SITE-DIRECTED MUTAGENESIS OF CD38 IN CHO TET-OFF CELLS
SHEN YIRU
NATIONAL UNIVERSITY OF SINGAPORE
2003
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SITE-DIRECTED MUTAGENESIS OF CD38
IN CHO TET-OFF CELLS
SHEN YIRU (B.Sc Hons, NUS)
A THESIS SUBMITTED
FOR THE DEGREE OF MASTER OF SCIENCE
DEPARTMENT OF BIOCHEMISTRY
NATIONAL UNIVERSITY OF SINGAPORE
2003
i
ACKNOWLEDGEMENTS
First and foremost, I would like to extend my deepest appreciation and gratitude to
my supervisor, Associate Professor Chang Chan Fong, who was always available to give his
expert advice and oversee my project.
Thanks also go out to Hussain, Fubo, Qian Feng and Angela and everyone else in the
lab for their valuable assistance and support. Finally, I would like to thank my family
members for their encouragement.
Last but not least to my examiners for taking their precious time off to mark this
thesis.
ii
CONTENTS
Title page
Acknowledgements i
Table of Contents ii
Summary iv
Abbreviations viii
Chapter 1 Introduction 1 1.1 General introduction of CD38 3
1.1.1 Structure of CD38 3
1.1.2 Tissue distribution of CD38 6
1.2 CD38 as an Enzyme 8
1.2.1 Enzymatic activities of CD38 8
1.2.2 Cyclic ADP-ribose 12
1.2.3 Other known enzymatic activities of CD38 13
1.3 Regulation of CD38 function 13
1.3.1 Topological paradox of CD38 14
1.4 CD38 is an adhesion molecule 15
1.5 The CD38 family and its related molecules 17
1.6 CD38 and diseases 18
1.6.1 Leukemia and myeloma 18
1.6.2 HIV infection 19
iii
1.6.3 Alzheimer’s disease 19
1.6.4 X-linked agammaglobulinemia (XLA) 19
1.7 Green Fluorescence Protein 21
1.8 Tet-Off system 23
1.8.1 Components of Tet-off system 23
1.8.2 Advantages of Tet-system 26
1.8.3 Tet-off vs Tet-on 27
1.9 Objectives of the Study 27
Chapter 2 Materials and Methods 29
2.1 Materials 30
2.1.1 Bacterial strains 30
2.1.2 Mammalian cell line 30
2.1.3 Plasmids 30
2.1.4 Synthetic primers 31
2.1.5 Media and solutions 31
2.1.5.1 Bacterial media 31
2.1.5.2 Media and reagents for mammalian cell culture 31
2.1.5.3 Supplier for reagents and other materials 32
2.2 Methods 34
2.2.1 Mammalian cell culture techniques 34
2.2.1.1 Maintenance of cells 34
2.2.1.2 Storage of frozen cells 34
iv
2.2.1.3 Revival of frozen cells 34
2.2.1.4 Stable Transfection of cells using Electroporation 34
2.2.1.5 Transient transfection using Liposome (Lipofectamine) 35
2.2.1.6 Ligands preparation for internalization studies 35
2.2.2 Methods for manipulation of nucleic acids 36
2.2.2.1 Polymerase Chain Reaction (PCR) 36
2.2.2.2 Cloning of full length rat CD38 cDNA into pEGFP-N1 37
2.2.2.3 Cloning of CD38-GFP fusion gene into pTRE2-hyg 37
2.2.2.4 Site-directed mutagenesis by QuickChange Mutagenesis 40
2.2.2.5 Restriction endonuclease digestion of DNA 40
2.2.2.6 Concentration of nucleic acids 42
2.2.2.7 DNA ligation 42
2.2.2.8 Agarose gel electrophoresis of DNA 42
2.2.2.9 Isolation of DNA from agarose gel 43
2.2.2.10 DNA sequencing 44
2.2.3 Methods for bacterial culturing and transformation 44
2.2.3.1 Preparation of competent cells 44
2.2.3.2 Transformation by electroporation 45
2.2.3.3 Transformation by heat shock 45
2.2.4 Preparation of plasmid DNA from Bacterial cells 46
2.2.4.1 Miniprep 46
2.2.4.2 Midi/Maxiprep 47
2.2.5 Expression, purification and analysis of CD38 48
v
2.2.5.1 Membrane protein harvesting 48
2.2.5.2 SDS-Polacylamide gel electrophoresis (SDS-PAGE) 48
2.2.5.3 Staining of SDS-PAGE gel with Coomassie Blue 49
2.2.5.4 Western Blot 50
2.2.5.5 Stripping and reprobing of membranes 50
2.2.5.6 Enzyme Assays for CD38 51
2.2.5.7 Affinity purification of biotinylated CD38 51
2.2.5.8 Confocal microscopy of CD38 expression and localization 52
2.2.5.9 Live-imaging of CD38 internalization 53
Chapter 3 Results 54 3.1 Cloning of full length rat CD38 cDNA into pEGFP-N1 vector 55
3.2 Cloning of CD38-GFP fusion gene fragment into pTRE2-hyg vector 55
3.3 Effect of amino acid residue on cADPR metabolism 56
3.4 Site-directed mutations of CD38 60
3.5 Transfection of CHO Tet-off cells 61
3.6 Tetracycline regulated expression of Wild-type CD38 63
3.7 Relative percentage of enzyme activities of mutants 67
3.8 Expression of CD38 and mutants in CHO Tet-off cells 72
3.9 Live-imaging of internalization with NAD+ ligand 72
3.10 Live-imaging of internalization with cADPR and 8-NH2-cADPR ligand 74
3.11 Cell Surface Biotinylation of Wild-type CD38 and W162G mutant 81
Chapter 4 Discussion and Conclusion 88
References 97
vi
SUMMARY
CD38 acts as a bifunctional ectoenzyme, synthesizing (ADP-ribosyl cyclase) and
degrading (cyclic ADP-ribose (cADPR) hydrolase) cADPR, a calcium mobilizer from
intracellular pool. CD38 internalization has been proposed as a mechanism by which the
ectoenzyme produced intracellular cADPR, and thiol compounds have been shown to induce
internalization.
Here, we show the function of ADP-ribosyl cyclase activity may be important for
its ligand- induced internalization of CD38. Previously, a plasmid construct, pCD38-GFP,
encoding a fusion protein of CD38-GFP (Green Fluorescent Protein) was generated. Several
mutants were constructed and cloned into pTRE2-Hyg response plasmid. Their expressions
were studied in established stable Chinese Hamster Ovary (CHO) Tet-off cells. Wild type
CD38, C122K, L149V and W162G mutants were expressed on the plasma membrane, but
the E229G mutant was retained in the cytoplasmic location as revealed via confocal
microscopy. Mutants (C122K and L149V), which exhibited ADP-ribosyl cyclase activity
only were able to be expressed on surface membrane and undergo ligand- induced
internalization. However, the W162G mutant which had shown significant reduction in the
cyclase activity but with intact hydrolase activity was not able to undergo ligand- induced
internalization. Glu-229 mutant was shown to be devoid of all its enzymatic activities and
was neither able to be expressed on surface membrane nor undergo ligand- induced
internalization.
CD38 and its mutants were expressed in CHO Tet-off cells and characterized by
enzymatic assays, confocal microscopy and immunoblot. We have affinity purified the
internalized CD38 from NAD+ treated and untreated cell lysates by a streptavidin/ biotin-
vii
based method. Altogether, these results demonstrated the important role of ADP-ribosyl
cyclase activity in ligand-dependent internalization and possible down-stream production of
intracellular cADPR to mediate calcium signaling.
viii
ABBREVIATIONS Ab Antibody Ag Antigen APS Ammonium persulfate ATRA All-trans-retinoic acid Bp Base pair BSA Bovine serum albumin cADPR Cyclic ADP-ribose CD Cluster of differentiation cDNA complementary DNA cGDPR Cyclic GDP-ribose CICR Ca2+-induced Ca2+ release cIDPR Cyclic IDP-ribose DEAE Diethylaminoethyl DMSO Dimethyl sulfoxide Dnase Deoxyribonuclease dNTP Deoxynucleoside triphosphate ECL Enhanced chemiluminescence EDTA Ethylene diamine-N,N,N’,N’-tetra-acetic acid FBS Fetal bovine serum GFP Green Fluorescent Protein GPI Glycosylphosphatidylinositol
ix
FITC Fluorescein isothiocyanate GPI Glycosylphophatidylinositide HRP Horseradish peroxidase hr Hour IgG Immunoglobulin G IP3 Inositol-1,4,5-trisphosphate kb Kilobase(s) kDa Kilodalton LB Luria-Bertani mAb Monoclonal antibody MES 2’-(N-morpholino)ethanesulfonic acid mHL-60 Monocytic HL-60 min minute ml Milliliter µl Microliter mM Millimolar NAADP+ Nicotinic acid adenine dinucleotide phosphate NAD+ Nicotinamide adenine dinucleotide NADP+ Nicotinamide adenine dinucleotide phosphate NGD+ Nicotinamide guanine dinucleotide phosphate nm Nanometer nM Nanomolar O.D. Optical density
x
PBS Phosphate-buffered saline PCR Polymerase chain reaction PI-PLC Phosphatidylinositol-specific phospholipase C PKC Protein kinase C PMSF Phenylmethylsulfonyl fluoride RA Rheumatoid arthritis Rnase A Ribonuclease A rpm Revolution per minute RT Reverse transcription RyR Ryanodine receptor SDS-PAGE Sodium dodacyl sulfate-polyacrylamide gel electrophoresis TAE Tris-acetate EDTA TBS-T Tris-buffered saline/Tween 20 TEMED N,N,N’,N’-Tetramethylethylenediamine TRITC Tetramethylrhodamine isothiocyanate UV Ultra-violet VD3 1α,25-dihydroxyvitamin D3
Chapter 1 Introduction 1
Chapter 1
Introduction
Chapter 1 Introduction 2
CD38 or T10 molecule, a membrane glycoprotein of molecular weight of 45-
kDa, was one of the first characterized but least understood human lymphocyte
differentiation antigen. As most lymphocyte surface markers, it was identified by means
of monoclonal antibodies. The cDNA encoding the human lymphocyte antigen CD38 was
isolated and sequenced from a mixture of four different lymphocyte cDNA libraries
expressed transiently in Cos-7 cells and screened by panning with CD38 monoclonal
antibodies (Jackson and Bell, 1990). By using antibodies, CD38 was found to have a
rather unique distribution pattern, being predominantly expressed by progenitors and early
hematopoietic cells, then lost during maturation, only to be re-expressed during cell
activation. Because of this curious pattern of expression, CD38 has been used primarily as
a phenotypic marker of differentiation in normal and leukemic blood cells. However,
interest in CD38 antigen beyond its use as a marker of cellular differentiation has grown
recently after a report that human CD38 has significant amino acid sequence similarity to
ADP-ribosyl cyclase (States et al., 1992), a 29 kDa cytosolic enzyme previously isolated
from a sea molluse, Aplysia califonica (Hellmich and Strumwasser, 1991; Lee and
Aarhus, 1991).
However, it is no longer just considered a cellular marker for lymphocyte
differentiation but this exclusive bi- functional ectoenzyme bears complex catalytic
properties, displaying an ADP-ribosyl cyclase and a cyclic ADP-ribose (cADPR)
hydrolase that catalyzes the synthesis from the metabolite nicotinamide adenine
dinucleotide (NAD+) and the hydrolysis of cADPR, respectively. CD38 proteins were
detected in humans, mice and rats (Reinherz et al., 1980; Howard et al., 1993; Koguma et
al., 1994).
Chapter 1 Introduction 3
1.1 General Introduction of CD38
1.1.1 Structure of CD38
CD38 is a type II transmembrane glycoprotein of 300 amino acids expressed in
many vertebrate cells. The deduced amino acid sequences of CD38 have shown that the
polypeptide consists of three domains, a short NH2-terminal cytoplasmic tail, a single
transmembrane region, and a large extracellular COOH-terminal catalytic domain (Figure
1.1). It is a bifunctional ectoenzyme that catalyzes both the synthesis of cyclic ADP-ribose
(cADPR) from NAD+ and the degradation of cADPR to ADP-ribose by means of its ADP-
ribosyl cyclase and cADPR-hydrolase activities, respectively (De Flora et al., 1997).
The gene encoding human CD38 protein is located on chromosome 4p15
(Nakagawara et al., 1995). The short cytoplasmic domain of CD38 contains no known
motifs (Src homology domain 2 or 3 [SH2 or SH3], antigen receptor activation motif
[ARAM], or pleckstrin homology [PH]) that could mediate interactions with other
signaling proteins and seems to have no enzymatic activity. The intracellular part of CD38
contains two conserved serine residues within the consensus sites recognized by cyclic
guanosine monophosphate (cGMP)-dependent protein kinases. cGMP - dependent serine/
threonine kinases in sea urchin eggs modulate the activity of ADP-ribosyl cyclase, an
enzyme that displays a functional homology to CD38 protein (Lund et al., 1996). Four
potential amino-terminal linked glycosylation sites and two to four high-mannose amino-
terminal linked oligosaccharide chain containing sialic acid residues contribute 25% of the
molecular mass of the CD38 protein. Extracellular part of CD38 may function in
attachment to the extracellular matrix. Human CD38 proteins contain three putative
hyaluronate-binding motifs (HA motifs), two of which are located in extracellular domain
Chapter 1 Introduction 4
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Chapter 1 Introduction 5
and one in the cytoplasmic part of the molecule (Nishina et al., 1994). In addition, four
asparagines residues in the extracellular part of CD38 serve as potential carbohydrate-
binding sites.
The human CD38 molecule contains 12 conserved cysteines, 11 of which are
located in the extracellular domain. It has been shown previously that four cysteines (Cys-
119, Cys-160, Cys-173, and Cys-201) play an essential role in the cADPR synthetic and
cADPR hydrolytic activity of human CD38 (Tohgo et al., 1994). Furthermore, the C-
terminal part of CD38, including the peptide sequence of residues 273-285 and in
particularly Cys-275, contributes to the NAD glycohydrolytic activity of CD38 (Hoshino
et.al, 1997). In particular, the conserved pair of amino acids Glu-146 and Asp-147 seems
to endow ADP-ribosyl cyclase activity to the CD38 protein (Grimaldi et al., 1995;
Okazaki and Moss, 1996). Reducing agents such as dithiothreitol, 2-mercaptoethanol, or
reducing glutathione inhibit the enzymatic activity of CD38, suggesting that the disulfide
bonds are important for the catalytic activity of the CD38 proteins (Tohgo et al., Zocchi et
al., 1995). A number of leucines within the transmembrane and extracellular domains
potentially form leucine zippers motifs that can provide associations of CD38 with other
proteins (Hurst, 1994).
The dileucine motifs in the CD38 molecule is, however, not accessible to the
intracellular targeting because of the extracellular location. Two dileucine (LL) motifs are
located in the middle of human CD38 proteins. Intracellular targeting and internalization
of different transmembrane proteins require the LL motif within the C terminus of the
cytoplasmic domains (Aiken et al., 1994). Binding of NADP+ at the active site of CD38
can facilitate the assembly of these putative leucine zippers for mutual interactions
Chapter 1 Introduction 6
between CD38 molecules on the membrane that in turn can lead to lateral movements and
clustering of CD38 and followed by internalization.
In addition to the membrane-anchored form (mCD38; 45KDa), a soluble form
of CD38 also exists (sCD38; 39KDa), probably as a result of enzymatic cleavage of the
cell-surface protein. Therefore, the soluble form of CD38 can be detected in vitro in
culture supernatants from alloactivated T- lymphocytes and CD38+ tumor cell lines. It is
also detectable in normal amniotic fluid and in serum and ascites from patients with
multiple myeloma (Funaro et al., 1995). Not long ago, a high molecular mass of CD38
(190kDa) was identified in the retinoic acid (RA)- induced human myeloid leukemia cells
(Umar et al., 1996). This high molecular weight form may be the result of
transglutaminase-catalyzed post-translational cross- linking of mCD38. A CD38-like
78kDa soluble protein was also identified in culture supernatants of Epstein-Barr virus
(EBV)-transformed B cells from patients with X-linked agammaglobulinemia (XLA)
(Mallone et al., 1998). The behavior of sCD38 appears to be similar to that of many other
receptors enzymatically cleaved from leukocyte membranes in response to interaction
with ligands or agonistic antibodies which mimic the natural ligands (Higuchi and
Arrgarwal, 1994; Bazil and Horejsi, 1992; Berg and James, 1990).
1.1.2 Tissues distribution of CD38
Although CD38 was initially discovered as a differentiation marker of T-
lymphocytes (T10), it is now clear that various levels of CD38 can be discovered in a
large number of cells and tissues. Thus, CD38 has been found on neurons of human brain
(Mizuguchi et al., 1995), pancreatic cells (Kato et al., 1995), epithelial cells of the prostate
(Kramer et al., 1995), and other cells (Mehta et al., 1996). Studies of the expression of
Chapter 1 Introduction 7
CD38 have been a focus of recent research; the results obtained suggest that the molecule
is widely distributed. An interesting observation is that the surface expression of human
CD38 varies significantly with age. In adults, the expression of CD38 protein is present on
the majority of natural killer cells, T cells, B cells, monocytes/macrophages (Malavasi et
al., 1994), and to some extent on platelets (Ramaschi et al., 1996) and erythrocytes
(Zocchi et al., 1993). Furthermore, high glucose-utilizing tissues such as the pancreas,
brain, spleen and liver express high amounts of CD38 (Koguma et al., 1994). CD38 plays
a particularly central role in glucose- induced insulin secretion from islet cells in the
pancreas.
In mouse, CD38 is also expressed by a significant portion of T- lineage cells,
natural killer cells and myeloid cells. T lymphocytes and myeloid cells do express variable
amounts of surface CD38, but its expression was never as high as in the human
counterparts (Lund et al., 1995). The difference in expression between species can be
explained by assuming that the murine CD38 belongs to the CD38 family but is not the
exact orthologue of the human antigen.
However in rat, the information available is very limited and does not allow
comparison with human or mouse molecule. Despite that, the mRNA for CD38 has been
detected in various rat tissues, namely, in the liver, heart, thymus, thyroid gland, adrenal
gland and pancreatic islets (Koguma et al., 1994).
A new CD38-like molecules, CD157 (BST-1/BP-3) has been discovered, and
appears to be a cell surface molecule. Two groups have isolated the human [bone marrow
stromal cell antigen 1 (BST-1)] and mouse (BP-3) analogs of a
glycosylphosphatidylinositol (GPI)-anchored membrane protein that is expressed by an
array of hematopoietic and non-hematopoietic cells. These proteins bear the same
Chapter 1 Introduction 8
structural relationship to Aplysia ADP-ribosyl cyclase that is exhibited by CD38 (Table
1.1).
1.2 CD38 as an Enzyme
1.2.1 Enzymatic activities of CD38
The first evidence for the enzymatic activity of CD38 was reported in the
murine model in 1993 (Howard et al., 1993) and later was confirmed in humans (Gelman
et al., 1993). CD38 is one of the first few cell surface signaling molecules shown to
possess ectoenzymatic activity. It is a bifunctional ectoenzyme that catalyzes both the
synthesis of cyclic ADP-ribose (cADPR) from NAD+ and the degradation of cADPR to
ADP-ribose by means of its ADP-ribosyl cyclase and cADPR-hydrolase activities,
respectively (De Flora et al., 1997) (Figure 1.2). The overall catalytic activity of CD38 is
similar to that of NAD+ glycohydrolase (NADase). NADases are a diverse group of
enzymes that have been grouped together because of their common activity; they cleave
the nicotinamide-ribose bond of NAD+ to adenosine diphosphoribose (ADPR) and
nicotinamide. To distinguish the Aplysia enzyme from the conventional NADases that
produce ADP-ribose, the name Aplysia ADP-ribosyl cyclase was proposed (Lee and
Aarhus, 1991).
Aplysia Californica ADP-ribosyl cyclase has been found to share considerable
structural homology with the mouse and human CD38 (States et al., 1992). In addition to
ADP-ribosyl cyclase activity, purified CD38 also exhibited the ability to hydrolyze
cADPR to ADPR (Howard et al., 1993). The overall reaction thus catalyzed by CD38 is
the conversion of NAD+ to ADPR and nicotinamide, a reaction identical to that catalyzed
Chapter 1 Introduction 9
by NAD-glycohydrolase (NADase). CD38 can also utilize nicotinamide guanine
dinucleotide (NGD) and nicotinamide adenine dinucleotide phosphate (NADP+) as
substrates, leading to the synthesis of cyclic GDP-ribose (cGDPR) and nicotinic acid
adenine dinucleotide phosphate (NAADP+), respectively. The Ca2+ release mechanism
activated by NAADP is totally independent of cADPR and inositol trisphosphate (IP3)
(Albrieux et al., 1998).
Chapter 1 Introduction 10
CD38 in humans Lymph node Lymphoblast germinal cells, plasma cells and interfollicular cells Thymus Paracortical and mainly medullary thymocytes Brain Perikaryal and dendritic cytoplasm of neurons Digestive tract Lamina propria lymphocytes Kidney Proximal tubuli Prostate Cytoplasmic membrane and secretory vacuoles Skeletal and cardiac muscles Sarcolemmin of myocites and cardiomyocites CD38 in mouse Lymphocytes Predominantly B cells and cell lines; variable proportions of T cells and thymocytes
Myeloid cells Variable proportions: MAC-1 macrophages from peritoneum are CD38+, unstimulated BM macrophages are CD38-; BM-derived cell lines in GM -CSF and CD38+
CD157 (BST-1/BP-3) in human and mouse
B lymphocytes 100% pre-B in BM; 35% circulating B;30% splenocytes; < 20% in lymph node, peritoneum and Beyer’s patches
Myeloid cells Almost absent in BM; relatively high in circulating nutrophilis and peritoneal macrophages
Vessels Constitutive in endothelial cells and not enhanced by IL-1 Stroma and synovia Cell lines constitutively expressing high levels Epithelial cells Bowel’s brush border, collecting tubules of the kidney Placenta, lung, liver BST-1 mRNA
Table 1.1 Tissue Distribution of CD38 and related molecules
Chapter 1 Introduction 11
Figure 1.2 Enzymatic pathways involved in the metabolism of Cyclic ADP-
ribose. CD38 is a lymphocyte antigen that is also a bifunctional enzyme, catalyzing both the synthesis and the hydrolysis of cyclic ADP-ribose.
Reproduce from Lee et al (1994), Vitamin & Hormones, Vol 48: 199-257
Chapter 1 Introduction 12
1.2.2 Cyclic ADP-ribose
cADPR is a naturally occurring metabolite of NAD+, which is involved in Ca2+
signaling (Lee et al.,1995). There is a growing recognition that it is an endogenous
modulator of cADPR-sensitive Ca2+ release mechanism (Lee et al., 1996). Ca2+ release by
cADPR was discovered in sea urchin eggs in 1987 (Clapper et al., 1987). cADPR
possesses many characteristics of a novel second messenger acting on a ryanodine-
sensitive Ca2+ release channel distinct from that mediated by inositol trisphosphate (IP3)
receptors. cADPR, a known endogenous modulator of ryanodine receptor Ca2+ releasing
channels, is also found in the nervous system. FK506, an immunosuppressant that
prolongs allograft survival, as well as cADPR, were found to induce the release of Ca2+
from islet microsome (Okamoto et al., 1999). In cells, Ca2+ -induced- Ca2+ -release
(CICR) mechanism requires calmodulin and Ca2+. Ca2+ release is also induced by the
CICR modulators, ryanodine and caffeine. The discovery of sensitizing effects of cADPR
on Ca2+ release provides a physiological mechanism for modulating its Ca2+ sensitivity
over a wide range. The cyclic nucleotide is synthesized from NAD+ by ADP-ribosyl
cyclase (ADPR cyclase) and is degraded by cADPR hydrolase to ADP-ribose. The
enzymes involved in the metabolism of cADPR are summarized in Figure 1.2.
Walseth and Lee (1993) have described several 8-subsituted analogs of cADPR
that antagonize the actions of cADPR. 8-Amino-, 8-Bromo-, and 8-Azido-derivatives of
cADPR were synthesized by incubating the appropriately substituted NAD+ analogs with
the purified ADP-ribosyl cyclase from Aplysia ovotestis. Studies using these analogs
indicate that the 8-position of cADPR is critically important for its Ca2+ - releasing
activity, since none of the analogs had the ability to release Ca2+ from sea urchin egg
microsomes. However, all three block cADPR from releasing Ca2+; 8-Amino-cADPR is
Chapter 1 Introduction 13
the most effective. The antagonistic effects of these cADPR analogs are competitive since
high concentrations of cADPR can overcome the inhibition, suggesting that the analogs
may bind to the same site as cADPR itself.
1.2.3 Other known enzymatic activities of CD38
Besides generating cyclic ADP ribose and ADP ribose, CD38 may also
function as a mono-ADP-ribosyl transferase. One group (Grimaldi et al., 1995) has
shown that recombinant murine CD38 exhibits autoribosylation and was able to
ribosylate a number of different proteins at the cysteine residue. Another group (Okazaki
and Moss, 1996) has observed that CD38 and CD38-related proteins shared similar
catalytic amino acid sequences to that found in eukaryotic mono-ADP-ribosyl transferases
and in bacterial toxin transferases.
1.3 Regulation of CD38 function
Various chemical agents do affect the enzymatic activity of CD38. The cyclase
activity of CD38 from human erythrocyte membranes was found to be stimulated by Cu2+
and Zn2+ (Zocchi et al., 1993). Besides, Zn2+ ion has also been shown to stimulate the
ADP-ribosyl cyclase activity of recombinant CD38, while dramatically inhibited the NAD
glycohydrolase activity of CD38 (Kukimoto et al., 1996). Gangliosides inhibited
glycohydrolase and ADP-ribosyl cyclase activity of CD38 (Yokoyama et al., 1996). ATP
has been shown to inhibit cADPR hydrolase of human CD38 but the ADP-ribosyl cyclase
activity was unaffected, leading to preferential accumulation of cADPR (Takasawa et al.,
1993).
Chapter 1 Introduction 14
The expression of CD38 in hematopoietic cells (Drach et al., 1994) was
increased by all-trans-retinoic acid (ATRA). After the synthesis, CD38 undergo
posttranslational modification into a higher molecular mass form (190kDa).
Monodansylcadaverine, a specific substrate inhibitor of trans-glutaminase, inhibits the
oligomerization of CD38 into a high-molecular-weight form in ATRA-treated HL-60
cells, suggesting that CD38 is cross- linked through a trans-glutaminase reaction (Umar et
al., 1996). Besides, all trans-retinoic acid, 1,25-dihydroxyvitamin D3 also regulate the
induction of CD38 expression in certain hematopoietic cells (Galibert et al., 1996). It has
been shown by another group, (Stoeckler et al., 1996), that in vitro exposure of CD38 to
1,25-dihydroxyvitamin D3 increased CD38 density on activated tonsillar B cells and
peripheral T cells. Furthermore, Interleukin-4 has been found to mediate transcriptional
down-regulation of CD38 expression in B cells by a serine/threonine kinase-dependent
mechanism (Shubinsky et al., 1996).
1.3.1 Topological paradox of CD38
The enzymatic activities of CD38 are displayed by the extracellular domain.
Despite the ectoenzyme nature of CD38, significant levels of cADPR were still observed
in CD38+ cells (Takahashi et al., 1995). The unusual properties of CD38/cADPR system
and the many still unanswered questions on its function are potential questions for future
research to be carried out. Internalization of membrane CD38 might be a solution to the
topological paradox. The apparent topological paradox (Figure 1.3) of ectocellular
synthesis and intracellular activity of cADPR might be explained: (a) influx of cADPR
across the plasma membrane to reach its target stores, as suggested by experiments on
cerebellar granule cells; and (b) NAD+ - induced internalization, following membrane
Chapter 1 Introduction 15
oligomerization, of CD38 with consequent partial import of cADPR metabolism to an
intracellular compartment, as recently observed in lymphoid B cells. Two different
processes which this paradox can be overcome were described by two groups: (1)
Incubation of Namalwa B cells with external NAD+ has been shown to result in an
extensive internalization of CD38 accompanied by an increase in intracellular ADP-
ribosyl cyclase activity and of intracellular cADPR concentration (Zocchi et al., 1996); (2)
the cADPR-transporting function of membrane-bound CD38, which behaves as a
catalytically active transporter responsible for generation and selective influx of cADPR
across membranes (Franco et al., 1998).
1.4 CD38 as an adhesion molecule
The initial clue concerning the involvement of CD38 in cell-cell contacts came
from distribution studies among cells of lymphatic origin. CD38 is present on the
membrane of cells for which heterotyp ic interactions are a crucial development step
(Mehta et al., 1996). The CD38-mediated adhesion is weak, allows lymphocyte rolling
over HEC monolayers and is overwhelmed by integrin function (Dianzani et al., 1994).
These features resemble those of selectins, which are responsible for dynamic interactions
in the bloodstream between leukocytes and endothelial cells. However, CD38 lacks
structural homologies or sequence similarities to selectins.
Chapter 1 Introduction 16
NAD+ cADPRE ADPR
Plasma membrane
CD38
Ca2+ Endoplasmic Recticulum
Ca2+
Ca2+
ADP + P Figure 1.3 Topological paradox of CD38. CD38-catalyzed extracellular formation of cADPR (cADPRE) and intracellular Ca2+-releasing activity of cADPR (cADPRI) on responsive stores. PM: Plasma membrane. ER: Endoplasmic reticulum.
cADPRI ATP
Chapter 1 Introduction 17
The molecule, Moon-1, a 120-kDa protein was first identified as a natural
ligand for human CD38 (Deaglio et al., 1996). It was shown to co-expressed with CD38
on vascular endothelium, lymphocytes, platelets, NK cells, T, B and myeloid cells. Thus
CD38 may be involved in the homotypic and heterotypic adhesiveness of lymphocytes
and in the cooperative interactions. Further studies have shown that the molecule
recognized by Moon-1 mAb was found to be CD31, which is a 130KDa member of the
immunoglobulin (Ig) superfamily (Deaglio et al., 1998). Another aspect which has
received attention is the novel intracellular signaling pathway initiated by CD38/CD31
binding. They share the ability to transduce extracellular signals, resulting in an increase
of cytoplasmic Ca2+ levels through IP3-dependent mechanisms. In addition, CD38/CD31
interaction plays a role in cytotoxicity. The results indicate that CD38 and CD31 are
almost constantly always co-expressed on the surface of the IL-2-dependent T cell line
TALL-104 (Cesano et al., 1998). Indeed, CD31 ligation dramatically increases
cytotoxicity, while CD38 induces cytokine synthesis and release.
1.5 The CD38 family and its related molecules
The identification of structural and catalytic homologies between CD38
molecule and Aplysia ADP-ribosyl cyclase has lead to the identification of several other
molecules displaying similar characteristics and sequences as CD38. One other family
member includes CD157, preciously designated BST-1 or BP-3 antigen by several
different investigators. This molecule has been recently been characterized as a
glycosylphosphatidylinositide (GPI)- anchored leukocyte surface protein (Kaisho et al.,
1994; Dong et al., 1994; Itoh et al., 1994; Ishihara et al., 1997). The expression patterns of
Chapter 1 Introduction 18
both molecules are overlapping in several tissues and hematopoietic systems or
compensating in certain microenvironments, suggesting that CD157 and CD38 are
functionally cooperating or compensating in distinct situations (Ishihara et al., 1997)
(Table 1.2).
1.6 CD38 and diseases
CD38 has been of interest clinically because its expression is upregulated on T
lymphocytes during acute viral infections and other pathological conditions (Akbar et al.,
1993; Callan et al., 1998). Previous studies (Ellis et al., 1995; Stevenson et al., 1991)
have found that CD38-specific antibodies is useful in the treatment of leukemia and
myeloma. Therefore, CD38 was passed from being a simple activation marker to a multi-
functional surface receptor, possessing both complex enzymatic and cell-surface adhesion
functions. Other reports stem from recent findings concerning its distribution (e.g.,
Alzheimer’s disease) or from its newly attributed functions (e.g., Bruton’s X-linked
agammaglobulinemia). The other results available in the literature reflect the use of CD38
molecule as a prognostic marker in several neoplastic diseases.
1.6.1 Leukemia and myeloma
CD38 has been widely used as a marker to study normal T and B cell
differentiation and also as an auxilium in the systematic classification of lymphocyte
malignancies. CD38 is highly expressed in leukemia blast cells of most patients with acute
leukemia. Although detection of CD38 does not play a critical role in modern leukemia
classification and does not clearly identify subtypes of leukemias with distinctive
treatment outcomes, the widespread expression of this surface molecule suggests the
potential use of anti-CD38 antibodies as antileukemic agents.
Chapter 1 Introduction 19
1.6.2 HIV infection
More than a decade ago, it was observed that CD8+ T-cells in AIDS patients
have an increased expression of CD38 (Salazar-Gonzales et al., 1985). It was subsequently
shown that the expansion of these cells preceded the decline in CD4+ T-cells and the
development of AIDS (Bofill et al., 1996). This observation was subsequently expanded
by some researchers who used CD38 as a useful and reliable prognostic marker for the
pathological development of AIDS (Liu et al., 1998). CD38 expression inhibits
lymphocyte susceptibility to HIV infection, probably by inhibiting gp120/CD4-dependent
viral binding to target cells (Savarino et al., 1996).
1.6.3 Alzheimer’s disease
The CD38 antigen was detected in the perikaryal and dendritic cytoplasm of
neurons and also in the neurofibrillary tangles that occur in the neuronal perikarya and
proximal dendrites that are the pathological hallmark of Alzheimer’s disease (Otsuka et
al., 1994). The neuronal localization of CD38 perfectly matches the predicted function of
the molecule, which ultimately leads to the mobilization of intracellular Ca2+ and
consequently may control certain brain functions such as neuronal plasticity.
1.6.4 X-linked agammaglobulinemia (XLA)
Bruton’s disease or XLA is characterized by a reduced concentration of serum
Ig secondary to a dramatic decrease of circulating B cells (Conley et al., 1992). The gene
responsible for this disease is Bruton’s tyrosine kinase (Btk), which encodes a protein
sharing some features with src tyrosine kinase family. It is likely that Btk is an integral
component of the CD38-induced signal transduction pathway in B cells.
Chapter 1 Introduction 20
Molecule Mol. Mass (kDa) Membrane % Amino acid Distribution Association similarity Human CD38 43.7 TM 100 Hemopoietic, other Murine CD38 42 TM 70 B,T and NK cells,
monocyte/macrophage Rat CD38 34.4 TM 76 Spleen, liver,
heart,thymus,ileum, colon, salivary gland
Human BST -1 43 GPI-anchored 33 Stromal, endothelia cells
Murine BP3-BST-1 38-48 GPI-anchored 30 Early B and T lymphocytes
Rat BST -1 32-35 GPI-anchored 33 Kidney, spleen, heart, thymus
Aplysia Cyclase 29-31 Cytosolic 25 Ovotestis
Table 1.2 CD38 family and other related molecules
Chapter 1 Introduction 21
1.7 Green Fluorescence Protein
Green fluorescent protein (GFP) is a stable and proteolysis-resistant single chain
of 238 amino acid protein isolated from jellyfish Aequorea victoria (Figure 1.4). It
maximally absorbs blue light at 395 nm, emits green light with a peak at 509 nm and a high
quantum yield of 0.72 to 0.85 (Kendall & Badminton, 1998). In A. victoria, light is produced
when calcium binds to the photoprotein, aequorin. The energy transfer from aequorin to GFP
results in the emission of green light. Formation of the fluorophore in GFP requires
molecular oxygen but once formed it absorbs and emits light without cofactors. GFP
fluorescence can also be detected by ultraviolet light excitation.
The most distinctive feature of the fold of GFP is an 11-stranded β barrel with a
coaxial helix, with the chromophore forming the central helix (Ormo et al., 1996). Almost all
the primary sequences are used to build the β-barrel and axial helix, so that there are no
obvious places where one could design large deletions and reduce the size of the protein. The
development of GFP mutants with red-shifted excitation and emission maxim would be
valuable for avoidance of cellular autofluorescence at shorter wavelength and for
simultaneous multicolour reporting of activity of two or more cellular processes. The protein
is stable and can be functionally expressed in a wide variety of organisms including
prokaryotes, plants, mammalian cells, parasites, fungi and yeasts.
GFP is used frequently as a reporter of gene expression (Charlfie et al., 1994) and
protein localization in live cells in real time. The fluorescence is an intrinsic property of the
protein, which does not require additional gene products, substrates or cofactors and occurs,
in a species independent fashion. Because fluorescence requires no cofactors, the GFP signal
can be recorded in living cells with no disruption of cellular integrity.
Chapter 1 Introduction 22
Figure 1.4 Green Fluorescent Protein. 238 amino acids make up the green fluorescent protein. Strands of -sheet (blue) form the walls of a cylinder. Short segments of -helices (red) cap the top and bottom of the 'b-can' and also provide a scaffold for the fluorophore which is near geometric center of the can. Reproduce from www.biochem.arizona.edu/dept/ GreenFlour-miesfeld.jpg
Chapter 1 Introduction 23
Besides, the fluorescence signal is highly resistant to photo bleaching and no apparent toxic
effects of GFP expression in bacteria or eukaryotes.
Despite all the advantages mentioned above, GFP also suffers from several
disadvantages. For example, the signal intensity may be too weak for some applications,
however brighter GFP mutants can overcome this limitation. Future directions for
improvement of GFP as a genetic reporter include efforts to increase the intensity of
fluorescence signal and thereby enhance detection sensitivity, and to reduce the variability of
signal between replicate samples.
1.8 Tet-Off system
In the Tet-Off system, gene expression is turned on when tetracycline (Tc) or
doxycycline (Dox; a Tc derivative) is removed from the culture medium. Maximal
expression levels in Tet systems are very high and compare favorably with the maximal
levels obtainable from strong, constitutive mammalian promoters such as CMV. Unlike other
inducible mammalian expression systems, gene regulation in the Tet Systems is highly
specific, so interpretation of results is not complicated by pleiotropic effects or nonspecific
induction.
1.8.1 Components of Tet-off system
The first component of the Tet system is the regulatory protein, based on TetR
(Tet repressor protein). In the Tet-off system, this protein is a fusion of amino acids of TetR
and the C-terminal of the Herpes simplex virus VP16 activation domain: The VP16 domain
Chapter 1 Introduction 24
converts the TetR from a transcriptional repressor to a transcriptional activator, and the
resulting hybrid protein is known as the tetracycline-controlled transactivator (tTA).
The second component is the response plasmid (pTRE) which expresses a gene of
interest (Gene X) under the control of the tetracycline-response element (TRE): The TRE
consists of seven direct repeats of a 42-bp sequence containing the tetO, and is located just
upstream of the minimal CMV promoter, which lacks the strong enhancer elements normally
associated with the CMV immediate early promoter (Figure 1.5).
Chapter 1 Introduction 25
Transcription
+ Tet/Dox
Transcription is switched off
Figure 1.5 Tet-off system. The TRE is located upstream of the minimal immediate early promoter of cytomegalovirus (PminCMV ), which is silent in the absence of activation. tTA binds the TRE—and thereby activates transcription of Gene X—in the absence of Tet or Dox.
TRE
TRE
Gene X
tTa VP16
tTa
VP16
Gene X
Chapter 1 Introduction 26
1.8.2 Advantages of Tet-system
The Tet-Off and Tet-On systems have several advantages over other regulated
gene expression systems that function in mammalian cells:
(1) The system is under extremely tight on/off regulation. The background, or
leaky, expression of Gene X in the absence of induction is extremely low.
(2) No pleiotropic effects. When introduced into mammalian cells, the prokaryotic
regulatory proteins (TetR or rTetR) act very specifically on the ir target sequences,
presumably because these regulatory DNA sequences are nonexistent in eukaryotic genomes
(Harkin et al.,1999).
(3) High inducibility and fast response times. With the Tet Systems, induction can
be detected within 30 minutes using nontoxic levels of inducer. Induction levels up to
10,000-fold have been observed. In contrast, other systems for mammalian expression exhibit
slow induction (up to several days), incomplete induction (compared to repressor- free
controls), low overall induction (often no more than 100-fold), and high (nearly cytotoxic)
levels of inducer (reviewed by Gossen et al., 1993; Yarronton, 1992).
(4) High absolute expression levels. Maximal expression levels in the Tet systems
can be higher than expression levels obtained from the CMV promoter or other constitutive
promoters. For example, Yin et al. (1996) reported that the maximal level of luciferase
expression in HeLa Tet-Off cells transiently transfected with pTRE-Luc is 35-fold higher
than that obtained with HeLa cells transiently transfected with a plasmid expressing
luciferase from the wild-type CMV promoter.
Chapter 1 Introduction 27
(5) Well-characterized inducer. In contrast to the inducer used in other systems,
such as in the ecdysone system, Tc and Dox are inexpensive, well characterized, and yield
highly reproducible results.
(6) Activation of a promoter, rather than repression, to control expression. To
completely shut off transcription, repression-based systems require very high—and difficult
to attain—levels of repressor to ensure 100% occupancy of the regulatory sites. Even if
suitably high levels of repressor can be obtained, the presence of high repressor levels makes
it difficult to achieve rapid, high- level induction (Yao et al.,1998) . For a more complete
discussion of the advantages of activation versus repression, see Gossen et al. (1993).
1.8.3 Tet-off vs Tet-on
Although the Tet-Off system has been studied more extensively than the Tet-On
system, the two systems are truly complementary. When properly optimized, both systems
give tight on/off control of gene expression, regulated dose-dependent induction with similar
kinetics of induction, and high absolute levels of gene expression. Thus, for most purposes,
there is no inherent advantage of using one system over the other. With the Tet-Off system, it
is necessary to keep Tc or Dox in the medium to maintain the native (off) state. Because Tc
and Dox have relatively short half- lives (see below), one must add Tc or Dox to the medium
at least every 48 hours to suppress expression of Gene X. Conversely, in the Tet-On system,
the native (off) state is maintained until induction. For this reason, Tet-On may be more
convenient in transgenic applications, because you need only add Dox to the animals' diet
when induction is desired.
Chapter 1 Introduction 28
1.9 Objectives of the Study
The objective of this study is to determine effect of site-directed mutations on rat
CD38 enzyme activity and dynamics in tet-off Chinese Hamster Ovary cells. The rationale
for this study is CD38 has been found to internalize upon NAD+ ligand-induced, therefore
from this study we hope to identify the amino acids which is important to induce
internalization of CD38. The particular emphasis is on the effect of the constructed CD38
mutants in ligand-dependent internalization of CD38 in mammalian cells. This was
investigated by using a variety of methods:
1) Confocal microscopy to view the expression and investigation of wild-type CD38 and
constructed mutants.
2) Expressed proteins characterized by Western blot and enzyme assays.
3) Real time-imaging of ligands induced internalization of CD38.
4) Quantitation of internalized CD38 using streptavidin-based method.
Chapter 2 Materials and Methods 29
Chapter 2
Materials and Methods
Chapter 2 Materials and Methods 30
2.1 Materials
2.1.1 Bacterial strains
The strain of Escherichia coli (DH5á) used in this study was prepared and
described in section 2.2.3.1. Long term storage of bacteria was carried out in LB-Broth with
20% glycerol at -70�C.
2.1.2 Mammalian cell line
Chinese Hamster Ovary cells (CHO) was obtained from the American Type
Culture Collection. For long term storage, cells were suspended in storage media (RPMI
1640 supplemented with 20% Fetal Bovine Serum and 10% DMSO) and kept in liquid
nitrogen. For short term storage, cells are suspended in the same storage media and kept at -
80�C. CHO Tet-off cells were established (see section 2.2.1.4) and stored in the same storage
media as described.
2.1.3 Plasmids
pEGFP-N1 (purchased from Clontech, USA) encodes a red-shifted variant of
wild-type GFP which has been optimized for brighter fluorescence and higher expression in
mammalian cells (Excitation maximum = 488 nm; emission maximum = 507 nm.). Genes
cloned into the MCS will be expressed as fusions to the N-terminus of EGFP if they are in
the same reading frame as EGFP and there are no intervening stop codons.
pTet-off is a regulatory vector for use in Tet- Off system. This consists of a fusion
of amino acids 1–207 of TetR and the C-terminal 127 amino acids of the Herpes simplex
virus VP16 activation domain.
Chapter 2 Materials and Methods 31
pTRE2-hyg is an expression plasmid, which expresses a gene of interest (Gene X)
under control of the tetracycline-response element, or TRE.
2.1.4 Synthetic primers
The primers used for this study were synthesized by GenSet. The sequences and
usage of these primers were described in relevant chapters.
2.1.5 Media and solutions
2.1.5.1 Bacterial media
All media for bacteria were sterilized by autoclaving at 15 Lb/in2 for 15 min.
Thermolabile supplements such as antibiotics were filter-sterilized through 0.2 µm filters and
added to the autoclaved media which have been cooled to 60°C.
LB-broth (Luria-Bertani medium): 1.0% Bacto-trypton, 0.5% Yeast-extract
1.0% NaCl. The pH value was adjusted to 7.0 with NaOH prior to autoclaving.
LB agar plate: LB-broth + 15 g/L agar. After autoclaving and cooling to 60°C,
the above medium was poured to 10 cm petri dish.
Antibiotics: Ampicillin 50 mg/ml
Kanamycin 25 mg/ml
The antibiotics were prepared as stock solutions, sterilized by filtration and stored in aliquots
in –20°C.
2.1.5.2 Media and reagents for mammalian cell culture
Chapter 2 Materials and Methods 32
Incomplete RPMI 1640 medium was purchased from the NUMI store (National
University of Singapore Medical Institute) store. Before use, add Penicillin/Streptomycin of
50 u/50 µg per ml, 10% FBS (sterile fetal bovine serum, inactivated) was added. For the
preparation of Tet-off media for CHO Tet-off cell line, the same approach as the above was
used except Tet System Approved Fetal Bovine Serum was used in the place of FCS.
Antibiotics: G418 (50 mg/ml). G418 was prepared as stock solutions, sterilized
by filtration and stored in aliquots at –20°C.
Mammalian cell freezing medium: Complete RPMI-1640 medium was used to
prepare mammalian cell freezing media, consisting of 60 % complete RPMI-1640 medium,
30% of FBS and 10% DMSO (Dimethyl Sulfoxide).
2.1.5.3 Supplier for reagents and other materials
Standard analytical grade laboratory chemicals for the preparation of general
reagents were obtained from Sigma, Merck and Clontech. Special reagents were obtained
from:
Amersham Pharmacia Biotech, USA
ECL TM Western blotting detection reagents Hyperfilm ECL.
Bio-Rad, USA
Gene Pulser curvette for electroporation Bio-Rad Protein Assay Kit Nitrocellulose membrane Polyacrylamide gel reagents SDS-PAGE standard markers
Clontech, USA
Chapter 2 Materials and Methods 33
Tet-off gene expression system kit (pTet-off plasmid and pTRE2 plasmid) Tet System Approved Fetal Bovine Serum Anti-GFP antibody
Invitrogen, USA LipofectamineTM
Promega, USA
T4 DNA ligase Restriction endonuclease 10x PCR buffer dNTP mix Taq DNA polymerase DNA markers
Qiagen, Germany
QIAEX II Gel Extraction Kit QIAGEN Plasmid Midi/Maxi Kit
Santa Cruz Biotechnology Inc, USA
QIAEX II Gel Extraction Kit QIAGEN Plasmid Midi
Sigma Chemical Co. Ltd, USA
QIAEX II Gel Extraction Kit QIAGEN Plasmid Midi
Stratagene, USA
Site-directed mutagenesis kit
Chapter 2 Materials and Methods 34
2.2 Methods
2.2.1 Mammalian cell culture techniques
2.2.1.1 Maintenance of cells
Cells were grown in RPMI 1640 supplemented with 10% heat inactivated FBS,
2mM L-glutamate, 50U/ml penicillin and 10µg/ml streptomycin. The cells were cultured at
37�C in a humidified 5% CO2 atmosphere.
2.2.1.2 Storage of frozen cells
Cells to be frozen were grown up to late log phase. Adherent cells were
trypsinized, centrifuged at 200g for 5 mins. Cells were harvested by centrifugation and
resuspended in the freezing medium (10% DMSO, 30% FBS, 60% complete medium) at a
density of 3 x 106 cells/ml, and portions (1 ml) are added to polypropylene freezing tubes.
The tubes are frozen slowly at 1�C/min. They were either stored in liquid nitrogen for long-
term or kept in -80�C for short-term storage.
2.2.1.3 Revival of frozen cells
Frozen stock of cell was thawed rapidly in a 37�C water bath. The cells were
transferred to a sterile tissue culture flask and appropriate volume of complete RPMI 1640
medium was added. The cells were then incubated in a 37�C incubator with 5% CO2.
2.2.1.4 Stable Transfection of cells using Electroporation
Chinese Hamster Ovary cells (CHO) of density (1 x 107 cells/ml of RPMI 1640
medium) were transfected with 40µg of pTet-off plasmid DNA by electroporation (960µF,
Chapter 2 Materials and Methods 35
750 V/cm) usng a Bio-Rad Gene Pulser. The cells were allowed to divide for 48 hrs before
addition of geneticin (400µg/ml) to the culture for selection. Stable clones were obtained
after 3 weeks growth in the drug-containing medium. The medium was replaced every 5 days
with fresh medium and selection antibiotics. Clones with the best signal-to-noise ratio were
chosen for the development of stable Tet-off cell line.
2.2.1.5 Transient transfection using Liposome (Lipofectamine)
Introduction of foreign DNA into CHO cells was done using
LIPOFECTAMINETM reagent (Gibco BRL)-mediated transfection method. CHO cells were
seeded at 15% confluency and cultured until 60%~70% confluncy. For the transfection of
cells in a 75cm2 flask, 12 µg of plasmid DNA was dissolved with 600 µl of OPTI-MEM
reduced serum medium (Gibco BRL). Then plasmid DNA was mixed with 30 µl
LIPOFECTAMINETM reagent which was also pre-diluted with 600 µl OPTI-MEM medium.
The mixture was incubated at room temperature for 30 mins to allow DNA-liposome
complex to form. CHO cells were rinsed twice with 1×PBS and one time with OPTI-MEM
medium just before transfection. The DNA-liposome mixture was diluted with 6 ml of the
OPTI-MEM medium and added to each of the 75 cm2 flask. The transfection was allowed for
10 hrs in a 37°C humidified incubator with 5% CO2. The medium was changed to complete
DMEM medium and cells were incubated for another 48 hrs. The CHO cells or culturing
medium were harvested to detect the expression of the targeted genes.
2.2.1.6 Preparation of ligands for internalization studies
Chapter 2 Materials and Methods 36
Different ligands were used in this study to induce internalization of wild-type
CD38-GFP and other constructed mutants from the surface membrane in transfected CHO
Tet-off cells. The ligands are CD38 substrate, â-NAD+, an analog of â-NAD+, á-NAD+
CD38-mediated second messenger which include cADPR and cADPR analogues including
8-Amino-cADPR. Milimolar concentration range was used for substrates like NAD+ (10mM)
and micromolar concentration range was used for cADPR and the analogues. Ligands were
prepared freshly with 1 x PBS on the day of experiment. The pH was then monitored within
the range of 7-8.
2.2.2 Methods for manipulation of nucleic acids
2.2.2.1 Polymerase Chain Reaction (PCR)
PCR was used to exponentially amplify, mutate or subclone target DNA
fragment. The thermostable Tag DNA polymerase (Promega) was used in the
cycling reactions. Reactions were typically carried out in a total volume of 50µl. The reaction
mixtures consisted of 1~20 ng DNA template, 1×PCR buffer, 2mM MgSO4, 0.2 mM dNTP
mix, 0.2 µM each primers, 1-2 units of Tag DNA polymerase. Mineral oil could be overlaid
or omitted to top of the reaction mixture prior to thermal cycling. Thermal cycling was
typically carried out for 25~35 cycles of the following profile: 94°C (DNA denaturation)
45~60 seconds, 45~60°C (primer annealing) 60 seconds, 72°C (Extension) 1~2 minutes. This
profile was adjusted dependent on the size of the amplified DNA, template and primer used.
The last cycles had an additional 5 minutes extension time.
2.2.2.2 Cloning of full length rat CD38 cDNA into pEGFP-N1 vector
Chapter 2 Materials and Methods 37
As depicted in Figure 2.1, full- length rat CD38 cDNA was amplified using Pfu
polymerase and cloned, in frame, into the pEGFP-N1 cloning vector (Clontech) at KpnI and
EcoRI restriction sites. The sequences of the sense and anti-sense primers used in the PCR
were 5’- CCGAATTCACCATGGCCAACTATGAATTTTC-3’ and 5’-
ACGGTACCACATTAAGTATACATGATGG-3’. The C-terminal of CD38 was fused to the
N-terminal of EGFP vector, thus leaving the targeting signal intact for localization and
internalization studies. A 922 bp fragment was amplified by using the two primers mentioned
above. DNA sequencing was carried out to ensure that no frame-shift has occur red in the
fusion. E.coli transformed with pGFP-CD38 was screened and positive colonies were
identified by PCR. The final construct allows the expression of CD38-GFP fusion protein.
2.2.2.3 Cloning of CD38-GFP fusion gene fragment into pTRE2-hyg vector
The plasmid from the first cloning (pCD38-GFP) was amplified using the
QIAGEN Maxi-kit. In order to clone this gene fragment (fusion CD38-GFP) into pTRE2-hyg
vector, we have identified two restriction sites (Nhe I and Not I). The fusion CD38-GFP, was
digested with Nhe I and Not I restriction enzymes and the fusion CD38-GFP was cloned into
the Nhe I-Not I site of pTRE2-hyg vector (Figure 2.2). The plasmids with CD38-GFP insert
were selected. DNA sequencing was carried out to ensure the gene was cloned in. The
constructed plasmid, pTRE2-CD38-GFP was used to construct mutants using the site-
directed mutagenesis kit mentioned in the next section
Chapter 2 Materials and Methods 38
EcoRI KpnI
pEGFP-N1 4.7kb
KpNI EcoRI
CD38
PCR
EcoRI + KpnI EcoRI + KpnI
HCMV Promoter
EcoRI CD38 KpnI
SV40 Poly A site
EGFP
pEGFP-N1 4.7kb Kanr/Neor
Figure 2.1 Cloning of CD38 into pEGFP-N1 vector. CD38 was first amplified and digested by EcoRI and KpnI and then inserted into pEGFP-N1 vector under the control of HCMV promoter.
Chapter 2 Materials and Methods 39
NheI NotI CD38-GFP
Not I
NheI
TRE PminCMV
Hygr CD38-GFP fusion
Ampr
pTRE2-hyg 5.3kb
NheI/NotI NheI/NotI
pTRE2-hyg 5.3kb
HCMV promoter
Figure 2.2 Cloning of CD38-GFP into pTRE2-hyg vector. Fusion gene (CD38-GFP) was digested with NheI and NotI enzymes before being cloned into the pTRE2-hyg expression vector under the control of HCMV promoter.
Chapter 2 Materials and Methods 40
2.2.2.4 Site-directed mutagenesis by QuickChange Mutagenesis Kit
This in vitro multiple sites mutagenesis method used the kit from Stratagene. The
three-step mutagenesis method is outlined in Figure 2.3. Step 1 used PCR procedure with one
or more synthetic single-stranded 5’ phosphorylated oligonucleotide primers and cloned
DNA in a vector as template. Double stranded DNA was generated with one strand bearing
mutations. The nicks were sealed by components in the enzyme blend. In step 2, the reaction
products were treated with restriction endonuclease Dpn I. The parental DNA template which
was methylated would be digested by Dpn I endonuclease. In step 3, the mutated single
stranded DNA was transformed into competent cells, where the mutant closed circle ss-DNA
is converted into ds-DNA in vivo. Double stranded plasmid DNA may then be prepared from
the transformants and analyzed by DNA sequencing to identify the clones bearing each of the
desired mutations.
2.2.2.5 Restriction endonuclease digestion of DNA
Digestion of DNA was normally carried out in a volume of 20 µl using
appropriate buffers as recommended by the manufacturers. A typical digestion mixture
consisted of: 2 µg of DNA in TE or water, 2 µl of restriction enzyme buffer (10×), 1 µl of
restriction enzymes (5-10 U/µl) and top up with sterile water to a final volume 20 µl. The
total amount of enzyme used did not exceed 10% of the total reaction volume so as to
prevent “star activity” by the enzyme. Restriction mixtures were incubated at the optimal
temperature from 1 hour to several hours. The digested DNA fragments may be separated by
agarose gel electrophoresis.
Chapter 2 Materials and Methods 41
Figure 2.3 Overview of Ouickchange site-directed mutagenesis
Step 1 Synthesis of the mutant strand
Thermal Cycles
Perform thermal cycles to:
1) Denature input DNA 2) Anneal mutagenic primer(s) 3) Extend primer(s)
which result in nicked circular strands.
(1) (2) (3)
Step 2 Dpn I digestion of template (parental) DNA
Step 3 Transformation
parental mutagenic template primer
Chapter 2 Materials and Methods 42
2.2.2.6 Concentration of nucleic acids
Ethanol precipitation was routinely used to concentrate and purify nucleic acids.
DNA or RNA at concentration of more than 20 µg/ml were readily precipitated with two
volumes of absolute ethanol and sodium acetate (pH4.6) at a final concentration of 0.3 M and
a 10 minute incubation at either room temperature or on ice. The precipitated nucleic acids
were then pelleted by microcentrifugation at top speed for 15 minutes. The pellet was then
washed twice with 70% ethanol at room temperature and dried. DNA or RNA at
concentration lower than 20 µg/ml need a longer incubation time, lower precipitation
temperature and longer centrifugation time to ensure efficiency of precipitation.
2.2.2.7 DNA ligation
T4 DNA ligase (Promega) was used for ligation of DNA fragments into vector.
Normally, ligation reaction was carried out at 23 °C for 4 hours or overnight at 16°C with
vector:insert molar ratio of approximately 1:3 to increase the ligation efficiency and to
reduce occurrences of multiple inserts. For blunt end ligations, the molar ratio of insert to
vector can be increased to 4:1 and a buffer containing PEG 6000 to a final concentration of
5% was used.
2.2.2.8 Agarose gel electrophoresis of DNA
Agarose gel electrophoresis separates DNA molecules according to the size of the
DNA fragments and the percentage of agarose in the gel used depends on the sizes of DNA
fragments to be separated. The smaller the DNA molecule is, the higher the agarose
concentration the gel should contain, and vice versa. For rountine gel electrophoresis, the
Chapter 2 Materials and Methods 43
gels were made with 0.7-1.5% (W/V) electrophoresis grade agarose melted in 30 ml 1×TBE
buffer. The gel was then cooled down to 60°C and added with 0.1 µg/ml ethidium bromide
and poured into a horizontal gel casting tray. A comb to create the wells for loading DNA
samples was inserted immediately into the molten agarose and left to set for 30 minutes.
Once the gel was set, the comb was removed and the gel was submerged in an
electrophoresis tank containing 1×TBE buffer. DNA samples were premixed with the
appropriate amount of 10× loading dye and loaded into the wells. Electrophoresis was
normally carried out at 5 V/cm field strength and the migrated DNA bands were visualized
under U.V. light.
2.2.2.9 Isolation of DNA from agarose gel
The QIAGEN Gel Extraction kit was used for the extraction and purification of
DNA fragments from agarose gel. The DNA band of interest was excised from the agarose
gel under the U.V. light with a clean scalpel. The size of the excised gel was minimized as
much as possible by trimming the excess gel. The gel slice was weighed and 3 volumes of
buffer QXI was added to 1 volume of gel for DNA fragments from 100 bp to 4 kb. After the
agarose gel was solubilized at 50°C, QXII solution which contained the DNA binding beads
were added and vortexed from time to time for 10 min. After washing, DNA fragments
bound to the beads was eluted with appropriate amount of TE buffer. The eluted DNA
sample is suitable for most downstream applications such as restriction digestion, ligation
and sequencing.
Chapter 2 Materials and Methods 44
2.2.2.10 DNA sequencing
Cloned DNA fragments were sequenced using the cycle-sequencing technique
and the sequencing carried out in an automated DNA sequencer. Sequencing
reaction is essentially a polymerase chain reaction (PCR) which employs ABI
PRISMTM Dye Terminator cycle sequencing kit, an appropriate primer and a good quality
double-stranded DNA template. Apart from Tag DNA polymerase and the deoxynucleotides,
the sequencing premix also contains fluorescence tagged dideoxy-nucleotides, which emit
different fluorescence (depending on which dideoxynuleotide), when hit by the laser.
Reactions were carried out in a volume of 20 µl and comprised of: 500 ng of double stranded
template DNA, 4 µl of Terminator mix, 4 µl of Half term, 3.2 pmol of Primer and add sterile
water to a final volume 20 µl.
The thermal cycling was carried out using the following profiles:
96°C (DNA denaturation) 30 seconds
50°C (primer annealing) 15 seconds
60°C (primer extension) 4 minutes
The reaction was subjected to 25 cycles.
2.2.3 Methods for bacterial culturing and transformation
2.2.3.1 Preparation of competent cells
Streak LB plate with the competent cells; a single colony was picked and
inoculated into 5 ml LB medium. After overnight growth at 37°C, 3 ml of the cultured cells
Chapter 2 Materials and Methods 45
was inoculated into 500 ml prewarmed LB broth in a 2 liter flask and let grow till OD 600
reached 0.5. The culture was chilled on ice for 10-15 min and transferred into a sterile,
round-bottom centrifuge tube. Cells were collected by centrifugation at 4000g for 20 min at
2°C. The cell pellet was then resuspended in 500 ml ice cold water and spun again. After
washing again with cold water, cells were washed with 40 ml ice cold 10% glycerol. The cell
pellet was finally resuspended in 1 ml ice cold 10% glycerol and aliquot 45 µl into pre-
chilled centrifuge tubes and stored in -80°C.
2.2.3.2 Transformation by electroporation
A BioRad MicroPulser was used for high efficiency electroporation of plasmid
into competent cells. Cell were thawed and stored on ice, then 1 µl of ligation
product was added and mixed with the cells. The mixture was transferred to a cold 0.2 cm
electroporation cuvette. This was then pulsed once at a voltage of 1.8 kV. 1 ml SOC medium
or LB medium was immediately added to the cuvette to resuspend the cells. Cells were then
transferred to a 10 ml culture tube and incubated at 37°C for 1 hr with shaking at 200rpm.
Different volumes of culture were plated onto LB plate containing appropriate antibiotics to
ensure selection of colonies.
2.2.3.3 Transformation by heat shock
45 µl of competent cells in polypropylene tubes were thawed on ice. After
adding 2 µl of β-ME mix into each tube of the cells, the mixture was swirled gently and
incubated on ice for 10 min with swirling gently every 2 min. Transfer 1.5 µl of the DNA to
be transformed into the cell mixture, swirl and incubate on ice for another 30 min. The tube
Chapter 2 Materials and Methods 46
was then heat-pulsed in a 42°C water bath for 30 sec, then incubated on ice for 2 min. 0.5 ml
of preheated (42°C) LB medium was added into each tube and incubated at 37°C for 1 hr
with shaking of 225-250 rpm. Appropriate volume of the transformation mixture was plated
onto agar plates containing the appropriate antibiotics fo r the plasmid vector. The
transformation plates were incubated at 37°C for >16 hr.
2.2.4 Preparation of plasmid DNA from Bacterial cells
Isolation of plasmid DNA was performed on either a small or ‘miniprep’ scale to
obtain sufficient material for analysis, or on a large, ‘maxiprep’ scale to provide a stock of
plasmid for long-term use.
2.2.4.1 Miniprep
This proceed was carried out using Wizard ®Plus SV Minipreps DNA
Purification System. Individual bacterial clones were picked and dispersed into separate
sterile tubes that contain 3 ml of LB and the appropriate antibiotics. The tubes were shaken
overnight at 37�C overnight. The culture was pelleted by centrifugation for 2 minutes at
10,000 × g in a microcentrifuge. The supernatant was poured off and blot the tube upside-
down on a paper towel to remove excess media. The cell pellet was resuspended in 250µl of
Cell Resuspension Solution (50mM Tris-HCl (pH 7.5), 10mM EDTA, 100µg/ml RNase A).
250µl of Cell Lysis Solution (0.2M of NaOH, 1%SDS) was added and mixed by inverting
the tube 4 times. The cell suspension should clear immediately. 10µl of Alkaline Protease
Solution was added and mixed by inverting the tube 4 times and incubated for 5 minutes at
room temperature. Alkaline protease inactivates endonucleases and othe r proteins released
Chapter 2 Materials and Methods 47
during the lysis of the bacterial cells that can adversely affect the quality of the isolated
DNA. 350µl of Neutralization Solution (4.09M guanidine hydrochloride, 0.759M potassium
acetate, 2.12M glacial acetic acid) was added immediately and mixed well. The bacterial
lysate was centrifuged at maximum speed (around 14,000 × g) in a microcentrifuge for 10
minutes at room temperature.
After centrifugation, the cleared lysate was transferred to the prepared Spin
Column. The supernatant was spun at maximum speed for 1 min and the flowthrough was
discarded. The Spin Column was washed with 750µl Column Wash solution twice by
centrifugation and the flowthrough was discarded. The Spin Column was transferred to a new
sterile microcentrifuge tube and the plasmid DNA was eluted by adding nuclease-free water
to the Spin Column and spun for 2 min. After eluting, the Spin Column can be discarded and
the purified plasmid DNA can be stored at -20�C for future use.
2.2.4.2 Midi/Maxiprep
Large-scale plasmid DNA preparations were carried out using QIAGEN Plasmid
Midi/Maxi Kit. The bacterial cells were harvested by centrifugation at 6,000 x g for 15 min at
4�C. The pellet was resuspended in Buffer P1 and mixed with the same volume of Buffer P2.
After incubation at room temperature for 5 min, Buffer P3 was added and mixed
immediately. The sample was incubated on ice for 20 min and then centrifuged at 12,000 x g
at 4�C for 30 min and 15 min once each. The supernatant was then applied to the pre-
equilibrated QIAGEN-tip 100/500 column and was washed with Buffer QC twice. After that,
the DNA was eluted with Buffer QF and precipitated with 0.7 vol of isopropanol. The
Chapter 2 Materials and Methods 48
precipitated DNA was centrifuged at 16,000 x g for 15 min. After washing with 70% ethanol
and air-drying, the DNA was re-dissolved in a suitable volume of TE buffer.
2.2.5 Expression, purification and analysis of CD38
2.2.5.1 Membrane protein harvesting
The transfected CHO Tet-off cells with the wild-type CD38 and its mutants were
harvested for its membrane proteins following 48hr post-transfection. Briefly, the cells were
scraped from the culture flasks and protease inhibitor cocktail containing leupeptin and
aprotinin (10µg/ml), 50µg/ml soybean trypsin inhibitor and 100µM phenylmethysulfonyl
fluoride were added immediately to the cells resuspended in 1 x PBS. The cell lysate was
then homogenized with a polytron homogenizer for a brief period of 1 min. The cells were
then pelleted at 1000 x g for 10min and the process was repeated again. The supernatants
collected were pooled and spun at a higher speed of 6000 x g for 30min. The pellet that was
recovered was resuspended in cold 1 x PBS stored at -20�C for future use. Protein
concentration was determined by Bradford protein assay (Bio-Rad) with bovine serum
albumin as a standard, according to manufacturer’s instructions.
2.2.5.2 SDS-Polacylamide gel electrophoresis (SDS-PAGE)
The preparation of SDS-PAGE was carried out following the well-established
Laemmli system (Laemmli, 1970). Briefly, to prepare 10% SDS-PAGE gel, 10 ml and 3 ml
of separating and stacking gel solutions were prepared according to the following menu,
respectively.
Chapter 2 Materials and Methods 49
Stock Solution Separating Gel (ml) Stacking Gel (ml)
dH2O 4 2.3
30% Acrylamide (29:1) 3.3 0.3
1.5 M Tris-HCl (pH8.8) 2.5
1 M Tris-HCl (pH6.8) 0.38
10% SDS 0.1 0.03
10% APS 0.1 0.03
TEMED 0.004 0.003
APS and TEMED were added to the solution immediately before casting the gel.
The separating gel solution was poured into the vertical gel caster (Bio-Rad) first and left to
set at room temperature before pouring in the stacking gel and placing the comb. Once the
stacking gel was set in the caster, it was fixed into the vertical electrophoresis unit. Protein
samples were mixed and boiled for 5 min with protein loading buffer and loaded into the
wells of the PAGE gel. Electrophoresis was carried out at 100 V until the protein sample
entered the separating gel then increased to 200 V. After the electrophoresis, the gel can be
stained with coomassie blue staining solution or proceed for Western blotting.
2.2.5.3 Staining of SDS-PAGE gel with Coomassie Blue
The electrophoresed SDS-PAGE gel was detached from the glass plates and was
submerged in Coomassie Blue dye (0.025% Coomassie G250, 0.025% Coomassie R250,
25% isopropyl alcohol, 10% acetic acid). Staining was carried out for at least 2 hours with
Chapter 2 Materials and Methods 50
gentle shaking. It was then transferred into destaining solution (10% acetic acid and 40%
methanol in water) and was allowed to destain for 2 hours to overnight with one or two
changes of the solution. The destained gels were visually examined.
2.2.5.4 Western Blot
Protein samples were separated on SDS-PAGE as described above in 2.2.5.3 and
transferred to nitrocellulose membrane using a wet transelectroblotting system (Bio-Rad
Inc., England) at a low voltage of 80 V for 2hr. The transfer buffer consist of 150mM Tris-
HCl, 39mM glycine and 20% methanol with a final pH of 8.3. The membrane was then
blocked in TBS-T (20mM Tris, 137 mM NaCl, pH7.5, 0.1% Tween 20) containing 5% non-
fat milk for 1 hour. After incubating in appropriate primary antibody for 1-2 hours at room
temperature, the membrane was washed three times with TBS-T buffer, 15 min each time to
remove the excess antibodies. Then, the membrane was incubated in horseradish peroxidase-
conjugated secondary antibody for 1 hour at room temperature. The unbounded secondary
antibodies were washed away with TBS-T buffer three times, 10 min each wash. The
immunoreactive bands were detected by enhanced chemiluminescence (ECL) reagent
exposed to a Fuji X-ray film and processed by a KODAK film processor.
2.2.5.5 Stripping and reprobing of membranes
After each immunodetection, the bound primary and secondary antibodies can be
removed from the membrane and the membrane can be reprobed using different antibodies
following the method below: Submerge the membrane in stripping buffer (100 mM 2-
mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH6.7) and incubated at 50°C for 30 min
with occasional agitation. The membrane was then washed with large volume of TBS-T
Chapter 2 Materials and Methods 51
buffer for two times at room temperature, 10 min each time. Block the membrane by
immersing in 5% non-fat milk in TBS-T for 1 hour at room temperature. The membrane is
ready to be reprobed by a different antibody with same protocol described above in 2.2.5.4.
2.2.5.6 Enzyme Assays for CD38
The ADP-ribosyl cyclase and cADPR hydrolase activities of wild-type or mutant
CD38 were measured with the alternative substrates of NGD and cIDPR, respectively.
Membrane fractions from wild-type and mutant CD38 were incubated for 15 min at 37�C in
20mM Tris-HCl (pH 7.4) with 100µM NGD or cIDPR. CD38 cyclizes a non-fluorescent
substrate NGD into a fluorescent product, cyclic GDP-ribose. Since cyclic GDP-ribose is
highly resistant to hydrolysis, we have used cyclic IDP-ribose, which can be readily
hydrolyzed by CD38 to indicate the hydrolase activity of wild-type CD38 and its mutants.
The fluorescence was measured using a high sensitivity Perkin Elmer LS 50B
spectrofluoremeter (excitation wavelength = 300nm; emission wavelength = 410nm).
2.2.5.7 Affinity purification of biotinylated CD38
In a typical experiment, CHO Tet-off cells were transfected with the wild-type
CD38-GFP and W163G mutant plasmid. After 48h transfection, the cells in culture medium
were washed in RPMI without serum and incubated in PBS containing 5 mM glucose, 0.1
mM CaCl2, 1 mM MgCl2, and 0.8 mM NHS-SS-biotin for 20 min at 4°C. Cells were then
washed in RPMI containing 0.2% bovine serum albumin (fatty acid free) and incubated in
complete medium in the presence or absence (control) of 10 mM NAD+ for 2 h at 37°C. Cells
were then incubated in RPMI with 50 mM GSH at 37°C for 5 min to remove the surface
Chapter 2 Materials and Methods 52
biotin from control and NAD+ -treated cells. To remove untreated GSH, cells were washed in
PBS containing iodoacetamide (5 mg/ml). Finally, cells were solubilized in lysis buffer [PBS
containing 1% Triton X-100, 5 mM EDTA and protease inhibitor cocktail containing
leupeptin and aprotinin (10 mg/ml), 50 mg/ml soybean trypsin inhibitor, and 100 mM PMSF]
at 4°C. The supernatants were obtained from cell lysates by centrifugation at 10,000g for 10
min at 4°C, and the supernatants were incubated with immobilized streptavidin, at 300 µl
resin/ml, for 2 h at 4°C with shaking. The flowthrough was recovered by centrifugation at
200g for 5 min. The resin was washed with lysis buffer and the adsorbed (i.e. biotinylated)
proteins were eluted with 0.1 M glycine buffer, pH 2.0. It was immediately neutralized with
1 M Tris–HCl, pH 8.3. The eluate and flowthrough from control and NADP+- treated cells
were dialyzed in 10 mM Tris–HCl, pH 7.2, containing 0.1% Triton X-100 for 16 h at 4°C,
and then concentrated using Centricon 30 (Amicon Inc., Beverly, MA).
2.2.5.8 Confocal microscopy of CD38 expression and localization
Confocal microscopy was performed using a Carl Zeiss LSM 510 confocal
microscope, equipped with a 40 x Fluar objective (NA = 1.3), 63 x Plan-Apochromatic
objective (NA = 1.4) and a 100x Fluar objective (NA = 1.3), oil immersion. CHO Tet-off
cells were grown on glass coverslip in culture medium and were transiently transfected with
the following plasmids, wild-type pTRE2hyg-CD38-GFP, pEGFP-N1 (positive control),
pTRE2hyg plasmid (negative control), mutated pTRE2hyg-CD38-GFP at Cys-122 site,
mutated pTRE2hyg-CD38-GFP at Glu-229 site, mutated pTRE2hyg-CD38-GFP at
Leu-149 site and mutated pTRE2hyg-CD38-GFP at Trp-162 site. The analysis was to
examine the distribution of wild-type CD38-GFP protein and its mutants in CHO Tet-off
Chapter 2 Materials and Methods 53
cells with the use tetracycline drug. Tetracycline was added initially to switch off the
expression of the protein and was removed by washing with 1 x PBS and the expression of
the different proteins were allowed to be studied. After 48h post transfection, the cells were
fixed with 3.7% paraformaldehyde for 10 min. The cells were then mounted for confocal
viewing.
2.2.5.9 Live-imaging of CD38 internalization
Different ligands including á-NAD+, â-NAD+, cADPR and 8-NH2-cADPR were
tested on the Tet-off CHO cells transfected with wild-type and mutation plasmids. Milimolar
concentration range was fixed for substrates like NAD+. For secondary messenger like
cADPR and the analogues, we have used micromolar concentrations, which is in line with
the physiological range. The above mentioned ligands were dissolved in 1 x PBS under
sterile condition and the pH was closely monitored within range of 7-8.
The transfected CHO Tet-off cells with the wild-type CD38 and its mutant
plasmids were subjected to 2 hr incubation with the above-mentioned ligands following the
48hr post-transfection and the images of the cells were taken at a few time intervals (0, 30,
60 and 120 min).
Chapter 3 Results 54
Chapter 3
Results
Chapter 3 Results 55
3.1 Cloning of full length rat CD38 cDNA into pEGFP-N1 vector
As mentioned in the previous section (2.2.2.2), full- length rat CD38 cDNA
was amplified using Pfu polymerase and cloned, in frame, into the pEGFP-N1 cloning
vector (Clontech) at Kpn1 and EcoR1 restriction sites. The sequences of the sense and
anti-sense primers were 5’- CCGAATTCACCATGGCCAACTATGAATTTTC-3’ and 5’-
ACGGTACCACATTAAGTATACATGATGG-3’. The C-terminal of CD38 was fused to
the N-terminal of EGFP vector, thus leaving the targeting signal intact for localization and
internalization studies. A PCR product of 922 bp fragment was amplified by using the two
primers mentioned above and a product of ~900bp was obtained (Figure 3.1, lane A).
Upon digestion of pEGFP-vector with EcoR1 and Kpn1, a fragment of ~4680 bp was
obtained (Figure 3.1, lane B). Sequences at the fusion point were confirmed by DNA
sequencing to ensure that no frame-shift has occurred. E.coli transformed with pGFP-
CD38 was screened and positive colonies were identified by PCR and the complete
sequence of rat CD38 insert sub-cloned into pEGFP-vector was verified by DNA
sequencing (data not shown).
3.2 Cloning of CD38-GFP fusion gene fragment into pTRE2-hyg vector
The plasmid from the first cloning (pCD38-GFP) was amplified using the
QIAGEN Maxi-kit (described in Section 2.2.4.2). In order to clone this gene fragment into
pTRE2-hyg vector, we have identified two restriction sites (Nhe 1 and Not 1). The fusion
CD38-GFP was digested with Nhe 1 and Not 1 restriction enzymes and a fragment of
~1674 bp was obtained and sub-cloned into the Nhe 1-Not 1 site of digested pTRE2-hyg
vector. The plasmids with CD38-GFP insert were selected. DNA sequencing was carried
out to ensure this fusion gene was cloned in. The constructed plasmid, pTRE2-hyg-CD38-
Chapter 3 Results 56
GFP was used to study site-directed mutagenesis of CD38 using the mutagenesis kit
mentioned in the next section. Figure 3.2 shows the detailed agarose gel analysis of the
PCR products and the constructed plasmid.
3.3 Effects of amino acid substitution of CD38 on cADPR metabolism
Previously, it has been reported that some amino acid residues affect the
enzymatic activities, ADP-ribosyl cyclase and cADPR hydrolysis, of human CD38 (Shan
et al., 1995; Lee 2000; Lee 2001). By this, we hope to obtain valuable information from it
to investigate the effect of enzymatic activities on ligand-dependent internalization in rat
CD38. To investigate on the importance of enzymatic roles during internalization, we
have compared the amino acid sequences of human, rat, mouse CD38s and Aplysia ADP-
ribosyl cyclase (Figure 3.3). The rat, human and mouse CD38s are known to possess both
ADP-ribosyl cyclase and cADPR hydrolase activity. Since it has been previously reported
that amino acid residues Cys-119 and Cys-201 in human CD38 play crucial roles in the
synthesis and hydrolysis of cADPR (Tohgo et al., 1994), we would like investigate if the
corresponding mutation of Cys-122 residue, corresponding to Cys-119 in human CD38
will affect internalization in rat CD38. Furthermore, the di- leucine motif has been reported
in some studies, which might play an important role in the process of membrane protein
internalization. Therefore from the sequence comparison, we have selected one of the
leucine residues, Leu-149, present in the extracellular domain of rat CD38 for site-directed
mutagenesis. Both amino acid residues in human CD38, Trp-140 and Glu-226, have also
been reported previously for their important role in enzymatic activities. In this study, we
have also selected Trp-162 and Glu-229 in rat CD38 for our mutation studies to see its
effect on ligand-induced internalization processes.
Chapter 3 Results 57
Figure 3.1: DNA gel. The rat CD38 cDNA was amplified by using the appropriate primers which gave a ~900 bp inserts. Lane A: PCR product from rat CD38 cDNA. Lane B: EcoR1 and KPN1 digested pEGFP-N1 vector. Lane M1: 100bp markers. Lane M2: Lambda Hind III markers.
� 922 bp
4361bp �� �� 4680bp
M1 A M2 B
500bp��
Chapter 3 Results 58
Figure 3.2 Agarose gel of PCR products and constructed plasmids. PCR product of rat CD38 was digested with EcoR1 and KpN1 and cloned into the EcoR1-Kpn1 site of pEGFP-N1 vector (Lane A). The fusion pCD38-GFP as shown in Lane A was excised at Nhe I and Not I sites (lane B, 1647 bp) and sub-cloned into the pTRE2-hyg vector. The constructed pTRE2-hyg-CD38-GFP (Lane C) was digested at both Nhe 1 and Not 1 sites. The final cloned plasmid (Lane D) was checked again by double-digestion (Nhe 1 and Not 1), which showed the corresponding bands. Lane M1 and M2: 100bp DNA marker and 1kb DNA marker respectively.
�� 1674 bp
�� 1674 bp
M1 M2 A B M1 M2 C D
Chapter 3 Results 59
MANCEFSPVSGDKPCCRLSRRAQLCLGVSILVLILVVVLAVVVPRWRQQ---WSGPGTTKRFPETVLARC MANYEFSQVSEDRPGCRLTRKAQIGLGVGLLLLVALVV-VVVIVLWPRSPLVWKGKPTTKHFADIILGRC MANYEFSQVSGDRPGCRLSRKAQIGLGVGLLVLIALVVGIVVILLRPRSLLVWTGEPTTKHFSDIFLGRC
VKYTEI-HPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFT LIYTQILRPEMRDQDCKKILSTFKRGFISKNPCNITNEDYAPLVKLVTQTIPCNKTLFWSKSKHLAHQYT LIYTQILRPEMRDQNCQEILSTFKGAFVSKNPCNITREDYAPLVKLVTQTIPCNKTLFWSKSKHLAHQYT
QVQRDMFTLEDTLLGYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSVFWKTVSRRFAEAACDVVHV WIQGKMFTLEDTLLGYIADDLRWCGDPSTSDMNYDSCPHWSENCPNNPVAVFWNVISQKFAEDACGVVQV WIQGKMFTLEDTLLGYIADDLRWCGDPSTSDMNYVSCPHWSENCPNNPITVFWKVISQKFAEDACGVVQV
MLNGSRSKIFDKNSTFGSVEVHNLQPEKVQTLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCK MLNGSLSEPFYRNSTFGSVEVFNLDPNKVHKLQAWVMHDIKGTSSNACSSPSINELKSIVNKRNMIFACQ MLNGSLREPFYKNSTFGSVEVFSLDPNKVHKLQAWVMHDIEGASSNACSSSSLNELKMIVQKRNMIFACV
NIYRPDKFLQCVKNPEDSSCTSEI DNYRPVRFLQCVKNPEHPSCRLNV DNYRPARFLQCVKNPEHPSCRLNT
Figure 3.3 Sequence homology of rat, mouse and human CD38. The predicted amino sequences were based on nucleotide sequences in the GenBank databases.
Human CD38 Rat CD38 Mouse CD38
Human CD38 Rat CD38 Mouse CD38
Human CD38 Rat CD38 Mouse CD38
Human CD38 Rat CD38 Mouse CD38
Human CD38 Rat CD38 Mouse CD38
117 69 70
136 139 140
206 209 210
276 279 280
300 303 304
Chapter 3 Results 60
3.4 Site-directed mutations of CD38
One of the site-directed mutations that we have performed was to replace Cys-
122 residue with Lysine (as found in Aplysia cyclase) in rat CD38 cDNA. The resulting
cDNA construct was expressed transiently in CHO Tet-off cells. We have constructed
several other CD38 mutants in which the mutations were identified to be critical for
catalysis as well as internalization. We made 3 other mutants in which the Leu-149, Glu-
229, Trp-162 were replaced with valine, glycine and glycine respectively, and introduced
the mutants into CHO Tet-off cells.
The site-directed mutants were constructed using the QuickChange Site-
directed Mutagenesis Kit obtained from Stratagene (as described in Section 2.2.2.4). This
method utilizes Pfu Turbo DNA polymerase and mutant oligonucleotide primers to
generate the desired mutations in the super-coiled plasmid vector containing the inserts of
interest. The following oligonucleotide primers were generated for the mutations:
E229G primer:
5’-PO4- GCACCTTTGGAAGTGTGGGAGTCTTTAATTTGGACCC-3’
L149V primer:
5’-PO4- TTCACCCTGGAGGACACAGTGCTGGGCTATATTGCAG-3’
C122K primer:
5’-PO4- GGTTACTCAAACCATACCAAAGAACAAGACTCTCTTTTG-3’
Chapter 3 Results 61
W162G primer:
5’-PO4- GCAGATGATCTCAGGGGATGTGGAGACCCCAGTACTTCC-3’
Following that, the product is treated with Dpn I endonuclease which specifically digest
the hemi-methylated parental DNA strand leaving behind the mutation containing
synthesized DNA. Plasmid DNA isolated from most E.coli strain is dam methylated which
can be digested with Dpn I enzyme. The desired mutant is transformed into XL1-Blue
super-competent cells. The mutant clones were selected and confirmed by DNA
sequencing.
3.5 Transfection of CHO Tet-off cells
Once the wild-type CD38 and the constructed mutants have been confirmed by
DNA sequencing and restriction enzyme analysis, CHO Tet-off cells (see section 2.2.1.4)
were transfected with the plasmids by Lipofectamine (Invitrogen). Transfected cells were
cultured for 48h and harvested for the preparation of total cell lysate. The membrane
proteins of the transfected cells were resolved on SDS-PAGE under reducing conditions
and subsequently immunoblotted with polyclonal rabbit anti-rat CD38 primary antibody.
The results are shown in Figure 3.4. The level of expression of all mutant proteins was
roughly comparable to that observed with the wild-type CD38-GFP protein. These data
suggest that site-directed mutations do not interfere with the level of expression of the
constructed mutants. The expression of GFP was also determined in the
CHO Tet-off cells by transfecting with pEGFP-NI control plasmid alone and resolving on
SDS-PAGE under reducing conditions. The GFP expression alone was confirmed by
immunoblotting with anti-GFP antibody, which shows a band of 29kDa (Figure 3.5).
Chapter 3 Results 62
Figure 3.4 Transfection of wild-type CD38 and mutants. Wild-type CD38 and mutants (C122K, E229G, L149V and W162G) were transiently transfected in the CHO Tet-off cells. 48h after post-transfection and removal of tetracycline, the membrane protein were harvested and analyzed by Western blot. 20µg of each sample was subjected to 10% SDS-PAGE under reducing conditions. The position of CD38-GFP fusion protein bands was indicated by an arrowhead, of size 74kDa. NT: Non-transfected CHO Tet-off cells.
NT WT C122K L149V E229G W162G
�� 74-kDa
Chapter 3 Results 63
The localization of GFP expression, wild-type fusion CD38-GFP protein and non-
transfected CHO-Tet cells (data not shown) were further confirmed with confocal
microscopy as shown in Figure 3.6.
3.5 Tetracycline regulated expression of Wild-type CD38
To study CD38 internalization with the Tet-Off system, it is particularly
important to know the kinetics of induction and inactivation of gene expression. A more
direct test for the regulated expression studies of CD38 would be transfecting CHO cells
with a Tet-off expression construct containing wild-type CD38. The expression system
can be regulated by treating the transfected CHO Tet-off cells with tetracycline. We first
tested the addition of tetracycline to the wild-type CHO cells to ensure that it has no effect
on the morphology changes of the cells. We have optimized the amount of tetracycline to
add in order to switch off the protein expression over a time period and confirmed that
tetracycline did not lead to any cell toxicity and rTA function remained stable in the
course of transfection. A concentration of 2µg/ml tetracycline was chosen (Figure 3.7).
This dosage was considered non-toxic since toxic effects. Changes in cell morphology
were only observed at concentrations higher that 6µg/ml (data not shown).
We attempted to express wild-type CD38 in the CHO Tet-off cells to check the
optimal time for the subsequent functional expression study. Figure 3.8 shows that in
Chapter 3 Results 64
Figure 3.5 Immunoblot analysis of GFP protein alone and wild-type CD38-GFP fusion protein. Detection of CD38-GFP and GFP alone from crude membranes of CHO Tet-off cells transfected with pCD38-GFP and pEGFP-NI alone with Western Blot using monoclonal antibody to GFP as the primary antibodies. The crude membrane fractions were subjected to 10% (w/v) SDS-PAGE under reducing conditions (0.04 M β-mercaptoethanol in the sample buffer), followed by blotting onto the nitrocellulose membrane and probing with mouse monoclonal anti-GFP primary antibody and secondary anti-mouse antibodies conjugated to horseradish peroxidase were used before subjected to ECL system. Lane 1, crude membranes from the wild-type pCD38-GFP transfected CHO-Tet cells. Lane 2, crude membranes from pEGFP-NI transfected CHO-Tet-off cells. Lane 3, crude membranes from the non-transfected CHO-Tet-off cells. The antibodies recognized a single band of approximately 74-kDa characteristic of the fusion protein of CD38-GFP (lane 1) and a 29-kDa band characteristic of GFP (lane 2).
74-kDa �� 29-kDa ��
1 2 3
Chapter 3 Results 65
A B
C D
Figure 3.6 Transfection of pEGFP-NI vector and wild-type pTRE2-Hyg-CD38-GFP into CHO Tet-off cells. The expression of wild-type CD38-GFP at the plasma membrane was detected by confocal microscopy. Phase contrast image (A) of CHO Tet-off cells transfected with pEGFP vector. Phase contrast image (C) of CHO Tet-off cells transfected with wild-type pTRE2-Hyg-CD38-GFP. GFP fluorescence as shown in B & D. Size of bar = 20 µm.
Chapter 3 Results 66
Figure 3.7 Determination of optimal concentration of tetracycline. CHO Tet-off cells were stably transfected with a plasmid expressing CD38-GFP under control of the TRE and grown in the presence of the indicated amounts of Tc. A Western blot containing 100 µg of total protein from each condition was probed with mouse anti-GFP specific monoclonal antibodies and secondary anti-mouse antibodies conjugated to horseradish peroxidase were used before subjected to ECL system. A concentration of 2µg/ml was chosen for subsequent experiments. The equal loading of membrane protein was determined by, â-actin (42-kDa) expression (shown in the bottom panel).
200ng/ml 100ng/ml 0.4µg/ml 0.6µg/ml 0.8µg/ml 2ìg/ml
�� 74-kDa �� â-actin
Chapter 3 Results 67
cells transfected with the wild-type CD38, with the removal of tetracycline, it has reached
optimal expression of the fusion protein, CD38-GFP (74–kDa) at 48h as shown by
Western-blot. The equal loading of lysate proteins was judged by the level of the house-
keeping gene, â-actin (42-kDa). This was further confirmed by enzymatic assay as shown
in Figure 3.9 (A) using wild-type CD38 membrane extracts obtained at 48h. At 0h, the
presence of tetracycline inhibited the protein expression. The extent of increase in CD38
cyclase activity after 48h which is induced by removal of tetracycline is significantly
higher than cells with tetracycline. We also observed the hydrolase activity of CD38 was
also optimal at 48h after the removal of tetracycline. Furthermore, we see that CD38-GFP
expression on the surface membrane reached its maximum level via confocal microscopy
within 48h after removal of tetracycline from the growth medium as seen in Figure 3.9
(B). These data support the notion that wild-type CD38 level of expression can be
regulated via the drug, tetracycline.
3.7 Relative percentage of enzyme activities of CD38 mutants
The relative enzymatic activities of each mutant expressed as a percentage of
both ADP-ribosyl cyclase and cADPR hydrolase activities of the wild-type rat CD38 are
shown in Figure 3.10. Wild-type CD38 exhibited both ADP-ribosyl cyclase and cADPR
hydrolase activities (Figure 3.10, wild-type, lane 1). This enzymatic activity is critical for
the cyclization of NAD+ which produces cADPR essentially for Ca2+ release. ADP-ribosyl
cyclase activity was detected not only in the mutant Cys-122 but also in the mutant having
one of the leucine residues (L149V) replaced. However, we could only detect low levels
of ADP-ribosyl cyclase in the W162G mutant. In contrast with ADP-ribosyl
cyclase, the cADPR hydrolase activity was not detected in C122K and L129V mutants
Chapter 3 Results 68
Figure 3.8 Regulation of wild-type CD38-GFP protein expression using tetracycline. Top panel: Wild-type CD38-GFP was transfected into tetracycline regulated Chinese Hamster Ovary (CHO) cells. CHO cells containing the Tet-off system, in the presence of (+) tetracycline, CD38-GFP expression is off (not expressed), whereas in the absence of (-) tetracycline, CD38-GFP is expressed. After 48 hours of incubation with or without 2µg/ml of tetracycline, membrane proteins were harvested at 12, 24, 36 and 48h and analyzed by Western blot for expression level of CD38-GFP (74-kDa) as described in Material and Methods. The equal loading of membrane protein was determined by the level of â-actin (42-kDa) expression (shown in the bottom panel). Maximum level of protein expression was reached at 48h.
�� 74-kDa �� 42-kDa
+ - + - + - + - 12h 24h 36h 48h
Chapter 3 Results 69
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Figure 3.9 (A) Enzyme assays. Top panel: The ADP-ribosyl cyclase activity of wild-type CD38-GFP was measured fluorometrically as the production of cGDPR from the substrate NGD+. At time 0h, the (+) presence of tetracycline inhibit the expression of CD38 resulted in no detectable enzymatic activity. At 48h after which tetracycline was removed, the presence of ADP-ribosyl cyclase was shown. Bottom panel: The cADPR hydrolase of the wild-type CD38-GFP was measured with the use of cyclic IDP-ribose, which can be readily hydrolyzed by CD38 to indicate the hydrolase activity. At 0h, the hydrolase activity was inhibited by the presence of tetracycline. At 48h after tetracycline was removed, the presence of hydrolase activity was detected.
Chapter 3 Results 70
A 0h B 0h
C 48h D 48h
Figure 3.9 (B) Effect of tetracycline on CD38-GFP expression. The effect of tetracycline was again confirmed by confocal scanning laser microscopy. At 0h, the presence of tetracycline has inhibited the expression of the green fluorescent protein (A) phase contrast image of CHO Tet-off cells transfected with pTRE2-hyg-CD38-GFP (B) fluorescent image. As shown in (C) phase contrast image of CHO Tet-off cells transfected with pTRE2-hyg-CD38-GFP, the surface membrane fluorescence of CD38-GFP (D) was detected only after 48h post-transfection with the removal of tetracycline. Scale bar = 20 µm.
Chapter 3 Results 71
0
20
40
60
80
100
120
WT C122K L149V E229G W163G
Rel
ativ
e ac
tivity
(%)
Cyclaseactivity
Hydrolaseactivity
Figure 3.10 Relative enzymatic activities of each mutant as a percentage of the activity of the wild-type rat CD38. Relative ADP-ribosyl cyclase and cADPR hydrolase activities of the mutants of CD38-GFP. The data show three independent experiments with different sets of transfected cells. Bar chart shows the average results from the three experiments. Error bar represent the standard errors from the mean. The cyclase and hydrolase activities are expressed as nmoles cGDPR produced/min/mg of protein and nmoles cIDPR hydrolyzed/min/mg protein, respectively. (*) represent tha t hydrolase and cyclase activities below detection (<1pmol/min/mg protein). The homogenate of CHO Tet-off cells transfected with pTRE2-hyg did not show any ADP-ribosyl cyclase or cADPR hydrolase (data not shown) activities.
* * *
Chapter 3 Results 72
(Figure 3.10, lane 2 and 3 respectively), but cADPR hydrolase activity was detected in
Trp-162 mutant as shown in (Figure 3.10, lane 5). Neither ADP-ribosyl cyclase activity
nor cADPR hydrolase activity was detected in the other mutant, E229G (Figure 3.10, lane
4).
3.8 Expression of CD38 and mutants in CHO Tet-off cells
The membrane surface expression of wild-type CD38, C122K, L149V and
W162G mutants were confirmed by transfecting the individual plasmids and expressing
them in CHO Tet-off cells. We observed that E229G mutant was the only one that was
found at the cytoplasmic location as shown in Figure 3.11. The wild-type CD38 and the
rest of the mutants (C122K, L149V and W162G) were found to be expressed n the plasma
membrane 48h after transfection.
3.9 Live-imaging of NAD+ -induced internalization of CD38
Previous studies have shown that GSH- and NAD+ could induce internalization
of plasma membrane CD38 in Namalwa B cells, HeLa, and 3T3 cells (Zocchi et al.,
1996). Here we have studied the internalization of wild-type CD38 and the constructed
mutants with the using three different internalization inducing agents, 10mM of â-NAD+,
10ìM of cADPR and 10ìM of 8-NH2-cADPR. cADPR has been reported to be powerful
Ca2+ mobilizers in different cell types. However the of â-NAD+ analog, á-NAD+ , failed to
induce internalization of wild-type CD38 and the rest of the mutants (data not shown).
Chapter 3 Results 73
A B
C D
E
Figure 3.11 Localization of the wild-typeCD38-GFP and mutants in CHO Tet-off cells. Wild-type CD38 and constructed mutants were transfected into CHO Tet-off cells. The expression of CD38-GFP was detected by confocal scanning laser microscopy. (A) WT CD38-GFP (B) C122K mutant (C) L149V mutant (D) W162G mutant (E) E229G mutant The expression was visualized with a confocal microscope after 48h post-transfection and removal of tetracycline. Scale bar = 20 µm.
Chapter 3 Results 74
The tetracycline was removed after 12h incubation and expression of wild-type
CD38-GFP and the mutants were allowed for the next 48h. After which, the ligand â-
NAD+ was added to the CHO Tet-off cells transfected wild-type CD38, C122K, L149V
and W162G mutants. The images were taken at a few time intervals (0, 30, 60, 120 min)
to observe the internalization of wild-type CD38-GFP and the mutants. We have excluded
the mutant, E229G for the live- imaging internalization study because the mutant was not
able to be expressed on the plasma membrane of CHO Tet-off cells. As shown in (Figure
3.12 A, B, C and D), the effect of â-NAD+ on the internalization of wild-type CD38 and
C122K, L149V and W162G, respectively, in CHO Tet-off cells were detected by the
confocal microscopy for a period of over 2h. The green fluorescence on the plasma
membrane of wild-type CD38, C122K and L149V mutants decreased over a period of
time and cluster of fluorescence were observed in the cytoplasm location (as indicated by
the arrows). However, the fluorescence on the plasma membrane of W162G mutant
remained after 2h incubation with â-NAD+. No detectable fluorescence of CD38 was
found in the cytoplasm (Figure 3.12 D).
3.10 Live-imaging of internalization of CD38-GFP with cADPR and 8-NH2-
cADPR ligand
Similar live- imaging experiments using cADPR and 8-NH2-cADPR on
internalization of wild-type CD38, C122K, L149V and W162G revealed that these had the
same effect as â-NAD+ (Figure 3.13 A and Figure 3.13 B). These results further confirmed
that the presence of functional ADP-ribosyl cyclase activity is needed for the
internalization of wild-type CD38 and with intact ADP-ribosyl cyclase activity in CHO
Tet-off cells. Thus, with a significant decreased in the cyclase activity in the W162G
Chapter 3 Results 75
mutant, it was unable to undergo internalization with the above-mentioned ligands (Figure
3.12 D, Figure 3.13A & B, Lane 3).
Chapter 3 Results 76
Figure 3.12 (A) CHO Tet-off cells were transfected with wild-type CD38-GFP. After 48h post-transfection and removal of tetracycline, the ligand â-NAD+ was added. The real-time internalization of the transfected cells were captured at a few time intervals (0, 30, 60 and 120 min). The arrows indicate the clustered fluorescence of CD38 being internalized, and localized in the cytoplasmic location of the cell and ultimately near the peri-nuclear region, while less green fluorescence was displayed on the surface membrane over the time period. Cells under ligand treatment was subjected to optical sections of 1µm thickness using a Plan-Apochromat 63X/1.4 Oil DIC Fluar Zeiss objective with a zoom of 2 times and visualized at a mid-height of the cell. Scale bar = 20 µm.
Chapter 3 Results 77
Figure 3.12 (B) CHO Tet-off cells were transfected with mutant CD38-GFP, C122K. After 48h post-transfection and removal of tetracycline, the ligand â-NAD+ was added. The real-time internalization of the transfected cells were captured at a few time intervals (0, 30, 60 and 120 min). The arrows indicate the clustered fluorescence of CD38 being internalized, and localized in the cytoplasmic location of the cell and ultimately near the peri-nuclear region, while less green fluorescence was displayed on the surface membrane over the time period. Cells under ligand treatment was subjected to optical sections of 1µm thickness using a Plan-Apochromat 63X/1.4 Oil DIC Fluar Zeiss objective with a zoom of 2 times and visualized at a mid-height of the cell. Scale bar = 20 µm.
Chapter 3 Results 78
Figure 3.12 (C) CHO Tet-off cells were transfected with mutant CD38-GFP, L149V. After 48h post-transfection and removal of tetracycline, the ligand â-NAD+ was added. The real-time internalization of the transfected cells were captured at a few time intervals (0, 30, 60 and 120 min). The arrows indicate the clustered fluorescence of CD38 being internalized, and localized in the cytoplasmic location of the cell and ultimately near the peri-nuclear region, while less green fluorescence was displayed on the surface membrane over the time period. Cells under ligand treatment was subjected to optical sections of 1µm thickness using a Plan-Apochromat 63X/1.4 Oil DIC Fluar Zeiss objective with a zoom of 2 times and visualized at a mid-height of the cell. Scale bar = 20 µm.
Chapter 3 Results 79
Figure 3.12 (D) CHO Tet-off cells were transfected with mutant CD38-GFP, W162G. After 48h post-transfection and removal of tetracycline, the ligand â-NAD+ was added. The real-time internalization of the trans fected cells were captured at a few time intervals (0, 30, 60 and 120 min). No internalization of CD38-GFP was observed in this case. The green fluorescence was displayed on the surface membrane. Cells under ligand treatment was subjected to optical sections of 1µm thickness using a Plan-Apochromat 63X/1.4 Oil DIC Fluar Zeiss objective with a zoom of 2 times and visualized at a mid-height of the cell. Scale bar = 20 µm.
Chapter 3 Results 80
Chapter 3 Results 81
1
2
3
Figure 3.13 (A) Real- time live imaging of ligand- induced internalization of wild-type CD38-GFP and mutants using cADPR. CHO Tet-off cells were transfected with wild-type CD38-GFP (1) C122K (2) and W162G (3). After 48h post-transfection and with the removal of tetracycline, 10ìM cADPR was added. The real-time internalization of the transfected cells were captured at 2 time intervals (0 and 120 min). The arrows indicate the clustered fluorescence of CD38 being internalized, and localized in the cytoplasmic location of the cell and ultimately near the peri-nuclear region, while less green fluorescence was displayed on the surface membrane over the time period. CHO Tet-off cells transfected with W162G mutant (3), cADPR has no effect on internalization of CD38. Surface membrane fluorescence remained. Scale bar = 20 µm.
Chapter 3 Results 82
1
2
3
Figure 3.13 (B) Real- time live imaging of ligand-induced internalization of CD38-GFP mutants using antagonist 8-NH3-cADPR. Both C122K and L149V mutants were able to internalize upon treatment with 8-NH3-cADPR. No effect of 8-NH3-cADPR on CHO Tet-off cells transfected with W162G mutant. The surface membrane fluorescence remained. Scale bar = 20 µm.
Chapter 3 Results 83
3.11 Cell Surface Biotinylation of Wild-type CD38 and W162G mutant
Membrane proteins were labeled with sulpho-NHS-biotin and streptavidin-
agarose (Sigma) was used to absorb biotinylated proteins which were eluted from the
agarose. This will determine if CD38 was able to internalize from the surface membrane
to the internal compartments upon â-NAD+ treated. We have affinity purified the untreated
(absence of â-NAD+ ) cell lysates (Figure 3.14A) and the internalized CD38 from â-NAD+
treated wild-type CD38 and W162G mutant (Figure 3.14B).
The flowthrough and the eluates from the purified samples which correspond
to surface and internalized, biotinylated proteins, respectively, were resolved on SDS-
PAGE under reducing conditions. Results are as shown in (Figure 3.14A and 3.14B). 74-
kDa band corresponding to CD38-GFP fusion was seen on the immunoblot. As for the
untreated cells (absence of â-NAD+), CD38-GFP fusion protein is only present in the
flowthrough of wild-type CD38 and W162G mutant (Figure 3.14A, Lane 1 and 3). These
results indicated in the absence of â-NAD+, all biotin- labeled CD38-GFP remained on the
surface after incubation and was debiotinylated during the subsequent treatment with
GSH. This resulted in the failure to bind to streptavidin matrix and thus no band was
observed in the eluates of both untreated wild-type CD38-GFP and W162G mutant cells.
The fusion protein band was present in the flowthrough (Figure 3.14B, lane 1) and the
eluate (Figure 3.14B, lane 2) of wild-type CD38-GFP cells treated with â-NAD+. As for
W162G mutant transfected cells, they failed to internalize upon â-NAD+ treatment and
thus remained on the surface membrane. Therefore, we can only observe fusion protein in
the flowthrough (Figure 3.14B, lane 3) of these cell lysates but not in the eluate of the
W162G mutant transfected cells treated with â-NAD+ (Figure 3.14B, lane 4).
Chapter 3 Results 84
Figure 3.14 (A) Western blot analysis of untreated (absence â-NAD+) of wild-type CD38-GFP and W162G mutant transfected cells using a biotin/streptavidin based method. Immunoblot analysis of CD38-GFP fusion protein (74-kDa) purified from CHO Tet-off cells. The flowthrough and eluate of untreated cells were concentrated with centricon-30 and 30µg of each sample was subjected to 10% SDS-PAGE under reducing conditions. The membrane proteins were harvested and analyzed by Western blot as described under Material and Methods Lanes 1 and 2 show the protein fraction of flowthrough and eluate of untreated wild-type CD38-GFP transfected cells, respectively. Lanes 3 and 4 show the protein fraction of flowthrough and eluate of untreated W162G mutant transfected cells, respectively.
1 2 3 4
� 74-kDa
Chapter 3 Results 85
Figure 3.14 (B) Western blot analysis of â-NAD+-treated internalized wild-type CD38-GFP and W162G mutant transfected cells using a biotin/streptavidin based method. Internalized CD38-GFP fusion protein (74-kDa) was purified from CHO Tet-off cells by a biotin/streptavidin based method, as described in Material and Methods. The flowthrough and eluate of â-NAD+-treated cells were concentrated with centricon-30 and 30µg of each sample was subjected to 10% SDS-PAGE under reducing conditions. The membrane proteins were harvested and analyzed by Western blot as described under Material and Methods. Lanes 1 and 2 show the protein fraction of flowthrough and eluate of â-NAD+-treated wild-type CD38-GFP transfected cells, respectively. Lanes 3 and 4 show the protein fraction of flowthrough and eluate of â-NAD+-treated W162G mutant transfected cells, respectively.
1 2 3 4
� 74-kDa
Chapter 3 Results 86
In the â-NAD+ - treated cells, a fraction of the biotinylated- labeled wild-type
CD38-GFP escaped de-biotinylation and translocated to the intracellular compartment.
Upon lysis, this fraction of internalized CD38 was bound to the streptavidin matrix and
appeared in the eluate of the â-NAD+ - treated cells. Therefore the cluster appears as a
fusion band under reducing condition on the Western blot. The streptavidin eluate from
the W162G mutant treated with â-NAD+ showed no visible band on the Western blot. This
result suggests the importance of ADP-ribosyl cyclase which might play an important role
in the internalization of CD38. CD38 internalization has been proposed as a mechanism
by which the ectoenzyme produced intracellular cADPR which may assist in Ca2+
signaling.
The internalized CD38 purified by biotin/streptavidin was found to be active in
terms of ADP-ribosyl cyclase and cADPR hydrolase activities. The respective internalized
CD38 cyclase and hydrolase activities are shown in (Figure 3.15A and Figure 3.15B). As
expected, the internalized wild-type CD38 exhibited both ADP-ribosyl cyclase and
cADPR hydrolase activities, which did not vary significantly from the surface CD38. As
seen in the same figure, W162G mutant was not able to internalize and thus no protein
was pulled down by immobilized streptavidin. Taken together, our findings suggested that
the importance of ADP-ribosyl cyclase in playing a role in CD38 internalization.
Chapter 3 Results 87
050
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0 50 100 150 200 250 300 350 400 450
Time (sec)
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Wild-type CD38-GFP W162G
Figure 3.15 (A) ADP-ribosyl cyclase and cADPR hydrolase activities of internalized molecule of wild-type CD38-GFP and W162G mutant purified from CHO Tet-off cells by a biotin/streptavidin based method. The membrane proteins were harvested and analyzed as described under Material and Methods. The ADP-ribosyl cyclase activities of both internalized both wild-type CD38-GFP and W162G mutant transfected cells were determined fluorometrically as the production of cGDPR from the substrate NGD+.
Chapter 3 Results 88
150
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Time (sec)
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Wild-type CD38 W162G
Figure 3.15 (B) ADP-ribosyl cyclase and cADPR hydrolase of internalized molecule of wild-type CD38-GFP and W162G mutant purified from CHO Tet-off cells by a biotin/streptavidin based method. The membrane proteins were harvested and analyzed as described under Material and Methods. The cADPR hydrolase of the wild-type CD38-GFP and W162G mutant were measured with the use of cyclic IDP-ribose, which can be readily hydrolyzed by CD38 to indicate the hydrolase activity.
Chapter 4 Discussion & Conclusion 88
Chapter 4
Discussion & Conclusion
Chapter 4 Discussion & Conclusion 89
The relationship between the ectocellular localization of the cADPR-
synthesizing CD38 and the intracellular site of Ca2+-mobilizing activity displayed by
cADPR is a challenging topic in the areas of membrane biochemistry and signal
transduction. In this study, we have cloned and expressed the construct, pTRE2-CD38-
GFP in mammalian cells. GFP was chosen because its fluorescence is an intrinsic
property of the protein, and does not require additional gene products. Besides, time and
effort can be saved by not having to raise antibodies to localize CD38 in live cells. The
GFP-tagged CD38 is labeled stoichiometrically, and may be used to follow
internalization in live cells in real time. A similar work expressing CD38-GFP fusion
protein in NIH-3T3 cell line has been reported previously (Adebanjo et al., 1999).
In addition, the conventional methodology used in the study of the cellular
localization of CD38 has always been immunostaining. The use of antibodies, have
several drawbacks such as artifacts introduced during the process of fixation and
permeabilization. The ease with which GFP-tagged CD38 may be observed means
significant timesaving over other methods. The GFP-tagged CD38 also enables studies
involving ligand-dependent internalization in live cells in real time to be performed.
However, at times, the presence of GFP may affect the integrity of the protein of interest.
Therefore, the GDP-ribosyl cyclase enzyme assay was carried out to see if the GFP
protein interfered with the catalytic activity of the ectoenzyme, CD38. The fusion protein
was shown to have GDP-ribosyl cyclase activity (Figure 3.9 A). This suggests that the
bulky GFP protein did not perturb the proper folding of CD38, as the gross misfolding
may likely affect the catalytic property of CD38. The disadvantage of having CD38-GFP
Chapter 4 Discussion & Conclusion 90
fusion protein was that the fusion protein may not behave the same as the wild-type (non-
GFP) protein and may give incorrect localization results. Another problem faced when
using GFP is that the intensity of the fluorescence is relatively low and sometimes
requiring special filters to be viewed. Furthermore, care should be taken when assessing
any results that incorporate GFP technology, so as to make sure that the proteins’
function and localization has not been compromised. Here, the presence of green
fluorescence has indicated that GFP protein was properly folded and CD38 is functional.
Previously, it has been shown that Human lymphoid Namalwa B cells (a
continuous B-cell line derived from Burkitt’s lymphoma) are constitutively expressing
CD38+ as shown by immunofluorescence staining and assay of CD38 ectoenzyme
activities. Incubation of these cells with either NAD+ or thiol compounds, e.g. GSH, was
found to elicit extensive internalization in non-clathrin-coated vesicles, as shown by
different experimental approaches including cytofluorimetric analyses, assays of total
versus ectocellular CD38 enzyme activities, transient cell surface biotinylation
experiments and cryoimmunoelectron microscopy (Zocchi et al., 1996).
The results of the site-directed mutagenesis presented in this study have also
identified the importance of the catalytic domain of CD38 ligand-dependent for its
internalization. The extracellular functional domain of CD38 catalyzes the synthesis of
cADPR from NAD+. It is possible that internalization of CD38 may increase the
synthesis of cADPR intracellularly, which in turn regulate intracellular Ca2+-induced Ca2+
release (CICR) mechanism in many cell types. Ligands- induced internalization of CD38
Chapter 4 Discussion & Conclusion 91
has been observed in a variety of cells (Zocchi et al., 1999) and was shown to associate
with cADPR-dependent Ca2+ increases (Han et al., 2000). It is possible that such
internalization mechanism may lead to an increase in the concentration of cADPR in the
nucleus, which in turn can result in Ca2+ release via the nuclear ryanodine receptor
(RyR). Previous studies have shown the presence of RyR in cardiac nuclear envelopes.
(Guihard et al., 1997; Gyorke et al., 1993). Ca2+ mobilization in the nucleus is known to
play an important role in nuclear events such as DNA replication and transcriptional
activation. Therefore, ligand-dependent internalization might represent a means for
switching on some intracellular functions of CD38.
Another system, which is similar to the ligand-enhanced endocytosis, is
growth factor receptor (EGF receptor) signaling. The messages, which promote growth,
come from outside the cell in the form of protein growth hormones. EGF binds to the
surface of cells by attaching to EGF receptor. The complex of hormone and receptor is
then carried inside the cell which induces further changes in the cytoplasmic domain of
the transmembrane receptor (i.e. self phosphorylation, dimerization etc) resulting in the
binding or clustering of components of the endocytotic complex.
Though the physiological ligand(s) for CD38 have yet to be identified, our
findings have shown some ligands (â-NAD+, cADPR, 8-NH2-ADPR) were able to induce
CD38 internalization. A proposed mechanism by which a topological problem of the
ectocellular active site of CD38/intracellular localization of cADPR receptor could be
overcome was suggested by the demonstration of a specific transporter system for NAD+
Chapter 4 Discussion & Conclusion 92
in plasma membrane and of CD38 itself as a cADPR transporter (Zocchi et al., 1999;
Franco et al., 2001). The exact mechanism of how extracellularly produced cADPR from
CD38 can be transported into the cells for signaling remains to be elucidated, but
whatever the mechanism, the prime pre-requisite for internalization is the ADP-ribosyl
cyclase activity of CD38, as shown in this study.
In this study, we have used GFP to follow internalization in live cells with
wild-type CD38 and the constructed mutants. Previous studies have shown that double
mutant of both Cys-119/Cys-201 of human CD38 has ADP-ribosyl cylcase that was equal
to that of the wild-type, but is impaired in terms of internalization. From their data, these
are indeed important sites for internalization and that a reducing agent has been identified
to enhance CD38 internalization (Han et al., 2002). However, our data has shown that a
single Cys-122 mutant (equivalent to human C119K) in rat CD38 was able to internalize
upon ligands induction (Figure 3.12 B), despite the absence of cADPR hydrolase activity.
Future work in this aspect should also include constructing another mutant, a rat
equivalent of the human CD38 C119K/201E double mutant.
On the other hand, it has been shown that internalization of different
transmembrane proteins which requires a di- leucine motif in the C-terminal domain
(Aiken et al., 1994). A number of leucines (residues 124–145, 150–164, 208–229, and
271–285) within the transmembrane and extracellular domains of CD38 can potentially
form leucine zipper motifs, as proposed by Hurst et al., 1994. The dileucine motifs in the
CD38 molecule is, however, not accessible to intracellular targeting because of its
Chapter 4 Discussion & Conclusion 93
extracellular location. We have mutated one of the leucine residues present in the di-
leucine motif in CD38 to study if such a mutant could still be internalize. Indeed, Leu-
149 mutant, lack of cADPR hydrolase activity, was still able to internalize (Figure 3.12
C), despite the lack of only. Therefore, the two mutants have one common property –
presence of ADP-ribosyl cyclase activity. Taken together, these data have shown that the
presence of ADP-ribosyl cyclase was able to induce internalization, which will enable
future studies of downstream processes.
Data on the Glu-229 mutation with no CD38 expression on the surface
membrane (Figure 3.11 E) and blockage of ligand- induced internalization (data not
shown) are consistent with the previous finding that Glu-226 residue is essentially
important for all enzymatic activities of CD38. As seen in (Figure 3.11 E), Glu-229
mutant was probably associated with the internal membranes, which appeared to be
associated with the endoplasmic reticulum (ER). This mutant may be grossly mis-folded
and could not exit the ER. Since CD38 was not expressed on the surface membrane, it
has suggested the importance of the catalytic domain for signal transduction.
To further substantiate that the presence of ADP-ribosyl cyclase will mediate
internalization of CD38, we have constructed another mutant (W162G), which has
significantly reduced ADP-ribosyl cyclase activity, but its cADPR hydrolase remains
intact. CD38-GFP expression of W162G was found to remain on the surface membrane
but it was not able to internalize upon the use of different inducing agents (Figure 3.12 D,
Figure 3.13 A, lane 3 and Figure 3.13 B, lane 3). Although the exact mechanism of
Chapter 4 Discussion & Conclusion 94
ligand- induction for CD38 internalization is yet to be determined, from the above
constructed mutants, the ADP-ribosyl cyclase activity of CD38 seems to be an important
factor for ligand- induced internalization. The brief time of exposure to the different
ligands (2h) is sufficient to elicit the changes in subcellular localization as seen in this
study. In this study, we are only interested in CD38 being internalized upon ligand-
induced. We were not clear which internal compartment CD38 and its mutants were
localized after being induced. Further work can be carried out using double labeling
experiments with early and late endosome markers, where it will be able to clearly
suggest the exact re-localization of CD38 and its mutants. So far, no report has shown or
addressed the nature of endocytic vesicles involved in ligand-dependent CD38
internalization.
In conclusion, we have presented evidence that the ADP-ribosyl cyclase
activity play an important role in ligand-induced CD38 internalization. It is worth noting
that internalization of CD38 may also result in the import of ADP-ribosyl cyclases
activity from the cell surface to internal membranes. We can postulate that once CD38 is
internalized, it brings along the cADPR produced, it is possible through this the cADPR
system may play an important role in Ca2+ mobilization. We can conclude from these
results that the decreased in the green fluorescence on the plasma membrane upon
treatment with the different internalizing agents is due to ligand-dependent internalization
of CD38 in CHO Tet-off cells. This is consistent with the previous study which had
shown that the internalized CD38 had similar catalytic activities as in normal surface
CD38 (Natesavelalar and Chang, 1999). This suggests that the internalized CD38 can
Chapter 4 Discussion & Conclusion 95
catalyze the intracellular synthesis of cADPR if it has an access to the cytoplasmic pool
of NAD+. Subsequently, the intracellular formed cADPR can also mobilize Ca2+ from
intracellular stores. Figure 4.1 summarizes the properties of the mutants and their
localization.
A more direct test for the regulated expression of CD38 was achieved by
transfecting CHO cells with Tet-off expression construct containing full- length wild-type
CD38 (Figure 3.8). The expression of CD38 can be activated by treating the transfected
CHO cells with tetracycline. We have seen that removal of tetracycline has enabled
expression of CD38 on surface membrane of CHO Tet-off cells and with different
internalizing agents added after 12hr expression, CD38 was able to internalize and its
expression was located around the perinuclear membrane. We have also confirmed the
presence of tetracycline has no effect on the morphology of CHO cells.
With the use of tetracycline system, we hope to mimic physiological
conditions of CD38 and unravel the precise mechanism of its internalization in further
studies. Will CD38 follow the ubiquitous pathway of degradation or will it be recycled
back to the cell surface for the same function to produce cADPR? Alternatively, it would
be of interest if we can study the difference, if any, in down-modulation of the wild-type
CD38 and the mutants. CD38 down-modulation by internalization might be, along with
shedding, another regulatory element in both functions. Although the enzymatic function
and the ability of signal transduction of CD38 have been extensively investigated, the
Chapter 4 Discussion & Conclusion 96
Figure 4.1 Diagrammatic representation of enzymatic activities and localization of wild-type CD38 and its mutants after ligands-induced.
Enymatic
activities
Wild-type
CD38
C122K
mutant
E229G
mutant
L149V
mutant
W163G
mutant
ADP-ribosyl
Cyclase
+ + - + Very low
cADPR
Hydrolase
+ - - - +
Localization
after ligand
induced
cytoplasm cytoplasm membrane cytoplasm membrane
Cytoplasmic
Extracellular
Transmembranee CD38
E229
W163
C122
L149
Chapter 4 Discussion & Conclusion 97
link between these two activities still awaits elucidation. Previous studies have provided
evidence that an adhesion molecule, CD31 (platelet endothelial adhesion molecule-1,
PECAM-1) functions as a putative CD38 ligand (Deaglio et al.,1998). It would be of
interest to study whether a recombinant CD31 can mediate internalization of CD38
similar to that induced by â-NAD.
Further studies on the level of cADPR intracellularly as compared with the
extracellular level may lead to a better understanding of exact mechanism of intracellular
trafficking and CD38 internalization. Nevertheless, our data has indicated the major
ADP-ribosyl cyclase action of CD38 is to enable internalization to occur. More research
has to be done on the signaling pathways of the ectoenzyme, CD38. An effort to unravel
the precise mechanism of CD38 internalization and the pathway of intracellular
trafficking has to be made. An approach using immuno electron microscopy may be
useful to provide information concerning the precise topology of CD38 in the nuclear
membrane, as previous study has shown that CD38 was found to be on the nuclear
membrane after internalization. A better understanding of all the mechanisms involved in
regulating CD38 function may be useful for understanding the mechanisms involved in
the regulation of Ca2+ homeostasis.
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