Isolation of cDNA Clones and Complete Amino Acid Sequence of ...

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From the $&borntoire de Bioehimie Genktique, Unite Institut National de la Santi et de Ia ... National de la Sante et de la Recherche Medicale U91, Hiipital Henri Mondor, 94010 Criteil, France, and the 7Laboratoire ..... Universitk Paris V) for computer facilities, and to Christophe Tour- ... Steck, T. L. (1974) J. Cell Bwl. 62,l-19.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Cbemistt%,IUC,

Vol. 261. No. 1, Issue of January 5, p. 229-233.1986 Frtinted in U.S.A.

Isolation of cDNA Clones and Complete Amino Acid Sequence of Human ErythrocyteGlycophorin C* (Received for publication, June 24, 1985)

Yves Colin$,C k i l e Rahuel$, Jacqueline London$, Paul-Henri Romhg,Luc d’Aurioll, Francis Galibertll, and Jean-PierreCartronSII From the $&borntoire de Bioehimie Genktique, Unite Institut National de la Santi et de Ia Recherche MGdicale U76, Centre National de Transfusion Sanguine, 6 rue Alerandre Cabanel, F-75015 Paris, France, the §Service deBiochimie, Unite Institut National de la Sante et de la Recherche Medicale U91, Hiipital Henri Mondor, 94010 Criteil, France, and the 7Laboratoire d ’Himatologie Expirimentale, Hiipital St-Louis, F-75010 Paris, France

Two cDNA clones for glycophorin C, a transmembrane glycoprotein of the human erythrocyte which carries the blood group Gerbich antigens, have been isolated from a human reticulocytecDNA library. The clones were identified witha mixture of 32 oligonucleotide probes (14-mer) which have been synthetized according to the amino acid sequence Asp-Pro-GlyMet-Ala present in the N-terminal tryptic peptide of the molecule. The primary structureof glycophorin C deduced from the nucleotide sequence of the 460 basepair insert of the pGCW5 clone indicates that thecomplete proteinis a single polypeptide chain of 128 amino acids clearly organized inthree distinct domains. The N-terminal part (residues 1-57, approximately) which is N - and 0-glycosylated is connected to a hydrophilic C-terminal domain (residues 82-128, approximately) containing 4 tyrosineresidues by a hydrophobic stretch of nonpolar amino acids (residues 58-81, approximately) probably interacting with themembrane lipids and permitting the whole molecule to span the lipid bilayer. Northernblot analysis usinga 265-basepair restriction fragment obtained by DdeI digestion of the inserted DNA shows that the glycophorin C mRNA from human erythroblasts is approximately 1.4 kilobases long and is present in the human fetal liver and thehuman K562and HEL cell lines which exhibit erythroid features. The glycophorin C mRNA, however, is absent from adult liver and lymphocytes, indicating that this protein represents a new erythrocyte-specific probe which might be useful to study erythroid differentiation.

The erythrocyte membrane provides a well-defined system which has been usedas a model for the study of the molecular and structural organization of biological membranes (1-4). According to the model of Singer and Nicolson (5), the integral and peripheral membrane proteins are asymmetrically arranged in a fluid matrix of lipids. In the red cell, a more fixed protein framework might result from interactions between spectrin and other skeletal proteins located at the

* This investigation was supported by the Institut National de la SantL. et de la Recherche Mbdicale and the Centre National de la Recherche Scientifique. The costs of publication of this article were defrayed in part by the payment of page charges. This article must n t ” with 18 therefore be hereby marked ‘ ‘ ~ u e ~ ~ ein~ accordance U.S.C. Section 1734 solely to indicate this fact. (1 To whom all correspondence should be addressed.

cytoplasmic side of the lipid bilayer and the transmembrane proteins (4-6). Both peripheral and integral red cell membrane proteins can be easily separated by polyacrylamide gel electrophoresis in thepresence of sodium dodecylsulfate either in continuous (7) or, with better resolution, in discontinuous (8) buffer systems. The sialoglycoproteinsare stained specifically bythe periodic acid-Schiff (PAS1) reagent and therefore are easily identified among the separated membrane proteins. They have been extensively studied, since they are receptors for viruses, parasites, blood group antigens, and lectins (9-12). The four main monomeric specieswhich are presently identified under different nomenclatures (2,9,10,12) are, respectively, (i) glycophorin A (synonym PAS-2, MN-glycoprotein, or glycoprotein CY), (ii) glycophorin B (synonym PAS-3, Ssglycoprotein, or glycoprotein 61, (iii) glycophorin C ( s y n o n ~ PAS-2‘, component D, glycoprotein P), and (iv) glycoprotein y (or component E) also referred to as glycophorin C by Furthmayr (10). These molecules are sialic acid-rich components which carry most of the negative chargeof the red cell and represent 85,15,4, and 1%,respectively, of the totalPAS staining intensity. In the red cell membrane, glycophorin A and B, and presumably the two other glycoproteins, form homo- and heterodimers that are detectable as distinct bands after polyacrylamide gel electrophoresis and PAS staining. The best studied sialoglycoproteins are the glycophorins A and B (13-17), whereas onlylittle isknown about glycophorin C. So far, only the amino acid sequence of a N-terminal tryptic peptide of 47 amino acid residues from glycophorin C (apparent M,= 32,000) has been published (18).Much interest has focused recently on this glycoprotein for several reasons: fi) glycophorirrG as well as the o t h e ~ a i ~ ~ l o g l y c o proteins are the putative receptors for the penetration of Plasmodiumfakiparum merozoites into human red cells(19), (ii) rare genetic variants which are devoid of bloodgroup Gerbich antigens lackglycophorin C (and glycoprotein y) (20-221, (iii) glycophorin C was proposed as a possible membrane attachment site, via protein 4.1, for the erythrocyte skeleton proteins, and (iv) red cells from homozygous patients with hereditary ellipocytosiswho lack protein 4.1 are deficient in glycophorin C (23) and have strongly depressed Gerbich antigens? In order to obtain the complete primary sequence of glycophorin C, we have clonedits cDNA from a human reticulocyte The abbreviations used are: PAS, periodic acid-Schiff, bp, base pair. ‘D. Sondag, N. Alloisio, D. Blanchard, M.T. Ducluzeau, L. Colonna, D. Bachir, C. Bloy, J. P. Cartron, and J. Delaunay, manuscript submitted for publication.

229

230 cDNA library by using a mixture of 32 synthetic oligonucleotides (14-mer) as hydridization probes. MATERIALS ANDMETHODS

RNA Isolation and Fractionation-RNA was isolated from human reticulocytes, spleen, liver, and cell lines as previously described (24). Poly(A)' RNA was then purified by oligo(dT)-cellulose chromatography (Collaborative Research, Inc., Lexington, MA) according to Aviv and Leder (25) and furtherfractionatedon a 5-20% (w/v) sucrose gradient. Briefly, 200 pg of the poly(A)+-containingRNA was heated for 3 min at 70 "C, cooled in ice, and layered on top of the 12mi sucrose gradient prepared in 10 mM Tris-HC1, pH 7.5, 0.1 mM EDTA. After centrifugation at 33,000 rpm for 16 h at 4°C in a Beckman SW 41 rotor, the gradient was collected (200 plftube) using an ISCO density gradient fractionator. Fractions with a sedimentation above 12 S were pooled, ethanol-precipitated, and used for cDNA cloning. Cloning of Double-stranded cDNA-The reticulocyte mRNA was used to direct the synthesis of double-stranded cDNA according to the procedure described by Wickens et al. (26). After treatment with SI nuclease (Boehringer-Mannheim, F. R. G.), the double-stranded cDNAwas size-fractionated by Sepharose CL-4B chromatography (Pharmacia, Uppsala, Sweden) and molecules longer than 400 bp were inserted in the PstI siteof the plasmid vector pBR322 using the homopolymeric tailing and hybridization method of Michelson and Orkin (27). Escherichia coli strain RR1 was transformed with the recombinant plasmids according to Hanahan(28) and colonies growing in the presence of tetracycline (15 pg/mi) were selected. A permanent, ordered collection of 18,000 recombinant clones was established and screened with a 32P-labeledcDNA complementary to 9 S mRNA from reticulocytes to identify the globin-containing clones. A final ordered collection of 12,000 recombinant plasmids without globin inserts was then obtained and stored at -30 "C in 96-microwell culture dishes containing freezing medium (29) until used. Oligonucleotide Probes-Four mixtures of 8 oligonucleotides (14-mer) deduced from the known sequence Asp-Pro-Gly-Met-Ala from tryptic fragment of glycophorin C (18) were synthesized according to the solid-phase phosphotriester technique (30, 31) using the "SAM one" automated DNA synthesizer system (Biosearch). Deprotected oligonucleotides were purified by electrophoresis in 20% (wtv) poIyacrylamide denaturating gels. The following 3'-5'-sequences were obtained: C T ~ G G ~ C C ~ T A C(probes C G mixture 11, C ~ G G ~ C ~ T A C(probes C G mixture 2), C ~ G ~ C ~ T A C (probes mixture 3),C ~ G ~ C C ~ T A C(probes C G mixture 4). For the screening of the library, a mixture of all the 32 probes was labeled at the 5'-end with [y3'P]ATP (3000 Ci/mmol, Amersham, England) using the T4 polynucleotide kinase (Amersham, England) according to standard procedures (32) and was finally purified by chromatography through a Sephadex G-50 column. The specific activity of the labeled oligonucleotides was 5 X lo6 cpm/pmol. Library Screening-Replicas of the recombinant DNA plasmids on Whatman 540 filter papers (11 X 11cm) were made from LB agar replicas of the ordered colonies in 96-well microtiter plates (Linbro) according to Gergen et al.(29), except that thefilters were additionally treated with 20 mg/ml proteinase K (Merck, Darmstadt, F. R. G.) in Tris-HC150 mM, pH 7.5, EDTA 5 mM, sodium dodecyl sulfate 0.5% (w/v) for 2 h at 37 "C followed by a final wash in 95% ethanol and drying. The filters were then hybridized with the 5'-end-labeled oligonucleotide mixture (lo6 cpm/filter) for 16 h at 40 "C in 10 ml/ filter Tris-HC1, 90 mM, pH 8.3, containing 900 mM NaCl, 6 mM EDTA, 0.5% (w/v) Nonidet P-40 (Shell Co.), 100 pg/ml yeast tRNA (Boehringer-Mannheim, F. R. G , ) , 100 pg/ml salmon sperm DNA (Sigma) and were washed four times, at room temperature, with 6 X SSC (1 X SSC = 150 mM NaCl, 15 mM sodium citrate, pH 7.0), for 20 min each, followed by twoadditional washes at 41 "C, 20 min each, in the same buffer. More stringent washes were performed in 1 X SSC at 45 'C for 15 min. The filter papers were finally dried and exposed to x-ray films (Fuji RX, Japan) with an intensifying screen (Cronex Lightning-Plus, E. I. du Pont de Nemours & Co.). Plasmid DNA I s o ~ t w nand DNA S e q ~ ~ i n g - P l a s DNA ~ d was isolated as described by Birnboim and Doly (33). Restriction fragments from two recombinant plasmids pGCA5 and pGCW5 were 3'end-labeled with E. coli DNA polymerase I, Klenow fragment (BiotecPromega, Madison, WI) and theappropriate [a-32P]dNTP (3000 Ci/ mmol, Amersham, England) or with the terminal deoxynucleotidyltransferase (Amersham, England) and [a-"P]ddATP (5500 Ci/mmol,

Amersham, England) (34,35). After a secondary cleavage with a restriction enzyme (see Fig. 1) or strand separation, the labeled DNA fragments were sequenced according to Maxam and Gilbert (35). RNA Blot Analysis-Size fractionation of RNA preparations on a denaturing methylmercury hydroxide/agarose gelwas carried out according to Chandler et a1 (36) except that samples were made up in 10 mM methylmercury hydroxide (Ventron GmBH, F. R. G.) and applied to a gel containing 1.3% (w/v) agarose and 10 mM methylmercury hydroxide. After electrophoresis (35 mA/gel, overnight) the gel was washed sequentially in 100 ml of 10 mM phosphate buffer containing 1.1 M formaldehyde and 5 mM 2-mercaptoethanol for 40 min, then in the same buffer with 7 mM iodoacetate instead of 2mercaptoethanol for 30 min, and finally in phosphate buffer with formaldehyde alone for 20 min (37). The RNA was then transferred onto a n i t r ~ l l u l filter ~ e (BA85, Schleicher & Schull, Dassel, F. R. G,) or onto a Gene-Screen membrane (New England Nuclear) according to Thomas (38) and hybridized to a nick-translated 52Plabeled 265-bp DdeI-DdeI restriction fragment (39) from recombinant plasmid pGCW5. Prehybridization of the filters was performed in a plastic bag at 42 "Cfor 6 h in 10 ml of 50% (w/v) deionized formamide, 5 X SSPE (1 X SSPE = 180 mM NaCl, 10 mM NaHzP04, pH7.7, I mM EDTA), 2 X Denhardt (1 X Denhardt = 0.02% (w/v) each ficoll, polyvinylpyrrolidone, and bovine serum albumin), 200 pg/ml denatured salmon sperm DNA, 0.1% (w/v) sodium dodecyl sulfate, and 10% (w/v) dextran sulfate (Pharmacia, M,= 500,000). Hybridization with the nick-translated probe (5 x 10' cpm/ml) was done in the same bag at 42 "C for 16 h. The filters were then washed twice in 2 X SSPE; 0.1% (w/v) sodium docecyl sulfate for 10 min at room temperature, twice in the same buffer at 65 "C fox 10 min, and then dried and exposed to x-ray films as above. RESULTS ANDDISCUSSION

Library Screening and Characterization of Glycophorin C cDNA Clones-One hundred and thirty Whatman 540 filter papers (96 colonies/filter) obtained by transfer of the human reticulocyte cDNA library depleted in globin clones (12,000 colonies) were hybridized with the synthetic probes as described under "Materials and Methods.'' A mixture of 32 '*Plabeled 14-mer oligonucleotideprobes was used,the structure of which was deduced from the amino acid sequenceAsp-ProGly-Met-Ala sitio ions 19-23) of the N-terminal peptide of C G glycophorin C (18). This sequence was selected since it is partially repeated in the N-terminal peptide (positions 3842) and it may be expected that the mixture of synthetic oligonucleotides will contain the probes reacting with each region of the correspondingcDNA,which, in turn, might favor the detection of a specific hybridization signal. Two colonies givinga strong radioactive signal even after the final washings at 45 "C in 1 x standard saline citrate were selected. Recombinant plasmids, pGCA5 and pGCW5, from these two colonies were prepared and shown to contain 450- and 460bp inserts, respectively. Preliminary restriction map analysis using PstI, LldeI, HpaII, RsaI, and MboII enzymes suggested that both clones were identical, and appropriate restriction fragments 32P-labeledat their 3'-end were used for nucleotide sequencing.Sequence determination confvmed that the pGCA5 and pGCW5 wereidentical and indicated that the 10bp difference in length between the inserted DNA from the two plasmids was related to the different number of nucleotides present in the tail of poly (C). The partial restriction map of the recombinant plasmid pGCW5 insert is shown in Fig. 1, and most of its nucleotide sequence was determined on both DNA strands (Fig. 2). The nucleotide sequence of the insert from plasmid pGCW5 was confirmed by sequencing s ~ u l ~ n e othe ~ linsert y from plasmidpGCA5 (not shown). It is clear that among the eight synthetic oligonucleotides present in probes mixture 3, one is fully complementary to the nucleotidesequence 55-68of the cDNA(Fig.2) and another one has only one mismatch with the sequence 111125. Interestingly, the former probe contains as much as 71%

pGC ws

FIG. 1. Partial restriction map analysis of the eDNA insert of pGCW5 clone and strategyused for DNA sequencing. Fdt arrows indicate direction of sequencing following S'-end-labeling of the restriction fragments.Partinl arrow indicates nucleotide sequence of the plasmid vector towards an AvaII restriction site. Most of the sequence was determined on both DNA strands. The 41-bp restriction fragment RsaI-PstI on the right side contains 15 cDNA nucleotides, among which 9 belong to the coding sequence, followedby a streteh of 26 C. The nucleotide sequence ofthis fragment was confirmed by sequencinga DdeI-AvaII fragmentof the recombinantplasmid pGCA5 (not shown).

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deduced amino acid sequence of glycophorin C is shown above the nucleotide sequence and is numbered beginning with 1 for the first aminoacid of the matureprotein. ~ ~ e r l positions i ~ d indicate regionsofpreviouslyunknownaminoacidsequence. X shows the unique site for N-glycosylation. X X indicates the positions where a single nucleotide exchange, possibly related to a polymorphism, modifies the amino acid sequence published by other investigators (18). Partial underline indicates the regions chosen for constructing the synthetic oligonucleotide probes.The first six nucleotides indicated in parentheses are deduced from amino acid sequence and were not present in thepGCW5 clone.

G+C, which presumably generates by hybridization a thermodynamically stable duplex. The amino acid sequence deducedfrom the nucleotide sequence data shownin Fig. 2 demonstrates the presence of an open reading frame over 378 nucleotides before a termination codon was found. Comparison betweenthese results and thepartially known amino acid sequence of glycophorin C indicates a very good agreement, suggesting that the pGCW5 clone effectively codes for this protein and notfor glycophorins A or B which havea different N-terminal sequence (13-17). However, the DNA sequence analysis reveals tryptophan residues at positions 12 and 44 instead of the serine and glycine found by protein sequencing of glycophorin C (18). Furthermore, an additional glycine residue was detected between the asparagine at position 46

and thearginine assigned to position 47 by protein sequencing (18). The differences in amino acids observed at position 12 and 44 might well result from a true polymorphism corresponding to a single base substitution in the DNA, thereby replacing a serine (codon TCN) and a glycine (codon GGN), respectively, by a tryptophan (codon TGG) residue. On the contrary, the omission of the glycine residue may probably be attributed to an error in protein sequencing. The nucleotide sequence shows (Fig.2) that the 5'-end region of the cDNA upstream from the protein codingsequence is lacking in pGCW5 DNA,since the first nucleotides identified are those coding forthe serine residue at position 3 in the protein. Since the two first amino acids of glycophorin C are methionine and tryptophan, respectively, the nucleotide sequence at the beginning of the cDNA coding forthe mature protein should be ATG-TGG.The 3'-end region of the pGCW5 insert is also incomplete,but enough nucleotidesare present in thecloned DNA to deduce that the complete glycophorin C molecule consists of a single polypeptidechain of 128 amino acids. Molecular Structure and Function of Glycophorin C in Membrane-There is a reasonable agreement between the amino acid compositionof a partially purified glycophorin C preparation (10) and primary sequence of the protein deduced from DNA sequencing. However amino acids such as Val, Met, and Ile were underestimated, whereas the Glu(G1n) and Gly content was slightly overestimated. The calculated molecular mass of the protein backbone is close to 14 kDa. Considering that glycophorin C carries as an average 12 0-glycosidically A ~ 6 T C linked sialotetrasaccharides and one asparagine-linked carbohydrate chain (1&), the calculated molecular mass of the glycoprotein should reach about 28 kDa. Examination of the complete amino acid sequenceof glycophorinC (Fig. 2) reveals an organi~tion of the molecule in three distinct domains, a finding previously described forthe overall structure of glycophorin A within the red cell membrane(4, 13,40). The first domain, ranging approximately from residues1 to 57 comprises the hydrophilic N-terminal glycosylated part of the molecule which includes the tryptic glycopeptide previously is an extracellular segment ~ Tcharacterized ~ ( ~ ~(18).~ This ' carrying several si~otetrasaccha~des 0-linked to serine and threonine residues as well as theunique consensus site of Nglycosylation Asn-X-Thr/Ser (41) to which the alkali-stable carbohydrate chain present in the molecule, as detected by sugar co~position(10) and lectin binding studies (42), is covalently linked via the asparagine residue at position 8. No other potential site for N-glycosylation was detected. It is likely that thisdomain carries the blood group Gerbich antigens (22) as well as the receptors for €'. fakiparum (19). A second domain is defined by a long stretch of nonpolar amino acids (residues 58-81, approximately) and presumably represents a hydrophobic intr~embraneoussegment. Secondary-structure prediction using the computer program described by Garnier et al. (43) indicates that this nonglycosylated hydtophobic segment should have the cy-helical structure. Moreover, calculation of the hydrophobicity plot according to Kyte and Doolittle (44) indicated clearly that glycophork C contains a single hydrophobic domain located between residues 58 and 81 that could span the membrane. These results are in striking contrast with a similar calculation predicting predominantly a @-sheetsecondary structure for the intramembraneous segment (residues 73-92)of glycophorin A. This unexpected difference between glycophorins A and C certainly reflects a difference in amino acid composition of the membrane-penetratingmgionas well as a difference in the hydrophobic environment of the helix (45). Very recently an insoluble tryptic peptide (about 45 amino acids

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FIG. 3. Northern blot analysis of human mRNA from different tissueand cell extracts. Poly(A)+RNA (5 pg) denatured in methylmercury hydroxide were electrophoresed in 1.3% agarose gel containing 10 mM methylmercury hydroxide essentially as described by Chandler et al. (36). After transfer to nitrocellulose (lanes 1-6) or to Gene-Screen membrane (lanes 7 and 8) according to Thomas (38), the RNA blots were hybridized with a 32P-labeled265-bp DM-DdeI fragment prepared from the pGCW5 clone (see Fig. 2), washed, and autoradiographed 48 h on Fuji-RX film. Lanes 1 and 2, erythroblasts from adult spleen (2 specimens); lane 3, fetal liver; lane 4, adult liver; lane 5,K562 erythroid cell line; lane 6, HEL erythroid cell line; lanes 7 and 8, T-lymphocyte cell lines (Jurkat and AG103).

long) containing this region has been isolated from a purified glycophorin C preparation (46). The amino acid sequence of this peptide was again in verygood agreement with the primary structure deduced from DNA analysis, adding further evidence that the pGCW5 clone was coding for glycophorin C. The C-terminal end of glycophorin C, ranging approximately from residue 82 to 128, defines the third domain of the molecule which is composed of many hydrophilic amino acid residues. Among the first residues following the hydrophobic intramembraneous domain, there is a cluster of several basic amino acids (Arg, Lys, His) which might be in close vicinity with the polar groups of the phospholipid molecules along the cytoplasmic side of the lipid bilayer. Examination of the amino acid sequence reveals also that the four tyrosine residues present in glycophorin C are located within the Cterminal domain and thisprobably explains why the molecule is not labeled whenintact red cells are enzymatically radioiodinated by the lactoperoxidase procedure (47). The fact that glycophorin C is, however, heavily labeled from membrane preparations or following membrane extraction with l-butanol? is in agreement with the expected position of the four tyrosines inside the cell and the transmembrane orientation of the polypeptide chain. Previous studies (18) have already provided evidence for some structural similarity between the N-terminal domains of glycophorin C (residues 25-28 and 29-33) and glycophorin A and B (residues 1-4 and 8-12, respectively) as well as for the identity of the amino acid residues at position 17-22 and 36-41of glycophorin C itself. In addition, it is shown here D. Blanchard and J. P. Cartron, unpublished data.

that the sequence Ala-Gly-Val-Ile occurs within the intramembraneous domains of both glycophorin A and C. Some rare individuals are deficient in glycophorin C (and glycoprotein y) and about 10% of their circulating red cells are elliptocytic (20). Further studies using our cDNA probe should clarify the geneticbackgroundresponsiblefor the Gerbich-negative phenotype in individuals with elliptocytic and nonelliptocytic red cells. RNA Transfer Blot Analysis-A265-bpDa!eI-DdeI fragment prepared from the insert of pGCW5 was usedas hybridization probe in the RNA transfer blot analysis of human poly(A)+ RNA from adult erythroblasts, adult andfetal liver, K562 and HELcell lines which exhibit erythroid features (48, 49), and two human T-lymphocyte cell lines (Jurkat and AG103). As shown in Fig. 3, a single band of about 1.4 kilobases was observed in all mRNA preparations except those from adult liver and T-lymphocyte cell lines. These results strongly suggest that the expression of glycophorin C is restricted to erythroid tissues. Obviously the mRNA is longer than theglycophorin C coding sequence (384 nucleotides) and further studies will establish whether it encodes for a leader peptide upstream from the mature membrane-bound protein and contains a long stretch of untranslated sequences. Acknowledgments-We are grateful to Dr. Andreas Tsapis (Hspital Saint-Louis, Pans) for the generous gift of lymphocyte mRNA from the T cell lines Jurkat and AG103, to Claude Mugnier (C. I. T. I. 2, Universitk Paris V) for computer facilities, and to Christophe Tournamille for valuable technical assistance. REFERENCES 1. Singer, S. J. (1974) Annu. Rev. Biochem. 43,805-833 2. Steck, T.L. (1974) J. Cell Bwl. 62,l-19 3. Marchesi, V. T., Furthmayr, H., and Tomita, M. (1976) Annu. Rev. Biochem. 46,667-698 4. Marchesi, V. T. (1979) Semin. Hematol. 16,3-20 5. Singer, S. J., and Nicolson, G. L. (1972) Science176,720-731 6. Marchesi, V. T. (1983) Blood 61,l-11 7. Fairbanks, G.,Steck, T. L., and Wallach, D. F. H. (1971) Biochemistry 10,2606-2617 8. Laemmli, U. K. (1970) Nature 227,680-685 9. Dahr, W., Uhlenbruck, G., and Knott, H. (1975)J.Zmmunogenet. OX^) 2.87-100 10. Furthmayr, H. (1978) J. Supmmol. Struct. 9,79-95 11. Anstee, D. J. (1980) in Zmmunobiobgy of the Erythrocyte (Sandler, S. G., Nusbacher, J., and Schanfield, M. s.,eds) pp. 6798, Alan R. Liss Inc., New York 12. Anstee, D. J. (1981) Semin. Hemutol. 18,13-31 13. Tomita, M., Furthmayr, H., and Marchesi, V. T. (1978) Biochemistry 17,4756-4770 14. Dahr, W., and Uhlenbruck, G. (1977) Hoppe-SeyZer’s 2. Physiol. Chem. 369,835-843 15. Lisowska, E., and Wainiowska, K. (1978) Eur. J. Biochem. 88, 247-252 16. Furthmayr, H. (1978) Nature 271,519-523 17. Dahr, W., Beyreuther, K., Steinbach, H., Gielen, W., and Kriiger, J. (1980) Hoppe-Seyler’s 2. Physwl. Chem. 361,895-906 18. Dahr, W., Beyreuther, K., Kordowicz, M., and Kriiger, J. (1982) Eur. J. Biochem. 126,57-62 19. Pasvol, G.,Anstee, D. J., and Tanner, M. J. A. (1984) Lancet I, 907 20. Anstee, D. J., Parsons, S. F., Ridgwell,K., Tanner, M. J. A., Merry, A. H., Thomson, E. E., Judson, P. A., Johnson, P., Bates, S., andFraser, I. D. (1984) Biochem.J. 218,615-619 21. Anstee, D. J., Ridgwell, K., Tanner, M. J. A., Daniels, G. L., and Parsons, S. F. (1984) Biochem.J. 221,97-104 22. Dahr, W., Moulds, J., Baumeister, G., Moulds, M., Kiedrowski, S., and Hummel, M. (1985)Bwl.Chem. Hoppe-Sey&r 366, 201-211 23. Mueller, T. J., and Morrison, M. (1981) in Erythrocyte Membranes 2: Recent Clinical and Experimental Advances (Kruckenberg, W. C., Eaton, J. W., and Brewer, G. J., eds) pp. 95112, Alan R. Liss, Inc., New York

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