membrane. This would also

allow us to determine the fate of protein 4.2 in red cells of HS patients where impaired AE1-

protein 4.2 interaction leads to protein 4.2 deficiency. CD34+ cells would be superior to

transformed erythroid cell lines, such as K562 erythroleukemia cells, since they represent the

most native erythrocyte environment.

4.3 Conclusions

In my thesis I have investigated the structure and conformational stability of three HS mutants of

cdAE1 and their interaction with protein 4.2.

I have expressed and purified the three HS mutants of cdAE1, E40K, G130R and P327R, in

E. coli. Through various biophysical methods I discovered that the mutations do not cause any

gross conformational changes in the protein other than a slight thermal destabilization caused by

P327R, which had a Tm 5 ºC lower than that of wild-type cdAE1. This supports the finding that

normal amounts of these mutants occur at the plasma membrane of red blood cells. Many

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disease-causing mutations result in misfolded and degradation-prone proteins, if they get

expressed at all. The three HS mutations at the centre of this thesis are part of a minority of

disease-causing missense mutations that are stably expressed in the cell, yet affect the functional

properties of the protein (Gregersen et al. 2000). In the case of these three HS mutations, the

disease mechanism is quite different from the straightforward mutation resulting in degradation

of protein.

I have expressed full-length HS mutants of AE1 in HEK-293 cells and have shown that their

expression levels are similar to that of wild-type AE1 and they localize to the plasma membrane

of these cells. I have also expressed full-length protein 4.2 in these cells and have shown that its

expression levels are similar in the absence or presence of AE1. I demonstrated the broad

localization pattern of protein 4.2 in HEK cells, including at the plasma membrane even in the

absence of AE1. I showed co-localization of protein 4.2 at the membrane with AE1, AE1HS

mutants, kAE1 and mdAE1 and its co-localization with AE1SAO at the ER. I showed that the

three AE1HS mutants had impaired binding to protein 4.2 as was expected. The partial, versus

complete, deficiency of protein 4.2 in HS patient red cells supports the moderate, but significant,

binding impairment seen in HEK cells. I also showed that kAE1 and AE1SAO bind to protein

4.2 as well as wild-type AE1. Fatty acylation of protein 4.2 was necessary for plasma membrane

localization in the absence of AE1, as demonstrated by the fatty acyl mutant G2A/C173A (GC)

protein 4.2. By subcellular fractionation analysis, fatty acylation was shown to have no effect on

the ability of protein 4.2 to associate with the cytoskeleton.

Based on the crystal structure of cdAE1, the three HS mutations are located far apart from

each other in the folded protein. Since all three mutations affect protein 4.2 binding, each most

likely contributes to a large binding surface. Protein 4.2 is a fairly large protein at 72 kDa and,

based on its homology model, is large enough to interact with each of the HS mutation sites on

cdAE1 at once. The deficiency of protein 4.2 in HS red cells caused by these mutations is, at

least in part, a result of this impaired interaction.

Protein-protein interactions have become intriguing drug targets in recent years, not for

inhibiting interactions, as has been the standard methodology, but for promoting them (Corson et

al. 2008). This can be done using bifunctional ligands that bind two different protein targets

together. These ligands are composed of two different protein-binding moieties that are joined

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together by linkers of appropriate length. The protein-binding moieties can be natural or

synthetic protein-binding small molecules, and the linkers can be polymers such as polyethylene

glycol chains of varying length. In a disease such as HS, which is caused by impaired protein-

protein interactions, patients may benefit from such a strategy. This type of therapy requires

specific knowledge of the binding surfaces of the proteins that are to be brought together. It is

my hope that my work has contributed to elucidating the mechanism of the cdAE1-protein 4.2

interaction. As well, I hope I have contributed to the understanding of the mechanism of

defective protein interactions in disease in general, and in HS in particular.

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Appendix: Structural characterization of the cytoplasmic domain of the

kidney chloride/bicarbonate anion exchanger 1 (kAE1)

Adapted with permission from Pang, A.J., Bustos, S.P. & Reithmeier, R.A. Structural

characterization of the cytosolic domain of kidney chloride/bicarbonate anion exchanger 1

(kAE1). Biochemistry, 47, 4510-4517. Copyright 2008 American Chemical Society

(http://pubs.acs.org/doi/abs/10.1021/bi702149b) (Pang et al. 2008). Jing Li contributed to this

work by generation of the cdAE1, cdkAE1, and cdΔ54AE1 cDNA vectors. I supervised and

trained summer student Allison Pang, who performed all experiments and assisted in the

experimental design and analysis of results. I designed all experiments and assisted in the

analysis of results.

Abstract

kAE1 is a membrane glycoprotein expressed in α-intercalated cells in the collecting ducts of the

kidney where it mediates electroneutral chloride/bicarbonate exchange. Human kAE1 is a

truncated form of erythroid AE1 that is missing the first 65 residues of the N-terminal

cytoplasmic domain, which includes a disordered acidic region (residues 1-54) and the first β-

strand (residues 55-65) of the folded region. Unlike erythroid AE1, kAE1 does not bind

deoxyhemoglobin, glycolytic enzymes, or ankyrin. To understand the effect of the N-terminal

deletion on the structure of the cytoplasmic domain, we performed an extensive biophysical

analysis on His6-tagged cdAE1, the cytoplasmic domain of kAE1 (cdkAE1), and a novel

truncation mutant (cdΔ54AE1) missing the first 54 residues, but retaining the β-strand. CD did

not reveal any major differences in secondary structure, and sedimentation equilibrium

experiments showed that all three proteins were dimeric. DSC revealed that cdAE1 and

cdΔ54AE1 had similar thermal stabilities, with apparent midpoints of transition higher than

cdkAE1. cdAE1 and cdΔ54AE1 underwent similar pH-dependent fluorescence changes, while

cdkAE1 exhibited a higher fluorescence at neutral and acidic pH. Urea denaturation resulted in

dequenching of tryptophan fluorescence in cdAE1, while tryptophans in cdkAE1 were already

dequenched in the native state. We conclude that the absence of the central β-strand in cdkAE1

results in a less conformationally stable and more open structure than cdAE1. This structural

change, in addition to the loss of the acidic N-terminal region, may account for the altered

protein binding properties of kAE1.

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Introduction

AE1, also known as Band 3, is a major integral membrane protein of the human erythrocyte

membrane. It is a 911 amino acid glycoprotein that is responsible for the electroneutral exchange

of bicarbonate for chloride (Lepke and Passow 1976). Human AE1 has a monomer molecular

weight of 95 kDa and exists as a dimer and a tetramer in the red blood cell membrane (Casey and

Reithmeier 1991b, Wang et al. 1993). Mild proteolytic cleavage of this protein in erythrocyte

membranes yields two functional domains. The 52 kDa C-terminal TM domain (Gly361-Val911)

spans the membrane up to 12 times (Fujinaga et al. 1999, Popov 1999, Reithmeier et al. 1996,

Zhu et al. 2003) and mediates the anion transport function (Jennings 1989a, Lepke and Passow

1976). The 43 kDa N-terminal cytoplasmic domain includes Met1-Lys360 and provides binding

sites for various red cell cytoskeletal and cytoplasmic proteins (Lepke and Passow 1976). The

cytoplasmic domain of AE1 (cdAE1) acts as an anchoring site for ankyrin and protein 4.2, both

of which are found in the membrane’s cytoskeleton (Hargreaves et al. 1980, Perrotta et al. 2005).

AE1 is therefore thought to play an important role in maintaining the shape, stability, and

flexibility of the red blood cell (Lux and Palek 1995, Peters et al. 1996). AE1 also binds and

regulates the function of deoxyhemoglobin and various glycolytic enzymes such as GAPDH,

aldolase, and phosphofructokinase (Low 1986, Perrotta et al. 2005, Walder et al. 1984). The

cytoplasmic domain is characterized by a highly acidic N-terminal region with an N-acetylated

terminal methionine (Wang et al. 1992). This region of the domain is involved in binding

associated proteins (Perrotta et al. 2005), although other parts of the cytoplasmic domain also

provide protein binding sites, such as the ankyrin-binding loop (Chang and Low 2003).

In erythrocytes, AE1 transports bicarbonate, which is produced by CAII, into the plasma in

exchange for chloride, thereby increasing the CO2-carrying capacity of the blood (Jennings

1989a). In the human kidney, a truncated form of AE1 (kAE1) catalyzes the exchange of

bicarbonate and chloride across the basolateral membrane of acid-secreting cells in the collecting

ducts of the kidney, resulting in bicarbonate reabsorption into the blood and promoting acid

excretion into the urine. Human kAE1 is missing residues 1-65 of the erythroid form (Wang et

al. 1995). Since the acidic residues of the N-terminal region, which electrostatically bind

cytoskeletal and cytoplasmic proteins, are absent in this protein, the cytoplasmic domain of

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kidney AE1 (cdkAE1) is thought to have a different function than the erythroid isoform,

potentially mediating the binding of specific kidney proteins. Indeed, kAE1 has been shown not

to bind to glycolytic enzymes, deoxyhemoglobin, protein 4.1, or ankyrin (Ding et al. 1994, Wang

et al. 1995a), but has been found to bind to integrin-linked kinase, a protein involved in actin

cytoskeletal interactions (Keskanokwong et al. 2007).

The crystal structure of erythroid cdAE1 has been elucidated (Figure A1) at low pH by X-

ray diffraction, revealing a tight, symmetric dimer stabilized by interlocking dimerization arms

contributed by each subunit (Zhang et al. 2000). The purified recombinant protein has been

shown to have the same secondary structure, Stokes radius, and display similar pH-dependent

conformational changes as the native cytoplasmic domain prepared from red blood cells (Wang

et al. 1992b). Except for a missing N-acetylated N-terminus, no significant differences were

observed between the recombinant and native proteins. The first 54 residues of AE1 were

unresolved in the crystal structure due to the strongly anionic and disordered nature of the region

(Zhang et al. 2000, Zhou et al. 2005). The crystal structure also shows that residues 55-65 in

AE1 form a β-strand that is present in the core of the protein. Since the kidney isoform is missing

this β-strand, it is possible that the kidney cytoplasmic domain has a significantly different

structure from the full-length erythroid form.

Figure A1: Crystal structure of human cdAE1.

The structure of the cdAE1 dimer was solved at a resolution of 2.90 Å at pH 4.8 (Zhang et al.

2000). One monomer is coloured dark gray and the other is coloured light gray. The β-strand

insert, which is absent in the kidney isoform, is shown in black. The N-terminal residues 1–54

are not visible in the crystal structure. The positions of the four tryptophan residues in cdAE1 are

shown in black.

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The goal of this work was to structurally analyze cdAE1 and cdkAE1, as well as a novel

truncation mutant, cdΔ54AE1, which lacks the first 54 residues but retains the core β-strand. A

schematic of these three constructs is illustrated in Figure A2.

Figure A2: Domain structure of AE1 isoforms and gel-separated AE1 constructs.

(A) Domain structure of AE1, kAE1, and the cdAE1, cdkAE1, and cdΔ54AE1 constructs.

Residues 1–54 are not visible in the crystal structure, and residue 356 is the last residue that was

visible in the crystal structure of cdAE1. Residues 55–65 encompass the first β-strand in cdAE1.

(B) 12 % SDS-PAGE gel of the purified constructs stained with Coomassie blue. Lane 1:

molecular weight markers. Lane 2: eluate of purified cdkAE1. Lane 3: eluate of purified cdAE1.

Lane 4: eluate of purified cdΔ54AE1.

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Materials and methods

Materials

The following is a list of materials used and their suppliers: pcDNA3 vector (Invitrogen, San

Diego, CA); mutagenic primers (ACGT Corp., Toronto, ON); pETBlue-1 vector and Tuner

BL21(DE3)pLacI E .coli competent cells (Novagen, Madison, WI); growth media for E. coli

(BD, Sparks, MD); chloramphenicol and carbenicillin (Sigma, St.Louis, MO); IPTG (Bioshop,

Burlington, ON); Ni-NTA agarose resin (QIAGEN, Germantown, MD); PD-10 gel filtration

columns (Amersham Biosciences); lysozyme from chicken egg white (Sigma, St.Louis, MO);

deoxyribonuclease from bovine pancreas (Sigma, St. Louis, MO); and Sequanal grade urea

(Pierce, Rockford, IL).

Plasmid construction and mutagenesis

The cdAE1 construct was amplified by PCR from full-length human AE1 on a pcDNA3 vector,

which was then cloned into a pETBlue-1 expression vector. This expression vector contained an

IPTG-inducible T7lacO promoter (Giordano et al. 1989). The reverse primer encoded six

histidine residues that were used as a C-terminal tag for purification purposes. cdkAE1 and

cdΔ54AE1 were also amplified by PCR from full-length human AE1 using primers that

corresponded to their respective N- and C-termini. The primary sequence of all three proteins

extended to the last visible residue in the crystal structure of cdAE1, which is serine 356.

Constructs were confirmed by sequencing by ACGT Corp.

Protein expression and purification

All constructs were expressed in E. coli Tuner BL21(DE3)pLacI competent cells at 37 °C. Large

cultures of cells were grown in one liter of LB medium containing 50 μg/ml carbenicillin and 34

μg/ml chloramphenicol until an A600 of 0.5-0.6. Expression of the constructs was induced by the

addition of 1 mM IPTG (Studier and Moffatt 1986). Cells were grown for an additional five h

and then harvested by centrifugation at 4 000 rpm for 30 min. Cell pellets were solubilized in 80

ml of lysis buffer (50 mM sodium phosphate, 300 mM sodium chloride, 10 mM imidazole, 0.2 %

βME, and 0.2 % Triton X-100 (pH 8.0)). The following protease inhibitors were used: 2 μg/ml

aprotinin, 1.6 mM PMSF, 0.7 μg/ml pepstatin A, and 10 μM leupeptin. DNase and lysozyme

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were added to the cell pellets. The solubilized cell pellets were sonicated at 40 % duty cycle for

1.5 min on ice. Purification of the protein constructs was carried out using 1 ml of Ni-NTA

agarose beads (QIAGEN) per 80 ml lysate at 4 ºC. Resin was equilibrated using the lysis buffer

and, following protein binding, was washed twice with 10 ml of wash buffer (50 mM sodium

phosphate, 300 mM sodium chloride, 20 mM imidazole, and 0.2 % βME, pH 8.0). Protein was

eluted three times using 1 ml of elution buffer (50 mM sodium phosphate, 300 mM sodium

chloride, 250 mM imidazole, and 0.2 % βME pH 8.0). The three elution fractions were

combined, filtered, and applied to a pre-equilibrated PD-10 gel filtration column in order to

exchange the buffer with 10 mM NH4HCO3. Proteins were lyophilized overnight and stored at -

20 °C. Protein purity was determined to be >95 % by SDS-PAGE and Coomassie blue staining.

The final concentration of the purified proteins was measured by the BioRad protein assay,

which is based on the Bradford assay.

Analytical ultracentrifugation

SE experiments were performed on an Optima XL-A/XL-I analytical ultracentrifuge (Beckman

Instruments, Palo Alto, CA) at 10 000, 13 000, 16 000 and 19 000 rpm at 20 °C. Samples were

prepared by dissolving lyophilized protein in 10 mM sodium phosphate and 50 mM sodium

chloride (pH 7.5). SE experiments were carried out on cdAE1, cdkAE1, and cdΔ54AE1, each at

three different concentrations with the corresponding A280 values of 0.3, 0.6, and 1.0. Data

analysis was performed using XL-A/XL-I software (Origin version 4.1) from Beckman

Instruments.

SV experiments were also performed on an Optima XL-A/XL-I analytical ultracentrifuge

(Beckman Instruments, Palo Alto, CA) at 30 000 rpm at 4 ºC. Samples were prepared by

dissolving lyophilized protein in 50 mM sodium phosphate and 100 mM sodium chloride (pH

7.5). SV experiments were carried out on cdAE1, cdkAE1, and cdΔ54AE1, each at three

different concentrations with the corresponding A280 values of 0.3, 0.6, and 1.0. Data analysis

was performed using XL-A/XL-I software (SedFit and Ultrascan) from Beckman Instruments.

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Circular dichroism

Lyophilized protein was dissolved in buffer containing 50 mM sodium phosphate and 100 mM

sodium chloride, and adjusted to either pH 5.0, 7.5, or 10.5. Samples were filtered through a 0.22

µm syringe filter. The final concentrations of the protein solutions are indicated in the figure

captions. CD was performed on a Jasco J-810 spectropolarimeter. All spectra were measured at

24 °C from 200 to 260 nm with a 1 nm data pitch in a 1 mm path length cell. The mean residue

ellipticity was measured as a function of wavelength. The secondary structure content of the

spectra was determined using the SELCON3 program from the CDPro software package.

Differential scanning calorimetry

Lyophilized protein was dissolved in buffer containing 50 mM sodium phosphate and 100 mM

sodium chloride, and pre-adjusted to either pH 5.0, 7.5, or 10.5. Samples were filtered through a

0.22 µm syringe filter. The final concentrations of the protein solutions are indicated in the figure

captions. Heat capacity measurements from 25 to 90 °C were obtained on a Microcal VP-DSC

differential scanning calorimeter. Samples were heated at a rate of 1.5 °C/min. The heat capacity

was plotted as a function of temperature. Origin 7.0 data analysis software was used to baseline

correct and to analyze the temperature denaturation data by fitting to a two-state transition

model.

pH dependence of intrinsic fluorescence

Stock solutions of each protein were made by dissolving lyophilized samples in 50 mM sodium

phosphate and 100 mM sodium chloride (pH7.5) and by filtering them through a 0.22 µm

syringe filter. The stock protein was then diluted 50 × into the same buffer preadjusted to the

desired pH, ranging from pH 5.0 to pH 10.5 in 0.5 increments. The final concentration of the

protein samples was ~0.1-0.2 mg/ml. Samples were equilibrated overnight at 4 °C and were

allowed to reach room temperature prior to fluorescence measurement. The excitation

wavelength was 290 nm and the fluorescence emission was measured from 300 to 420 nm for

each protein sample at each pH value. Measurements were taken at 24 °C by a Fluorolog-3 FL3-

22 spectrofluorometer. The average emission wavelength for each sample, which is equal to

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Σ(λ*intensity)/Σ(intensity), was plotted as a function of pH. Fluorescence intensity was also

plotted against wavelength for samples at pH 5.0, 7.5, and 10.5.

Urea denaturation measured by intrinsic fluorescence

Stock solutions of cdAE1 and cdkAE1 were made by dissolving lyophilized samples in 50 mM

sodium phosphate and 100 mM sodium chloride (pH7.5) and filtering them through a 0.22 µm

syringe filter. The stock protein was diluted 50 × into the same buffer preadjusted to the desired

urea concentration ranging from 0 – 8 M. Samples were equilibrated overnight at 4 °C and were

allowed to reach room temperature prior to fluorescence measurement. The excitation

wavelength was 290 nm and the fluorescence emission was measured from 300 to 420 nm for

each protein sample at each urea concentration. Measurements were taken at 24 °C by a

Fluorolog-3 FL3-22 spectrofluorometer. The maximum peak fluorescence intensity was plotted

against urea concentration. The average emission wavelength was also plotted as a function of

urea concentration.

Results

Expression and purification of cdAE1, cdkAE1, and cdΔ54AE1 proteins

The purification of His6-tagged cdAE1, cdkAE1, and cdΔ54AE1 was performed by Ni-NTA

affinity chromatography as described in Methods. This method of purification yielded

approximately 15-20 mg of protein per liter of cell culture, the kidney isoform routinely having

the highest expression. All three proteins were more than 95 % pure as determined by SDS-

PAGE, and they ran as monomers with molecular masses of roughly 43 kDa (cdAE1), 32 kDa

(cdkAE1), and 35 kDa (cdΔ54AE1). The predicted molecular weights of cdAE1, cdkAE1, and

cdΔ54AE1, including their His6 tag, are 40 866 Da, 33 269 Da, and 34 738.5 Da, respectively.

The identity of the constructs were confirmed by mass spectrometry analysis which gave

molecular weights of 40 942.301 Da (cdAE1), 33 345.699 Da (cdkAE1), and 34 814.801 Da

(cdΔ54AE1), all of which were ~76 Da higher than the predicted values. This extra molecular

mass was most likely due to the formation of a βME adduct (+76 Da) on one of the cysteine

residues during purification.

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Analytical ultracentrifugation: sedimentation equilibrium and sedimentation

velocity experiments on cdAE1, cdkAE1, and cdΔ54AE1 proteins

SE experiments were carried out to determine the oligomeric state of the proteins and to see if

the missing 65 (cdkAE1) or 54 residues (cdΔ54AE1) affects the dimerization of the cytoplasmic

domain of AE1. It was previously shown by gel filtration combined with disulfide bond cross-

linking experiments (Appell and Low 1981), and SE experiments (Bustos and Reithmeier 2006)

that cdAE1 exists as a dimer. It was predicted that the missing N-terminal residues of cdkAE1

and cdΔ54AE1 would not distort the native structure of the C-terminal dimerization arm. SE

experiments indicated MWapp values that were approximately twice that of their MWseq. The

ratios of MWapp versus MWseq are listed in Table A1. The plots of ln(Abs) versus the radius

squared were linear for all samples, indicating that the samples were primarily composed of one

major species. A representative plot from the SE experiments on cdAE1 is shown in Figure A3,

as well as the residuals from fitting the data to a single-ideal species model. The results show that

the three proteins existed mainly as stable dimers in solution.

SV experiments were carried out to compare the relative conformations of cdAE1, cdkAE1,

and cdΔ54AE1 based on their S20,w values. The sedimentation coefficient of the erythroid

cytoplasmic fragment has been previously estimated to be ~4.1 S (Appell and Low 1981), in

agreement with our own result for this construct. cdAE1 and cdΔ54AE1 had sedimentation

values of 3.8 S and 3.9 S, respectively. Smaller S20,w values indicated that the truncated proteins

moved more slowly in response to the centrifugal force, likely due to their lower molecular

weights and perhaps more extended structures.

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Table A1: Summary of biophysical properties of cdAE1, cdkAE1, and cdΔ54AE1 at pH 7.5

Property cdAE1 cdΔ54AE1 cdkAE1

MWapp/MWseq

oligomeric state (SE)

1.83 dimer 1.77 dimer 1.93 dimer

S20,W (SV) 4.1 S 3.9 S 3.7 S

Secondary structure (CD) % α-helix = 30.5 ± 1.2;

n = 3

% α-helix = 34.7 ± 1.8;

n = 3

% α-helix = 32.0 ± 0.5;

n = 3

Apparent midpoint of

transition (Tm (°C)) (DSC)

pH 5 = 73.7 ± 1.7; n = 4

pH 7.5 = 65.3 ± 1.2; n = 12

pH 10.5 = 56.1 ± 2.7; n = 6

pH 5 = 73.1 ± 2.9; n = 5

pH 7.5 = 65.8 ± 1.2; n = 11

pH 10.5 = 56.0 ± 2.0; n = 6

pH 5 = 62.4 ± 5.3; n = 5

pH 7.5 = 60.1 ± 2.2; n = 11

pH 10.5 = 50.7 ± 3.0; n = 5

Figure A3: Analytical ultracentrifugation of cdAE1.

Absorbance at 280 nm is plotted as a function of centrifuge cell radius. The residuals from fitting

the data to a single ideal species model are shown. The SE experiment was done at 20 °C at a

speed of 13 000 rpm. The protein was dissolved in 50 mM sodium chloride and 10 mM sodium

phosphate at pH 7.5.

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Secondary structure content of cdAE1, cdkAE1, and cdΔ54AE1: analysis by

circular dichroism spectroscopy

The secondary structures of cdAE1, cdkAE1, and cdΔ54AE1 were examined by CD

spectroscopy. A representative spectrum of each protein at pH 7.5 is shown in Figure A4. A

comparison of the CD spectra, however, revealed only minor variations in secondary structure

content between the constructs. Each spectrum exhibited a negative maximum at 208 nm and a

shoulder at 222 nm, which are dominant features of an α-helical protein.

Figure A4: CD spectra of purified cdAE1, cdkAE1, and cdΔ54AE1 at pH 7.5. Lyophilized

proteins were dissolved in 100 mM sodium chloride and 50 mM sodium phosphate (pH 7.5) and

scanned at 24 °C in a 1 mm cell in a Jasco J-810 spectropolarimeter. The final concentrations of

cdAE1 ( — ), cdkAE1 (– – –), and cdΔ54AE1 (−×−), were 0.36 mg/ml, 0.38 mg/ml, and 0.34

mg/ml, respectively. The spectra are expressed as the mean residue ellipticity (θ) as a function of

wavelength.

The helical content of cdAE1 based on its crystal structure was 26 %. The deconvolution of

the CD spectra revealed similar helical content between cdAE1, cdkAE1, and cdΔ54AE1 of

approximately 30-35 % α-helix (Table A1). Since the crystal structure of cdAE1 was elucidated

at pH 4.8 (Zhang et al. 2000b), it was important to determine if the secondary structure differs at

physiological and acidic conditions. For this reason, pH values of 5.0, 7.5, and 10.5 were used to

detect possible pH-dependent structural changes. Based on previous fluorescence and gel

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filtration experiments, there is evidence that the global conformation of cdAE1 elongates as pH

is increased (Appell and Low 1981). However, there were no changes detected in the CD spectra

of the three constructs at the different pH values tested, indicating that the secondary structural

features were maintained.

Thermal denaturation of cdAE1, cdkAE1, and cdΔ54AE1 by differential scanning

calorimetry

Calorimetry can detect thermal stability changes in proteins, which appear as perturbations in the

midpoint of thermal denaturation transitions (Tm). Thermal denaturation by DSC was used to

compare the thermal stabilities of the cdAE1, cdkAE1, and cdΔ54AE1 proteins. Representative

plots of the thermal denaturation of cdAE1, cdkAE1, and cdΔ54AE1 at pH 7.5, as performed by

DSC, are shown in Figure A5. Cooling and reheating of the samples revealed that the thermal

denaturation was irreversible. The data were fit to a two-state transition model for the purpose of

obtaining the midpoints of the thermal denaturation transitions (Tm) so that they could be

compared to one another. Because the transitions were irreversible, these Tms were relative and

were considered to be the apparent Tms of the transitions. These Tms agree with the temperature

corresponding to the maximum excess heat capacity (Cp), which was used in other studies of

proteins with irreversible thermal denaturation profiles (Idakieva et al. 2005, Nielsen et al.

2003). It was found that cdkAE1 had the lowest Tm (60 ºC) and that cdAE1 (65 ºC) and

cdΔ54AE1 (66 ºC) had similar Tms. The results indicate that the thermal stability of cdAE1 was

unaffected by removal of the disordered N-terminal extension (residues 1-54) but was thermally

destabilized by removal of the core β-strand.

It was also observed that the thermal stability of the three proteins was pH-dependent; the

proteins are the least thermally stable at alkaline pH and the most thermally stable at acidic pH.

The effect of pH on the Tm values of cdAE1, cdkAE1, and cdΔ54AE1 is recorded in Table A1.

cdAE1 and cdΔ54AE1 behaved similarly, while cdkAE1 had a lower Tm at all pH values. These

observations corresponded with other studies that have reported the Tm of cdAE1 to be pH-

dependent with a value of 67 ºC at pH 7.51 (Appell and Low 1981). It was also noticed that the

peaks at low pH were broader, most likely due to aggregation of the samples. This may be

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Figure A5: Thermal denaturation of purified cdAE1, cdkAE1, and cdΔ54AE1 by DSC at

pH 7.5.

All DSC scans were baseline-subtracted. The proteins were dissolved in 100 mM sodium

chloride and 50 mM sodium phosphate (pH 7.5) to final protein concentrations of 0.9 mg/ml

(cdAE1 —– ), 1.2 mg/ml (cdkAE1 – – –), and 1.0 mg/ml (cdΔ54AE1 −×−). The specific heat

capacity (Cp) was measured as the temperature was increased from 40 to 80 °C. No transition

was observed once the proteins were thermally denatured, cooled, and reheated.

explained by the fact that at pH 5.0, the proteins approach their isoelectric points, the pI of

cdAE1, cdkAE1, and cdΔ54AE1 being 4.70, 5.29, and 5.30, respectively. These thermal

denaturation studies show that the β-strand plays a structural role in the thermal stabilization of

the cytoplasmic domain. Furthermore, the cytoplasmic domains containing the β-strand are the

most thermally stable at low pH. cdkAE1 has only a slightly higher midpoint of transition at pH

5 than at neutral pH suggesting that it is less well-packed under acidic conditions compared to

cdAE1.

Effect of pH on the intrinsic fluorescence of cdAE1, cdkAE1, and cdΔ54AE1

Differences in tertiary structure between cdAE1, cdkAE1, and cdΔ54AE1 were further studied

by carrying out intrinsic tryptophan fluorescence studies. It has been previously shown that

cdAE1 undergoes a pH-dependent conformational change, characterized by a dramatic increase

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in tryptophan fluorescence at alkaline pH (Appell and Low 1981) and an increase in its Stokes

radius (Wang et al. 1992b). Since a cluster of four tryptophan residues is located C-terminal to

the 65 residue amino extension, the local environment of these tryptophans in cdkAE1 may

differ from that of cdAE1, and the tryptophans would therefore exhibit different fluorescence

properties and sensitivities towards pH titration. As the pH increased, cdAE1, cdkAE1, and

cdΔ54AE1 experienced an increase in fluorescence intensity due to dequenching of their

tryptophan residues (Figure A6). cdAE1 and the novel mutant cdΔ54AE1 both showed a similar

increase in intensity, doubling in value from pH 5 to 10.5. At acidic and neutral pH, however,

cdkAE1 exhibited a higher intrinsic fluorescence than cdAE1, which suggests that it has a more

open structure under these conditions compared to the cdAE1 protein.

Figure A7 shows representative plots of the average emission wavelength of all three

proteins as a function of pH. A red-shift in peak wavelength at alkaline pH was observed,

indicating that the tryptophans were becoming exposed to a more polar environment. Even at

neutral pH the fluorescence spectrum of cdkAE1 was more red-shifted, indicating a higher

degree of exposure of tryptophans to a more polar environment. At alkaline pH, the three

constructs exhibited similar fluorescence properties, consistent with a similar exposure of

tryptophans to solvent.

Urea denaturation of cdAE1 and cdkAE1 by intrinsic tryptophan fluorescence

Urea denaturation was used to examine the conformational stability of cdAE1 and cdkAE1, and

to further understand the consequence of truncating the N-terminus of AE1’s cytoplasmic

domain. The observed fluorescence emission intensity of cdAE1 peaked at 4 M urea due to the

dequenching of its tryptophans, as seen in Figure A8 A. This increase was then followed by a

decrease in intensity at higher urea concentrations due to further exposure of the Trp residues to

the aqueous solvent. In contrast, cdkAE1displayed a higher intrinsic fluorescence under native

conditions and a continual decrease in fluorescence as urea increased. Both constructs, however,

eventually denatured to a similar unfolded state as seen by the convergence of their fluorescence

intensities at 6 M urea.

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Figure A6: Intrinsic tryptophan fluorescence emission spectra of (A) cdAE1, (B) cdkAE1,

and (C) cdΔ54AE1 at various pH values. The intensity is plotted as a function of wavelength.

Lyophilized proteins were dissolved in 100 mM sodium chloride and 50 mM sodium phosphate

preadjusted to the desired pH. The intrinsic fluorescence emission was measured from 300 to

420 nm following excitation at 290 nm. pH 5.31(—), pH 7.48 (− − − ), and pH 10.04 (−×−).

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The average emission wavelength red-shifted as the amount of urea increased (Figure A8 B).

This observed red-shift indicated that the tryptophans were progressively becoming exposed to a

more polar environment. As shown in Figure A8 B, cdAE1 and cdkAE1 underwent similar

conformational changes; however, cdkAE1 was less resistant to urea at lower concentrations of

the denaturant.

Figure A7: Average emission wavelength of purified cdAE1, cdkAE1, and cdΔ54AE1.

The average emission wavelength is plotted as a function of pH. Lyophilized proteins were

dissolved in 100 mM sodium chloride and 50 mM sodium phosphate preadjusted to the desired

pH. The intrinsic fluorescence emission was measured from 300 to 420 nm following excitation

at 290 nm. cdAE1 (—), cdkAE1 (− − −), and cdΔ54AE1 (−×−).

Discussion

In this study, the biophysical properties of cdAE1, cdkAE1, and the novel mutant cdΔ54AE1

were compared to determine the effect of loss of the central β-strand on the structure and

conformational stability of the cytoplasmic domain. As shown by SE experiments, removal of

residues 1-65 had no effect on the dimerization of the cytoplasmic domain of AE1, indicating

that cdkAE1 was still able to retain a dimeric structure in solution. CD analysis showed that the

amino-truncations (either Δ1-65 or Δ1-54) did not have a significant effect on the secondary

structure content of the cytoplasmic domain. The loss of the short β-strand alone (residues 55-65)

would not be expected to produce dramatic changes in the CD spectrum.

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Figure A8: Urea denaturation of cdAE1 and cdkAE1 monitored by intrinsic tryptophan

fluorescence.

Lyophilized proteins were dissolved in 100 mM sodium chloride and 50 mM sodium phosphate

preadjusted to the desired urea concentration. The intrinsic fluorescence intensity was measured

from 300 to 420 nm following excitation at 290 nm. The vertical bars represent the standard

deviation; n = 4. (A) Intrinsic fluorescence emission intensity of the proteins at 341 nm is plotted

as a function of urea concentration. (B) Average emission wavelength was calculated from each

spectrum and plotted as a function of urea concentration. cdAE1 (—), cdkAE1 (− − −).

DSC compared the thermal stability of cdkAE1 to that of cdAE1. cdkAE1 consistently had

lower Tm values regardless of the pH, indicating a less thermally stable structure. The

calorimetric values of the novel mutant cdΔ54AE1 were similar to those of cdAE1,

strengthening the contention that the β-strand (residues 55-65) helps stabilize the cytoplasmic

domain of AE1. These results were predicted because the crystal structure of AE1 shows that the

β-strand is located within the core of the domain. When the β-strand was removed, as in cdkAE1,

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there was a significant effect on the Tm. As expected, removal of residues 1-54 did not alter the

thermal stability of the domain as these residues form a flexible extension that was not observed

by X-ray crystallography. Varying the pH had a noticeable effect on the thermal stability of all

three proteins. At acidic pH, the Tm value increased compared to neutral and alkaline pH. This

indicated that the proteins acquired a greater thermal stability at low pH, perhaps adopting a

more compact conformation as revealed by the crystal structure. cdkAE1 was always the least

thermally stable at all pH values, however, the difference in Tm was greatest (~10 °C) at low pH.

The intrinsic fluorescence of a folded protein is primarily dependent on the local

environment of its tryptophan residues. Normally, when tryptophan is exposed to a hydrophobic

environment, the fluorescence emission is of high intensity and is blue-shifted. Upon exposure to

a more polar environment, tryptophan fluorescence decreases and its emission spectrum

experiences a red-shift. However, this is often not the case for multi-tryptophan proteins, such as

cdAE1, where there are four tryptophan residues found at position 75, 81, 94, and 105. At

alkaline pH, the fluorescence intensity of cdAE1 increased and there was a red-shift in average

emission wavelength. These observations indicated that an opening of the structure had occurred,

thereby dequenching fluorescence and exposing the tryptophan residues to a more polar

environment. Both cdAE1 and cdΔ54AE1 underwent similar pH-dependent conformational

changes, indicating structural similarity. cdkAE1 was not as structurally sensitive to pH titration

as were the other two protein constructs because of its more open structure at low pH. In

comparison, cdAE1 and the novel mutant had a more compact and folded structure at low pH,

and gradually adopted a more open conformation as pH increased. Although the secondary

structure content between proteins is comparable, the intrinsic fluorescence data suggest that

cdkAE1 has a less compact structure, especially at low pH.

Intrinsic fluorescence was also used as a probe for the urea denaturation experiments, once

again revealing information about the local environment of the N-terminal tryptophan cluster.

The tryptophan residues of cdAE1 first become dequenched, and then quenched at higher urea

concentrations. During denaturation, the tryptophan residues in cdAE1 move away from

quenching residues and become dequenched, but as the protein continues to denature, the

tryptophan residues become further exposed to the more polar, aqueous environment, which in

return causes a decrease in fluorescence. Under native conditions, the intrinsic fluorescence of

cdkAE1 was high, indicating that the tryptophan residues were dequenched. The tryptophans in

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this construct would already be more apart from quenching residues so that denaturation would

only cause the tryptophans to become even more exposed to the solvent, hence the decrease in

fluorescence that was observed. At 6 M urea, the fluorescence intensity of both cdAE1 and

cdkAE1 converge and plateau, indicating a similarly unfolded state.

In conclusion, the biophysical techniques employed in this study were useful in structurally

characterizing cdkAE1. It was shown through SE experiments and CD experiments that cdkAE1

retains a dimeric structure and has similar secondary structure content compared to the erythroid

isoform. The analyses made by SV experiments, DSC, and intrinsic tryptophan fluorescence

experiments established that removal of the acidic N-terminal extension (residues 1-54 only) had

little effect on the folding and conformational stability of cdAE1, whereas deletion of the β-

strand (residues 55-65), as in cdkAE1, produced a less thermally stable protein with a more open

structure.

The crystal structure of the cytoplasmic domain of AE1 has been elucidated for the erythroid

isoform in acidic conditions. In addition, structural features of specific regions of cdAE1 at

neutral pH, as investigated by SDSL in combination with EPR and DEER spectroscopies, were

in agreement with those from the crystal structure (Zhou et al. 2005a). We plan to further

characterize the structure of cdkAE1 by X-ray crystallography or NMR analysis. Based on the

results presented in this study, NMR would perhaps be the technique most amenable to solving

the structure of cdkAE1, which seems to adopt a less compact and a more open structure. NMR

would also be an ideal technique to study the protein dynamics of cdkAE1 and cdAE1.

The deletion of residues 1-65 has two effects on the cytoplasmic domain: 1) the loss of the

acidic N-terminal extension that is involved in protein binding, and 2) the loss of the β-strand,

which causes changes to the structure and thermal stability of the domain. Both of these effects

could account for the inability of kAE1 to bind cytoskeletal proteins and glycolytic enzymes.

The changes in the structure of cdkAE1 may also allow kAE1 to interact with different proteins

than AE1. In order to fully understand the function and regulation of kAE1 it would be important

to identify its interacting protein partners in kidney cells.