Miltenberger blood group antigen type III (Mi.III) enhances the

RED CELLS, IRON, AND ERYTHROPOIESIS

Miltenberger blood group antigen type III (Mi.III) enhances the expression ofband 3Kate Hsu,1 Naiwen Chi,1 Marjan Gucek,2 Jennifer E. Van Eyk,2 Robert N. Cole,2 Marie Lin,1 and D. Brian Foster2

1Mackay Memorial Hospital Transfusion Medicine Laboratory, Tamsui, Taiwan; and 2Johns Hopkins University School of Medicine, Baltimore, MD

The special blood group antigen Mi.IIIexhibits a characteristic hybrid structureof glycophorin A (GPA) and glycophorinB, termed Gp.Mur. This phenotype hasexceptionally high occurrence rates inseveral indigenous tribes in Taiwan(�21.2%-88.4%). Because glycophorin/Miltenberger begins interaction with an-ion exchanger-1 (AE1) in the endoplasmicreticulum, we hypothesized that the AE1-

based macrocomplexes on erythrocytemembranes obtained from Mi.III� peoplecould be differentiated from those ob-tained from non-Miltenberger people.Quantitative mass spectrometric compari-son of the AE1-based complexes byiTRAQ™ (Applied Biosystems) revealed25% to 67% higher expression of AE1 inMi.III� erythrocytes. In accordance withthe higher AE1 level, the Mi.III� erythro-

cytes exhibited superior HCO3� capaci-

ties, pH homeostasis, and osmotic resis-tance. Cotransfection experiments inHEK293 cells showed that Gp.Mur, likeGPA, enhanced trafficking of AE1 to theplasma membrane. In summary, the in-creased surface expression of AE1 inMi.III� erythrocytes could be attributed tothe additive effect of GPA and Gp.Murcoexpression. (Blood. 2009;114:1919-1928)

Introduction

Miltenberger antigens belong to the complex MNS blood groupsystem.1 They most likely evolved from specific gene mutationor crossover events of homologous glycophorin A (GYPA),glycophorin B (GYPB), and glycophorin E (GYPE). The occur-rence frequencies of the Miltenberger blood group antigens suchas type III (Mi.III) are extremely low in white2 (0.0098%) andJapanese (0.006%) people. However, the frequencies are excep-tionally high in several Taiwanese aboriginal tribes (up to 90%),compared with 2% to 3% in Han Taiwanese (Minnan).3 Mi.IIIexhibits a unique glycophorin hybrid peptide (Gp.Mur), thelikely product of specific gene insertion of GYPA into GYPB(denoted BAB as in Figure 1A).4 Because transfusion withincompatible Miltenberger blood could result in severe hemo-lytic diseases,5-8 blood bank screening of the Miltenbergerphenotypes before transfusion is required in Taiwan.

Previous work has shown that the glycophorin A (GPA)interacts with glycophorin B (GPB) and band 3 (also known asanion exchanger-1 [AE1]), as well as Rh polypeptides, aquaporin,glucose transporter type I, 4.1R, and spectrin, among other proteinsin large macromolecular complexes.9-11 Bruce et al have suggestedthat these individual protein components or complexes functioninteractively as a “gas-exchange metabolon,”11 in which AE1 actsas the central pillar for the entire assembly. The “macrocomplex”concept implies that variations among individual components orcomplexes are interconnected.

GPA exerts a stabilizing effect on AE1 expression, specificallyby assisting the folding and maturation, and by enhancing the anionexchange activity of AE1.12-14 GPA and AE1 begin coassembly inthe endoplasmic reticulum and Golgi, and their protein expressionis tightly coupled based on the studies of AE1 knockout mice andhuman GPA transgenic mice.10,15 In contrast, GPB that is highlyhomologous to GPA, but lacks a cytoplasmic domain,16 does not

seem to influence AE1 activities when expressed in Xenopusoocytes.12 The function of GPB remains unclear.17

In this study, we sought to identify the structural and functionalimpact of the Mi.III blood type commonly observed among Taiwanese.We reasoned that the hybrid structure of Gp.Mur might engendercompositional or structural differences in the AE1-based complexes,which, in turn, might manifest differences in erythrocyte membranefunctions. By comparing the protein compositions of AE1-basedcomplexes in erythrocyte ghosts obtained from Mi.III� and non-Miltenberger (control) people, we found a significant increase ofAE1 onMi.III� membrane. Their higher AE1 level was correlated with func-tional changes, including superior HCO3

�-transporting capacities, acid-base homeostasis, and osmotic resistance, which contrast with thephenotype of certain kinds of hereditary spherocytosis characterized bya marked reduction of AE1 expression. By unveiling the functionalrelevance of the Miltenberger antigen, our work suggests that itsevolutional emergence is beneficial.

Methods

Red blood cell samples

The Mackay Memorial Hospital Institutional Review Board has approved thecollection of human blood from consented donors free of infectious diseases. Alldonors provided informed consent in accordance with the Declaration ofHelsinki. The Mi.III phenotype was verified serologically using anti-Mia,anti-Mur, anti-Hil, and anti-Anek antisera (Table 1). Mi.III homozygosity(Mi.III��) was identified by the presence of Gp.Mur and the absence of GPB.

Immunoprecipitation

Anti-AE1 monoclonal antibodies used include AE12-M (Alpha Diagnos-tic), BRIC170, and BRIC71 (Bristol Institute for Transfustion Sciences

Submitted December 18, 2008; accepted June 9, 2009. Prepublished online asBlood First Edition paper, June 29, 2009; DOI 10.1182/blood-2008-12-195180.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 USC section 1734.

© 2009 by The American Society of Hematology

1919BLOOD, 27 AUGUST 2009 � VOLUME 114, NUMBER 9

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[BITS]). Anti-GPA and GPB monoclonal antibodies include E318 (Sigma-Aldrich) and R1.319 (BITS). E3 recognizes a nonglycosylated, homologousregion close to the transmembrane segment (residues 61-64 or 64-67[GPA]; residues 32-35 [GPB]),18 whereas R1.3 recognizes the N-terminalnonsialylated residues common to GPA and GPB.19 Erythrocyte ghostswere solubilized with an equal volume of the doubly concentrated lysisbuffer containing 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate, 2% Nonidet P-40, 0.05% sodium dodecyl sulfate (SDS),phosphate-buffered saline, and complete protease inhibitor mixtures (Cal-biochem). Anti-AE1 was dimethyl pimelimidate dihydrochloride cross-linked to Dynabeads; equal quantities of the ghost lysates (usually 1 mg persample) were mixed with the beads for immunoprecipitation at 4°C for12 to 16 hours, as described previously.20

Quantitative mass spectrometry by iTRAQ™

For iTRAQ™, 4 mg of the ghost lysates per sample was used as the startingmaterials for immunoprecipitation (IP). To facilitate mass spectrometry–basedprotein identification, coimmunoprecipitated carbohydrates were removed bychemical and enzymatic deglycosylation (supplemental Figure 1A-B, availableon the Blood website; see the Supplemental Materials link at the top of the onlinearticle). The samples were subsequently trichloracetic acid precipitated, individu-ally resolubilized, reduced, alkylated, and digested with trypsin, followed byiTRAQ™ labeling (Applied Biosystems; see supplemental Figure 1). Proteinsfrom the Mi.III samples (tagged with 116- and 117-Da reporter ions) whose ratiosrelative to the control samples (tagged with 114- and 115-Da reporters)consistently exceeded 1.2 or were less than 0.8 were deemed targets of interest.Further details are in the supplemental Methods.

The DIDS labeling of intact red blood cell surface

Equal numbers of intact erythrocytes were labeled with 5 �M DIDS(4,4�-di-isothiocyanato-2,2�-disulfostilbene) at room temperature for 20 min-utes, followed by 2 washes. The amount of DIDS bound to cell surface wasmeasured by a microplate spectrofluorometer (SpectraMAX Gemini XS;Molecular Devices) at 450 nm emission.

Measurement of HCO3�/Cl� transport capacities

HCO3�/Cl� transport across red blood cell (RBC) membrane was assessed

by the concentration changes of intracellular Cl� ([Cl�]in) with respect tothat of extracellular Cl� ([Cl�]out). Fresh erythrocytes were labeled with5 mM Cl�-sensitive dye 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ;Invitrogen), as previously described.21 SPQ fluorescence from wet erythro-cytes was excited at 350 nm, and its emission collected at 430 nm. [Cl�]in

was calculated based on individual calibration equations.21 Further detailsare provided in the supplemental Methods.

Intracellular pH measurement by flow cytometry

Fresh erythrocytes were loaded with 1 �M fluorescent pH indicatorcarboxy SNARF-1 (Invitrogen) for 10 minutes, followed by Hanks bal-anced salt solution wash. For intracellular pH (pHi) calibration, SNARF-1–loaded cells were incubated with nigericin-containing, high K� buffer.SNARF-1 fluorescence was excited at 488 nm, and its emission at yellowand red fluorescence channels was collected by FACSCalibur. BecauseSNARF-1 exhibits a pH-dependent spectral shift, pHi was calculated fromthe ratios of fluorescence intensities.22 Further details are provided in thesupplemental Methods.

Osmotic fragility test

A modified osmotic fragility test was performed to determine the range oftolerable osmotic stresses on erythrocytes. Equal quantities of fresh RBCswere incubated in 0.2% to 1% NaCl for 30 minutes at room temperature.Percentage of hemolysis was recorded after 5 minutes of 100g centrifugation.

Assessment of AE1 expression by FACS

The coding region of human AE1 (GenBank accession M27819) wassubcloned into expression vector pCIG (pIRES2-EGFP) to generateTa

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1920 HSU et al BLOOD, 27 AUGUST 2009 � VOLUME 114, NUMBER 9




pCIG-AE1. Gp.Mur (EU338225) and GPA (NM002099) were individuallysubcloned into pcDNA3.1. HEK293 cells were transiently transfected withpCIG-AE1 alone, or together with pcDNA-GPA or pcDNA-Gp.Mur, atequimolar concentrations. To assess AE1 surface expression, intact cellswere stained with anti-AE1 antibody BRIC71 at 36 to 72 hours posttransfec-tion, followed by secondary staining with Alexa Fluor 660–conjugatedanti–mouse antibody (Invitrogen). For total AE1 production, intact cellswere fixed and permeabilized first, followed by BRIC71 labeling. Trans-fected cells were selected for green fluorescent protein expression, and AE1levels were assessed using FACSCalibur.

Results

Comparison of the AE1-based complexes from Mi.III� and thecontrol erythrocytes

To characterize the Mi.III blood group proteome, 6 Mi.III (including2 homozygous Mi.III) and 6 control samples were collected. Theirglycophorin profiles were assessed by immunoblot. The E3 monoclonalantibody, which recognizes both GPA and GPB (anti-GAB), showedmonomeric and oligomeric GPA, GPB, and Gp.Mur in the ghost lysates

(Figure 1B). Most oligomers, that is, GPB-GPB, GPB-Gp.Mur, Gp.Mur-Gp.Mur, GPA-GPA, and GPA-GPB, could be deduced from aligning theGAB and GPA immunoblots. GPB expression in heterozygous Mi.III�

was reduced relative to the control. Homozygous Mi.III�/� cells weredevoid of GPB, but expressed more Gp.Mur than heterozygous Mi.III.This complementarity between the levels of GPB and Gp.Mur suggeststhat the combined level of GPB and Gp.Mur in Mi.III (or the Gp.Murlevel in Mi.III�/�) is probably equivalent to that of GPB in the control.Their GPA levels also appeared consistent.

To study the Mi.III blood group proteome comparatively, weused 2 different anti-AE1 antibodies (AE12-M and BRIC170),opting against using glycophorin/Miltenberger antisera in thepulldown experiments to avoid possible selection biases towardglycophorin variants. AE1 immunoprecipitates were deglycosy-lated and then pooled for iTRAQ™-based quantitative massspectrometry. The proteins identified in our immunoprecipitates arepresented in Table 2. Our results confirm the components of theband 3–based macromolecular complexes identified previously,including Rh polypeptides, �- and �-spectrins, �-hemoglobin,ankyrin-1, glucose transporter type I, 4.1R, and EPB42.11 Among

Figure 1. The expression levels of GPB and Gp.Mur in Mi.III� RBCs were complementary. (A) Mi.III-specific Gp.Mur probably evolved from homologous generecombination between GYPA and GYPB, and shows a unique glycophorin B-A-B structure and the antigenic Mur (marked as checkered). The protein sequences of full-lengthGp.Mur and GPB, and GPA lacking a cytoplasmic domain, were aligned by the CLUSTALW program. (B) Glycophorin immunoblot of ghost lysates from 6 Mi.III� and 6 non-Mi.III(control) samples (30 mg/lane). Solubilized ghosts were resolved on 10% SDS–polyacrylamide gel electrophoresis. (Left) GPA immunoblot by E4 antibody. (Right) Immunoblotagainst GAB by E3 antibody. Homozygous Mi.III samples are marked �/�.

Mi.III ENHANCES AE1 EXPRESSION 1921BLOOD, 27 AUGUST 2009 � VOLUME 114, NUMBER 9




them, �-tropomyosin, band 4.9, aquaporin-1 (AQP1), flotillin-1,flotillin-2, and stomatin were previously unreported (Table 2 andsupplemental Table 1). The first 2 components, �-tropomyosin andband 4.9, are established members of the submembranous cytostruc-tural junctional complexes, which additionally include spectrintetramers, short actin filaments, 4.1R, myosin, and adducin.23 Theother newly identified components, flotillins and stomatin, belongto distinct erythrocyte lipid rafts.24,25 Some of these cytoskeleton-associated proteins might be interconnected rather than directlybinding to AE1. The mass spectrometry (MS)–identified proteinswere validated by immunoblot analysis (Figure 2). We note thatcertain proteins, like Rh-associated glycoprotein and glycophorins,were present in our experiments (Figures 2-3), but proved recalci-trant to MS/MS identification. Despite our best efforts to deglyco-sylate the proteins (as shown by glycosilver stain in supplementalFigure 1), residual glycosylation might yet have been a confounder.

The major quantitative difference revealed by iTRAQ™ was25% to 67% increase of AE1 in Mi.III, for which a representativespectrum is presented in Figure 3A. This observation was con-firmed by immunoblot (Figure 3B). To confirm that the increasedAE1 levels in these experiments represented a true change inprotein expression and not confounding issues related to immuno-precipitation, we further assessed AE1 levels by comparativebinding of AE1 inhibitor, DIDS, to the surface of fresh intact RBCs(Figure 3C), and by immunoblot analysis of deglycosylated ghost(supplemental Figure 2).

There was general concordance between iTRAQ™ and immu-noblot analyses of the pulldown experiments. A notable exceptionwas AQP1, for which immunoblot revealed greater amounts inpulldowns from Mi.III, which in this case is most likely more

reliable given that MS/MS identified AQP1 by only one peptide(2.6% sequence coverage in Table 2). Whereas the overall AQP1levels were similar between Mi.III and the control, AQP1 was moresubstantially associated with AE1 in Mi.III (Figure 2D). Thissuggests that the AE1-AQP1 complex in Mi.III� RBCs is unique,and might result from their significantly higher AE1 expression.

Higher anion exchange capacity of Mi.III� erythrocytes

Erythrocyte AE1 has 2 important physiologic roles, each correspondingto a structural region: (1) its multispan transmembrane primarily servesas a Cl�/HCO3

� exchanger for blood CO2 transport; and (2) itsN-terminal cytoplasmic domain binding to ankyrin, 4.1R and EPB42,provides a mechanical support for RBC membranes and shapes.

The fact that more AE1 was expressed on Mi.III� RBCs hints thattheir anion exchange capacity should be larger. We evaluated thecapacity of Cl�/HCO3

� exchange by loading erythrocytes with afluorescent probe for Cl�, SPQ. When challenging the SPQ-loadedRBCs with different [Cl�]out, we observed small changes of [Cl�]in withrespect to changes of [Cl�]out in the milieu of 5 mM HCO3

� (Figure4A). When extracellular bicarbonate increased to 15 mM, the changesof Cl� content ([Cl�]in/[Cl�]out) were still small for the control, butwere significant for Mi.III. The intracellular Cl� concentrations were57.1 plus or minus 10.6 mM (control) and 84.0 plus or minus 5.5 mM(Mi.III) in the milieu of 15 mM HCO3

� and 90 mM Cl�, compared with62.6 plus or minus 13.4 mM (control) and 58.3 plus or minus 5.9 mM(Mi.III) in the milieu of 5 mM HCO3

� and 90 mM Cl� (Figure 4A).Mi.III� erythrocytes showed significantly higher Cl� permeability athigh [HCO3

�]out.

Table 2. Components of glycophorin/band 3 macrocomplexes identified and quantified by iTRAQ™

No. % Sequence Coverage Accession Name

115:114 116:114 117:114

Ratio P Ratio P Ratio P

1 33.83 P02549 SPTA1_HUMAN Spectrin � chain, erythrocyte 1.09 .005 1.06 .003 1.11 .005

2 30.93 P11277 SPTB1_HUMAN Spectrin � chain, erythrocyte 1.15 .005 1.08 .005 1.12 .005

3 25.57 P16157 ANK1_HUMAN Ankyrin-1 (erythrocyte ankyrin) 1.08 .006 1.15 .005 1.22 .005

4 29.42 P02730 B3AT_HUMAN Band 3 anion transport protein (anion exchange

protein 1)

0.97 .361 1.67 .005 1.25 .005

5 31.40 P16452 EPB42_HUMAN Erythrocyte membrane protein band 4.2 1.02 .797 1.14 .009 1.32 .005

6 32.00 P60709 ACTB_HUMAN Actin, cytoplasmic* 1.21 .000 1.08 .101 1.26 .005

7 27.16 Q08495 DEMA_HUMAN Dematin (erythrocyte membrane protein band 4.9) 1.20 .158 1.11 .211 1.00 � .999

8 24.07 P11171 41_HUMAN Protein 4.1 (band 4.1) 0.87 .002 1.08 .018 1.13 .016

9 29.51 P27105 STOM_HUMAN Stomatin (protein 7.2b) 0.98 .689 0.94 .141 1.10 .014

10 34.30 Q14254 FLOT2_HUMAN Flotillin-2 1.27 .026 1.02 .730 1.08 .372

11 9.35 P11166 GTR1_HUMAN Solute carrier family 2, facilitated glucose

transporter member 1

0.83 .009 1.07 .060 1.26 .000

12 47.57 P62805 H4_HUMAN Histone H4† 0.96 .520 0.51 .067 0.65 .021

13 13.73 P04406 G3P_HUMAN Glyceraldehyde-3-phosphate dehydrogenase 0.90 .420 0.94 .261 1.07 .158

14 26.46 O75955 FLOT1_HUMAN Flotillin-1 0.98 .794 0.89 .313 1.02 .689

15 22.45 P68871 HBB_HUMAN Hemoglobin‡ 1.06 .859 1.06 .448 0.89 .289

16 27.78 Q99877 H2B1N_HUMAN Histone H2B† 1.17 .302 0.33 .011 0.57 .020

17 56.58 P62988 UBIQ_HUMAN Ubiquitin 1.27 .151 1.22 .159 1.16 .200

18 30.77 Q6FI13 H2A2A_HUMAN Histone H2A† 0.25 0.19 0.40

19 6.71 Q02161 RHD_HUMAN Blood group Rh§ 1.23 .659 1.00 � .999 1.54 .032

20 50.00 Q71DI3 H32_HUMAN Histone H3† 1.17 .147 0.53 .003 0.60 .004

21 25.00 P06753 TPM3_HUMAN Tropomyosin �� 1.00 � .999 0.97 .677 0.89 .356

22 6.51 P35611 ADDA_HUMAN �-Adducin 0.76 .127 1.20 .141 0.91 .419

23 2.60 P29972 AQP1_HUMAN AQP1 0.78 0.84 1.41

*The � and � isoforms could not be differentiated.†Multiple isoforms could not be differentiated.‡The � and isoforms could not be differentiated.§Rh polypeptide 1 and Rh polypeptide 2 could not be differentiated.�The �-3 and �-4 isoforms could not be differentiated.





Figure 2. iTRAQ™ validation by immunoblot. Equal quantities of ghost lysates were pooled from 6 to 8 donors per group (control vs Mi.III) for IP, and one-tenth of thepulldown (vol/vol) was loaded onto 4% to 12% SDS-polyacrylamide gel electrophoresis for immunoblot comparison. (A) Immunoblots confirmed that glucose transporter type I,ANK1, EPB42, � spectrin, and � spectrin were not quantitatively different in AE1 immunoprecipitates from both groups. The pulldown experiments were repeated 2 to 8 times,and IP was also confirmed using another anti-AE1, BRIC170 (data not shown). (B) Both anti-4.1R and anti-Rh antibodies pulled down GPA, GPB, Gp.Mur, and AE1, indicatingthat 4.1R and Rh polypeptides were part of the AE1-based complexes. (C) Rh-associated glycoprotein was coimmunoprecipitated with AE1. AE1 IP was repeated andconfirmed with both anti-AE1 (AE12-M and BRIC170). (D) AQP1 was more substantially associated with AE1 on Mi.III� membrane. (Left) AQP1 immunoblot of the pooled ghostlysates showed similar expression levels for both Mi.III and the control. (Right) Both anti-AE1 antibodies coimmunoprecipitated more AQP1 from Mi.III. Thecoimmunoprecipitation experiment has been repeated and confirmed 7 times.





100 200 300 400 500 600 700 800 900 1000 1100 1200m/z, amu

0

50

100

150

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250

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350

400

450

500

550

588478.21

331.15

216.13787.43

450.22

145.10720.33

591.30817.91

848.39

563.31 1044.54259.17233.16

588

550

500

450

400

350

300

250

200

150

100

50

0

100 200 300 400 500 600 700 800 900 1000 1100 1200

b1

b2

b3

b6

b4/y5

y7

y8

b9/y10

b8/y9

b5

y3

y1Inte

nsi

ty

m/z (Da)

113.0 113 .5 114 .0 114 .5 115 .0 115 .5 116 .0 1 16 .5 117 .0 117 .5 118 .0m /z, am u

0

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11 7 .1 0

11 6 .1 0

11 4.1 0

11 5 .1 0

114 115

116 117

Control

Mi.III

A D F L E Q P V L G F V Rb -series

y-series 123567891013

1 2 3 5 6 7 8 9Band 3

m/z, amu

0

50

100

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200

250

300

350

400

450

500

550

588478.21

331.15

216.13787.43

450.22

145.10720.33

591.30817.91

848.39

563.31 1044.54259.17233.16

588

550

500

450

400

350

300

250

200

150

100

50

0

100 200 300 400 500 600 700 800 900 1000 1100 1200

b1

b2

b3

b6

b4/y5

y7

y8

b9/y10

b8/y9

b5

y3

y1Inte

nsi

ty

m/z (Da)

113.0 113 .5 114 .0 114 .5 115 .0 115 .5 116 .0 1 16 .5 117 .0 117 .5 118 .0m /z, am u

0

10

20

30

40

50

60

70

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90

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110

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130

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150

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170

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190

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11 7 .1 0

11 6 .1 0

11 4.1 0

11 5 .1 0

114 115

116 117

Control

Mi.III

113.0 113 .5 114 .0 114 .5 115 .0 115 .5 116 .0 1 16 .5 117 .0 117 .5 118 .0m /z, am u

0

10

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160

170

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11 7 .1 0

11 6 .1 0

11 4.1 0

11 5 .1 0

114 115

116 117

Control

Mi.III


y-series 123567891013

1 2 3 5 6 7 8 9


y-series 123567891013

1 2 3 5 6 7 8 9b -series

y-series 123567891013

1 2 3 5 6 7 8 9Band 3

LC-MS/MSLC-MS/MS

A

B C

0.0

0.3

0.6

0.9

DID

S f

luo

resc

ence

inte

nsi

ties

(a.

u.)

Control Mi.III

*Band 3

WB: AE12-M

IP: Band 3 (AE12-M)

GAB (E3)GPA (E4)Silver Stain

GPA GPB Gp.Mur

variousoligomers

Contl Mi.III Contl Mi.III Contl Mi.III Contl Mi.III

Band 3Band 3

WB: AE12-M

-M)

GAB (E3)GPA (E4)Silver Stain

GPA GPB Gp.MurGPA GPB Gp.Mur

variousoligomers

Contl Mi.IIIContl Mi.III Contl Mi.IIIContl Mi.III Contl Mi.IIIContl Mi.III Contl Mi.IIIContl Mi.III

62

49

38

28kDa

100

62

49

38

28kDa

Figure 3. AE1 expressed more in Mi.III� RBCs. (A top panel) An outline for the iTRAQ™-based quantitative proteomic method. To compare the composition of AE1-basedcomplexes in Mi.III versus the control, RBC samples were collected from 6 Mi.III� and 6 control donors, and each subjected to immunoprecipitation and then deglycosylation.The deglycosylated samples were independently digested with trypsin, and then combined for labeling with 1 of the 4 iTRAQ™ reagents. Two pools of 3 control samples and2 pools of 3 Mi.III� samples were randomly formed from the 6 donors from each group. The iTRAQ™-labeled peptides from all 4 pools were mixed, fractionated by strong cationchromatography, and analyzed by liquid chromatography–MS/MS. (Bottom panel) A representative fragmentation spectrum on iTRAQ™-labeled AE1. A high-scoring spectrumwith overlapping b- and y-series fragment ions assigned to the peptide ADFLEQPVLGFVR (99% confidence) from AE1. (Inset) Expansion of x-axis demonstrates theabundance (area under the curve) of the isobaric tags at 114, 115, 116, and 117 Da. (B) A representative immunoprecipitation experiment using AE12-M antibody. Equal proteinquantities of ghost lysates (m/m) from 6 to 8 donors per group (control vs Mi.III) were pooled for immunoprecipitation. One-tenth of the immunoprecipitate (vol/vol) was loadedfor immunoblot comparison. The IP experiment has been repeated 7 times and confirmed by another anti-AE1, BRIC170 (data not shown). (Left) Silver stain of the AE1immunoprecipitates from Mi.III and the control groups. (Right) Immunoblots for AE1, GPA, and GAB (E3). More AE1 was expressed by Mi.III� cells, whereas the levels of GPAand GPB/Gp.Mur were not significantly different between the 2 groups. (C) DIDS labeling of AE1 was significantly higher in Mi.III� than the control erythrocytes. Fresherythrocytes from 3 donors per group were labeled with DIDS. The background fluorescence intensities from the unlabeled erythrocytes were subtracted. Data are expressedas mean � SE; *P .01.





If Donnan equilibrium is assumed, [HCO3�]in could be approxi-

mated from the conserved Donnan ratio between [Cl�]in/[Cl�]out

and [HCO3�]in/[HCO3

�]out. [HCO3�]in are estimated 3.5 plus or

minus 0.7 mM (control) and 3.2 plus or minus 0.3 mM (Mi.III) inthe milieu of 5 mM HCO3

� and 90 mM Cl�, and 9.5 plus or minus1.8 mM (control) and 14.0 plus or minus 0.9 mM (Mi.III) at 15 mM[HCO3

�]out (Figure 4B). Mi.III� RBCs again showed higher HCO3�

permeability than the control at high bicarbonate. The better Cl� andHCO3

� permeability of Mi.III� RBCs could be attributed to their higherAE1 expression. However, how their transport capacity would expand athigh [HCO3

�]out remains unclear.

Greater pHi-buffering capacity of Mi.III� erythrocytes

In blood pH homeostasis (CO2 � H2O N H� � HCO3�), H� is

buffered by the high concentration of Hb� inside RBC, and HCO3�

is considered the major determinant for pHi.26 Because Mi.III�

erythrocytes showed higher HCO3�/Cl� exchange capacities (Fig-

ure 4), we tested whether their differences were reflected in pHi

homeostasis. Erythrocytes were loaded with fluorescent pH indica-tor carboxy SNARF-1, and the elicited pHi responses werequantified by flow cytometry.

At pHout 7.5, the pHi values for Mi.III� and control erythrocyteswere both approximately 7.2 (7.26 � 0.032 [Mi.III] vs 7.15 � 0.038[control]). Depletion of extracellular bicarbonate drove HCO3

�

efflux, resulting in pHi drop. Notably, pHi drop was more substan-tial for the control cells (6.75 � 0.088) than Mi.III (6.95 � 0.047)when [HCO3

�]out was depleted (Figure 5). Thus, Mi.III� erythro-cytes showed greater pHi-buffering capacity upon changes ofHCO3

� gradients.To verify the role of Cl�/HCO3

� exchange in pHi regulation,we replaced extracellular Cl� with membrane-impermeablegluconate�, which halted the coupled transport of Cl� influx andHCO3

� efflux. As the coupled transport of Cl� efflux andHCO3

� influx remained possible and the chemical gradient forCl� was substantial ([Cl�]out: 14330 mM), bicarbonate was forced toaccumulate intracellularly. Consequently, the intracellular pH increasedfrom approximately 7.2 to 7.71 plus or minus 0.037 (control) and to

0 30 60 90

30

60

90

[Cl- ] in

(m

M)

[Cl-]out

(mM)

Control (n=11) Mi.III (n=8) Homozygous Mi.III (n=4)

*

15 mM [HCO3-]out

A

B

0 30 60 90

30

60

90

[Cl-]out

(mM)

[Cl- ] in

(m

M)

5 mM [HCO3-]out

Control (n=11) Mi.III (n=8) Homozygous Mi.III (n=4)

control Mi.III0

5

10

15

20

[HC

O3- ] in

(m

M)

pre

dic

ted

fro

m D

on

nan

rat

io

15 mM HCO3

-

out

90 mM Cl-out

*

control Mi.III0

5

10

15

20

5 mM HCO3

-

out

90 mM Cl-out

[HC

O3- ] in

(m

M)

pre

dic

ted

fro

m D

on

nan

rat

io

Figure 4. Mi.III� RBCs exhibited higher Cl�/HCO3� exchange capacity upon HCO3

� stimulation. (A) Intracellular Cl� was labeled by fluorescent dye SPQ, and theintracellular Cl� concentration ([Cl�]out) was measured by the degree of SPQ quenching inside erythrocytes. When the extracellular bicarbonate ([HCO3

�]out) was 5 mM, [Cl�]inincreased little with respect to [Cl�]out. [Cl�]in was similar between the control and Mi.III� erythrocytes. In the milieu of 15 mM HCO3

�, Mi.III� erythrocytes contained more Cl�

and showed higher Cl� permeability than the control cells. Data are expressed as mean � SE; *P .05 at 90 mM [Cl�]out. The numbers of donors tested were indicated next tosample labels. (B) The concentrations of intracellular bicarbonate ([HCO3

�]in) were predicted according to Donnan equilibrium. In the milieu of 15 mM HCO3�, Mi.III�

erythrocytes also contained more HCO3� and showed higher HCO3

� permeability.





7.70 plus or minus 0.044 (Mi.III), whereas pHout remained 7.5 (Figure5). The fact that complete depletion of extracellular Cl� resulted in thesame maximal pHi for both groups of cells indicates that theirHCO3

�-buffering capacities could be forced to expand to the samemaxima. The asymmetry of their pHi responses from Cl� depletion andHCO3

� depletion experiments suggests that other erythrocyte transport-ers, like Na�/H� exchanger, may also contribute to the differences inpHi buffering. Future experiments are required to elucidate theseasymmetric pHi responses between Mi.III and the control RBCs.

Functional implication in membrane resistance to osmoticstress

From osmotic fragility tests, Mi.III� erythrocytes exhibited superiorresistance toward osmotic pressure. Osmotic stress was induced bydecreasing NaCl concentrations in the saline. Control cells beganhemolysis at 0.600 plus or minus 0.05% NaCl, whereas Mi.III� cellsbegan at 0.557 plus or minus 0.03% NaCl (Figure 6). We did not findsignificant alteration in their cytoskeletal densities, arrangements, orspectrin lengths by electron microscopy (supplemental Figure 3). Ourresults support the previous finding that AE1 directly interacts with cellmembranes to prevent surface loss and to maintain cell morphology.27

Conceivably, higher AE1 expression in Mi.III could increase contactswithin the AE1-cytoskeletal network and thereby strengthen membraneresistance to osmotic stress.

Gp.Mur enhanced the biosynthesis and surface expression ofAE1

To determine how the glycophorin complement of Mi.III blood groupengenders increased AE1 expression, we tested the hypothesis thatGp.Mur, essentially a variant of GPB containing an element of GPA,might mimic GPA with respect to its ability to act as a chaperone forAE1.12,13 We cloned Gp.Mur (GenBank accession EU338225) andGPA, and cotransfected them individually withAE1 into HEK293 cells.We found that GPA promoted the overall production of AE1 by1.18- plus or minus 0.045-fold, compared with the singly transfectedAE1 (Figure 7). GPA enhanced the surface expression of AE1 moresubstantially (1.58- � 0.10-fold). Coexpression of Gp.Mur showedsimilar functionality to GPA. Gp.Mur also enhanced AE1 biosynthesisby 1.17- plus or minus 0.093-fold, and surface expression by 1.43- plusor minus 0.05-fold (Figure 7). Notably, the GPA levels were similarbetween Mi.III and the control; the GPB levels (or that combiningGp.Mur and GPB) were similar, too (Figure 1B). Thus, the increasedsurface expression of AE1 in Mi.III is best explained by the additiveeffect of trafficking-enhancing GP.Mur that replaces GPB, while retain-ing levels of GPA.

Discussion

In this study, we identified a major function of blood group antigenMi.III in promotion of AE1 expression. Quantitative proteomics byiTRAQ™ revealed 25% to 67% more AE1 protein in Mi.III�

erythrocytes (Table 2 and Figure 3). GPA, a chaperone for AE1, isexpressed at similar levels in Mi.III� and the control erythrocytes(Figure 1B). Because Mi.III additionally expresses the functionallyequivalent Gp.Mur, its elevated AE1 level could be attributed to theadditive effect of Gp.Mur and GPA.

The functional equivalency between Gp.Mur and GPA is, itself,intriguing in light of our current understanding of the role ofglycophorin domains in AE1 trafficking (Figure 7). Previousstructural-functional studies on GPA and GPB attribute the en-hanced AE1 trafficking to the cytoplasmic region of GPA.12 ButGp.Mur, like GPB, also lacks a cytoplasmic domain that GPA has.Therefore, the extracellular and the transmembrane domains whereGp.Mur and GPA share homologies are important for facilitatingthe expression of AE1.

Could the unique interaction between Gp.Mur and AE1 modifythe intrinsic kinetics of AE1 antiport? From protein and transportstudies, both homozygous and heterozygous Mi.III� erythrocytesshowed similar AE1 levels (supplemental Figure 2) and similarCl�/HCO3

� exchange capacities (Figure 4). However, there wasmore Gp.Mur expressed in homozygous Mi.III (Figure 1B). If thedirect effect of Gp.Mur on the catalytic activity of AE1 were

14

6

8

14

6

8

HBSS HCO3

--free Cl--Free

6.8

7.2

7.6

*pH

i Control Mi.III

Figure 5. The pHi-buffering capacities of Mi.III� erythrocytes were superior.Fresh RBCs were loaded with fluorescent pH indicator SNARF-1, and its intracellularpH measurement at pHout 7.5 was measured by flow cytometry. In the absence ofextracellular bicarbonate, the control cells became more acidified than Mi.III.Depletion of extracellular Cl� maximized HCO3

� loading for both Mi.III� and thecontrol cells, and diminished their pHi differences. The number of donors tested wasindicated next to each bar. Data are expressed as mean � SE; *P .05.

0.0

0.2

0.3

0.4

% N

aCl a

t w

hic

h h

emo

lysi

s te

rmin

ates

Control Mi.III

7

7

0.5

0.6

Control Mi.III

% N

aCl a

t w

hic

h h

emo

lysi

s b

egin

s

*

Figure 6. Mi.III� erythrocytes were more resistant to osmoticstress. Osmotic fragility tests showed that Mi.III� erythrocytesbegan and completed hemolysis at more hypotonic concentra-tions than the control cells. *P .05. Data are expressed asmean � SE. The number of donors tested was indicated nextto each bar.





substantial, the homozygous and heterozygous Mi.III� erythro-cytes would have differential Cl�/HCO3

� exchange capacitiesbecause of their different Gp.Mur levels. Thus, greater surfaceexpression of AE1 in Mi.III appears to be the primary factor for itssuperior anion exchange.

The higher level of AE1 of Mi.III� erythrocytes has dualimpacts: (a) it expands anion exchange and pH-buffering capacities(Figures 4-5); (b) it strengthens cell membranes toward osmoticstress (Figure 6). The majority of blood CO2 is transported in theform of HCO3

�. CO2 is converted into HCO3� primarily by

carbonic anhydrase II inside erythrocytes. Although CO2 canpermeate through RBC membranes easily, HCO3

� is membraneimpermeable and must cross membranes via AE1. Because AE1-mediated transport is at least one order of magnitude slower thanthe enzymatic activity of carbonic anhydrase II, it is the rate-limiting step for CO2 transport.28 Increase of AE1 expression couldlift the limit and expand the capacity of CO2 respiration. Incontrast, blood bicarbonate and CO2 capacities have been shownmuch reduced in the AE1-deficient cattle.29 Thus, one might predictthat those with the Mi.III phenotype are capable of enduring higherphysical stresses (eg, during exercise; at altitudes). Indeed, as ananecdote, a disproportional number of the world-class athletesin Taiwan, including the “iron man of Asia,”30,31 are from theaborigine tribe with the highest Mi.III occurrence (80%-90%). Itwould be intriguing to determine whether the high degree ofathleticism can be correlated with the Mi.III phenotype.

Although AE1 was elevated in Mi.III, most of its binding partnerswere not elevated accordingly in the pulldowns from Mi.III� RBCs(Figure 2). One exception is AQP1, whose interaction with AE1 wasmore substantial in Mi.III (Figure 2D). Interestingly, Cho et al showedthat AE1 and AQP1 diffuse laterally at different rates on erythrocytesurface, and concluded that the 2 belong to distinct complexes.32 It istherefore conceivable that theAE1-AQP1 complex found more substan-tially on Mi.III� cells is distinctly different from the band 3 complex11 orthe 4.1R complex33 previously delineated. Their unique interaction inMi.III might further facilitate CO2 respiration, presumably throughcoupling between H2O transport and intracellular bicarbonate forma-tion, which requires H2O.

The glycophorin gene family is probably the second fastest evolvinggene following immunoglobin.34 Although the function of glycophorin/Miltenberger seems auxiliary, glycophorins are the major host receptorsfor Plasmodium.35,36 The speedy evolution of glycophorin was proposedas a decoy or an evasion mechanism against malaria infection.34,37 But itis unclear whether there was a correlation between malaria and theemergence of Mi.III in the coastal lowland aborigines in Taiwan.34,38

Conceivably, the expression of Mi.III could tilt the host-pathogeninteraction with its unique glycophorin structure, and/or with a higherAE1 level. It remains an intriguing question as to whether the higherAE1 level of Mi.III could help alleviate major malarial symptoms suchas acidosis.39,40 Perhaps this blood group antigen has evolved not toavoid encountering malaria,34,37,38 but to assist host survival uponinfection.

8

8 8

AE1 AE1+GPA AE1+Gp.Mur0.0

1

1.2

1.4

1.6

n.s.

To

tal

AE

1 P

rod

uct

ion

(fo

lds

of

the

sin

gly

-exp

res

sed

AE

1)

A B

9

9

8

AE1 AE1+GPA AE1+Gp.Mur0.0

1.0

1.2

1.4

1.6

AE

1 s

urf

ace

exp

ress

ion

(fo

lds

of

the

sin

gly

-exp

ress

ed A

E1)

n.s.

pCIGpCIG-AEpCIG-AE + GPApCIG-AE + Gp.Mur

101

102

103

104

0

2

4

6

8

101

102

103

0

6

12

18

24

cou

nt

BRIC71-staining intensity BRIC71-staining intensity

cou

nt

Total AE1 Production AE1 Surface Expression

Figure 7. Gp.Mur exhibited similar functionality as GPA in promoting AE1 expression. (A) Both GPA and Gp.Mur promoted AE1 biosynthesis. AE1 was subcloned inpCIG, a bicistronic construct containing the reporter gene green fluorescent protein. pCIG-AE1 was expressed alone, or together with GPA or Gp.Mur, in HEK293 cells. Thetransiently transfected cells were fixed, permeabilized, and stained with anti-AE1, BRIC71. The degrees of BRIC71 staining in the fixed and permeabilized cells reflected therelative expression levels of AE1 on intracellular and plasma membranes. (B) Both GPA and Gp.Mur promoted similar degrees of AE1 surface expression. Intact HEK293 cellswere directly stained with BRIC71 after scraped off from culture plates. (Top) A representative BRIC71 histogram from flow cytometry. (Bottom) The intensities of BRIC71staining for different coexpression groups were compared with that for the singly expressed AE1 (set at 1). Data were averaged from 8 to 9 independent experiments (asindicated next to each bar), and expressed as mean � SE. P � .05 was deemed not significant (n.s.).





Acknowledgments

We thank Mackay Memorial Hospital blood bank specialists forguidance on the identification of Miltenberger blood group anti-gens; S. Chen for electron microscopy services (Mackay MemorialHospital); Y. Yang, S. Huang, Y. Deng, Z. Chen, and director DrC.-P. Chen for providing research assistance (Mackay MemorialHospital); Prof D. Chandanayingyong for providing anti-Mur andanti-Anek antibodies (Siriraj Hospital); Dr M. Uchikawa forproviding anti-Hil antibodies (Japan Red Cross Central BloodCenter); Prof D. Anstee for providing the R1.3 and BRIC71antibodies (BITS); and J. Rucker for technical assistance (JohnsHopkins University).

This work was supported by Taiwan National Science Council(95-2320-B-195-002-MY2; 97-2314-B-195-021) and by MackayMemorial Hospital intramural grants to K.H. Support for proteomicanalysis was provided through a grant from the National Heart,

Lung, and Blood Institute (contract N01-HV-28180) to J.E.V.E.and R.N.C.

Authorship

Contribution: K.H. designed and performed the research, analyzed thedata, and wrote the paper; N.C. performed the research and analyzed thedata; M.G. performed the mass spectrometry research and analyzed thedata; R.N.C. and J.E.V.E. designed the research and helped write thepaper; M.L. helped conceive the thesis; and D.B.F. designed andperformed the research, analyzed the data, and wrote the paper.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Kate Hsu, Mackay Memorial Hospital Trans-fusion Medicine Laboratory, 45 Min-Sheng Rd, Research Bldg616, Tamsui, Taiwan 251; e-mail: [email protected].

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online June 29, 2009 originally publisheddoi:10.1182/blood-2008-12-195180

2009 114: 1919-1928

FosterKate Hsu, Naiwen Chi, Marjan Gucek, Jennifer E. Van Eyk, Robert N. Cole, Marie Lin and D. Brian expression of band 3Miltenberger blood group antigen type III (Mi.III) enhances the

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Miltenberger blood group antigen type III (Mi.III) enhances the