Cloning of glycoprotein D cDNA, which encodes the ... - Europe PMC

Proc. Natl. Acad. Sci. USA Vol. 90, pp. 10793-10797, November 1993 Medical Sciences

Cloning of glycoprotein D cDNA, which encodes the major subunit of the Duffy blood group system and the receptor for the Plasmodium vivax malaria parasite ASOK CHAUDHURIt, JULIA POLYAKOVAt, VALERIE ZBRZEZNAt, KENNETH WILLIAMSt, SUBHASH GULATI§, AND A. OSCAR POGOt¶ tLaboratory of Cell Biology, Lindsley F. Kimball Research Institute of the New York Blood Center, New York, NY 10021; *Protein and Nucleic Acid Chemistry Facility, Howard Hughes Medical Institute, Yale University, New Haven, CT 06510; and §Department of Medicine, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021 Communicated by Louis H. Miller, August 16, 1993 (received for review July 27, 1993)

units. A glycoprotein, named gpD, of 35-45 kDa is the major subunit of the protein complex and has the antigenic determinants defined by anti-Fya, anti-Fyb, and anti-Fy6 antibodies (5, 6). The characterization, at the molecular level, of this protein will be crucial in finding its function on the erythrocyte membrane, in understanding the parasite-erythrocyte recognition process, and eventually in resolving the molecular mechanism of parasite invasion. This report describes the isolation, sequence analysis, and tissue expression of a mRNA encoding gpD. 1

cDNA clones encoding the major subunit of ABSTRACT the Duffy blood group were isolated from a human bone marrow cDNA library using a PCR-amplifled DNA fragment encoding an internal peptide sequence of glycoprotein D (gpD) protein. The open reading frame of the 1267-bp cDNA clone indicated that gpD protein was composed of 338 amino acids, predicting a Mr of 35,733, which was the same as a deglycosylated gpD protein. Portions of the predicted amino acid sequence, matched with six CNBr/pepsin peptides obtained from affinity-purified gpD protein. In ELISA analysis, an anti-Duffy murine monoclonal antibody reacted with a synthetic peptide deduced from the cDNA clone. Hydropathy analysis suggested the presence of 9 membrane-spanning a-helices. In bone marrow RNA blot analysis, the gpD cDNA detected. a 1.27-kb mRNA in Duffy-positive but not in Duffynegative individuals. It also identified the same size mRNA in adult kidney, adult spleen, and fetal liver; in brain, it detected a prominent 8.5-kb and a minor 2.2-kb mRNA. In Southern blot analysis, gpD cDNA identified a single gene in Duffypositive and -negative individuals. Duffy-negative individuals, therefore, have the gpD gene, but it is not expressed in bone marrow. The same or a similar gene is active in adult kidney, adult spleen, and fetal liver of Duffy-positive individuals. Whether this is true in Duffy-negative individuals remains to be demonstrated. A GenBank sequence search yielded a significant protein sequence homology to human and rabbit interleukin-8 receptors.

MATERIALS AND METHODS Partial Amino Acid Sequence Analysis of gpD Protein. The purified protein from Fy(a-b+) human erythrocytes (5) was alkylated and cleaved with cyanogen bromide (CNBr) as explained (7). Pe-1 peptide was obtained by sequencing the nonfractionated CNBr digest using the o-phthalaldehyde blocking reagent (8) (see legend, Fig. 1B). Pe-5 peptide was the partial sequence of the onily fragment (-4 kDa) that separated very well from the CNBr digest run on the threelayer SDS/PAGE system (9). After the run, the peptide fragment was electroblotted onto ProBlott (Applied Biosystems) and sequenced (7). Another aliquot was digested with pepsin (50: 1 ratio) at 37°C overnight, and the fragments were separated by reverse-phase HPLC using a Vydac C18 column. Pe-2, Pe-3, Pe-4, and Pe-6 peptides, which were the few pepsin peptides yielded by reverse-phase HPLC, were sequenced. Applied Biosystems protein/peptide sequencer, model 470 or 477, was used according to the manufacturer's recommendations. Primer Design and PCR. The nucleotide sequence of the primers (23-mer each) was deduced from the N-terminal and C-terminal amino acid sequences of Pe-5 peptide (see legend, Fig. 1B). Bases were chosen according to the codon preference described by Lathe (10), and deoxyinosine (I) was incorporated at the position where degeneracy exceeded >3-fold, except toward the 3' end. Primer A (sense) was specific for residues 245-252 (see Fig. 1B) and consisted of 12-fold degeneracy 5'-ATGAAYATHYTITGGGCITGGTT (where Y = C or T; and H = C, T, or A). Primer B (antisense) was specific for residues 261-268 (see Fig. 1B) and consisted of 32-fold degeneracy 5'-ACIAGRAARTCIAGICCIARNAC (where R = A or G; and N = G, A, T, or C). First-strand cDNA was synthesized from Fy(a-b+) phenotype mRNA using the preamplification kit from BRL and oligo(dT) as primer. For enzymatic amplification, cDNA, primer A, primer B, and Taq polymerase (Stratagene) were

The Duffy blood group system consists of two principal antigens Fya and Fyb produced by FY*A and FY*B alleles. Antisera anti-Fya and anti-Fyb defined four phenotypes, Fy(a+b-), Fy(a-b+), Fy(a+b+), and Fy(a-b-) (1). Neither antiserum agglutinates Duffy Fy(a-b-) cells, the predominant phenotype in Blacks. Antisera defining the other phenotypes, Fy3, Fy4, and Fy5, are very rare. A murine monoclonal antibody, anti-Fy6, defined another Duffy antigenic determinant present in all Duffy-positive cells but absent in Fy(a-b-) cells (2). Blacks with Fy(a-b-) erythrocytes cannot be infected by the human malaria parasite Plasmodium vivax (3). These cells are also resistant to the in vitro invasion by Plasmodium knowlesi, a simian parasite that invades Fy(a+b-) and Fy(a-b+) human erythrocytes (4). Receptors for erythrocyte invasion by these parasites, therefore, are related to the Duffy blood group system. Using the anti-Fy6 monoclonal antibody, we have developed a procedure for purification of Duffy antigens in human erythrocytes (5). Duffy antigens appear to be multimeric erythrocyte-membrane proteins composed of different sub-

Abbreviation: gpD, glycoprotein D. lTo whom reprint requests should be addressed. 'The sequence reported in this paper has been deposited in the GenBank data base (accession no. U01839).

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Human mRNA and DNA Isolation. Poly(A)+ RNA was isolated as explained (11) and by using the Invitrogen isolation kit. mRNAs from Caucasian adult liver, spleen, kidney, brain, and fetal liver, as well as erythroleukemia cells K-562, were obtained from Clontech. DNA was obtained from peripheral blood leukocytes of the four Duffy phenotypes by a slight modification of a published procedure (12). RNA-Blot Analysis (Northern). Poly(A)+ RNAs were run on formaldehyde/agarose gel and transferred onto Hybond-N+ nylon membranes (Amersham). They were hybridized in QuickHyb (Stratagene) and washed according to the manufacturer's instructions. DNA-Blot Analysis (Southern). All restriction enzyme digestions were done according to the conditions suggested by the supplier (New England Biolabs). Digested DNA was size-fractionated on 0.8% agarose gel and blotted as described for Northern analysis. Hybridization in QuickHyb solution was carried out at 68°C for 1 hr according to the manufacturer's instructions. Construction and Screening of a cDNA Human Bone Marrow Library. A mixture of mRNA of several Fy(a-b+) individuals, the BRL Superscript Choice system, and oligo(dT) as a primer was used to prepare cDNA. The cDNA was ligated into AZAP II vector and packaged with Gigapack Gold (Stratagene) extract. About 1.9 x 106 unamplified cDNA clones were screened with the 32P-labeled probe described above. cDNA inserts in pBluescript were isolated by the plasmid-rescue method according to manufacturer's protocol. Both DNA strands were sequenced by using vector primers and by primers designed from the sequenced regions of the transcript. Primer Extension. A 32P-labeled 24-mer antisense primer from nt 57-80 of the coding strand (Fig. 1B) was extended on Fy(a-b+) mRNA using a preamplification kit (BRL), and the products were separated on a 6% sequencing gel. The M13 sequence ladder was used to determine sizes of products.

1267

FIG. 1. (A) Schematic representation and partial restriction sites of the two longest gpD protein cDNA clones. The overlapping of Fyb81 and Fyb71 and the combination of the two clones (Fyb7l-81) are shown. An, poly(A) tail. (B) Nucleotide and amino acid sequences of the combined Fyb71-81 cDNA encoding gpD protein. Amino acid residues are numbered on left; nucleotide positions are numbered on right. Positions of peptides that match predicted amino acids are shown by solid single lines. The two potential carbohydrate-binding sites to asparagine residues are shown by arrows. The third glycosylation site, the asparagine at position 37, is unlikely to occur because it is followed by aspartic acid (14). Double underlining at the 5' end indicates the sequence used to primer-extend the 5' end; double underlining at the 3' end is the consensus poly(A) addition sequence. The CNBr peptide (Pe-1) sequenced with the o-phthalaldehyde reagent was P(L/A)FRIQL(S/C)PGXPVLAQ. Sequences of pepsin peptides separated on HPLC were FSIVV (Pe-2), FAQAL (Pe-3), XXVGIX (Pe-4), and PSLPLPEGR (Pe-6). X denotes illdefined residues in the corresponding cycle, and L/A and S/C mean either residue. The sequence of CNBr peptide separated on SDS/ PAGE (Pe-5) was [M]NILWAWFIFWWPHGVVLGLDFLV (residues used to design primers A and B are in italics; residues used to design the probe for cDNA-library screening are in boldface letters; the methionine added to CNBr Pe-5 peptide is in brackets).

incubated in a Perkin-Elmer thermal DNA cycler. The amplification product of expected size (72 bp) was subcloned in pBluescript-SK vector (Stratagene). The deduced amino acid sequence of the insert matched the sequence of Pe-5 peptide (Fig. 1B). From the sequence WFIFWWPH of peptide Pe-5,

RESULTS AND DISCUSSION Partial Amino Acid Sequence of gpD Protein. In this study the gpD protein was purified from Fy(a-b+) erythrocytes. The N terminal of this highly hydrophobic protein was blocked. The obstacles to obtain internal sequences were its insolubility at neutral pH without detergents and its tendency to aggregate. After trial and error, the protein was digested with CNBr or pepsin, and three approaches were performed for amino acid sequencing. (i) The unfractionated CNBr peptide mixture was sequenced by blocking with the ophthalaldehyde reagent those peptides that lacked a proline in the earliest cycles of sequencing; the procedure yielded Pe-1 peptide of 16 residues (see legend for Fig. 1B). (ii) A fragment of the CNBr cleavage mixture (of =4 kDa) that resolved well on SDS/PAGE was sequenced and produced Pe-5 peptide of 23 residues. (iii) Four short pepsin fragments that eluted as single peaks from a reverse-phase HPLC column were sequenced and yielded Pe-2 and Pe-3 of five residues each, Pe-4 of three residues, and Pe-6 of nine residues (pepsin digestions at 100: 1 ratio and 4°C for 30 or 60 min did not generate larger peptides). RNA Amplification by PCR. Pe-5 peptide was the most promising for generating a probe for the selection of gpD protein clones. Pe-2, Pe-3, Pe-4, and Pe-6 peptides were too short for PCR amplification, whereas the Pe-1 peptide was larger, but it had three ill-defined residues (see legend for Fig. 1B). Because the Pe-5 peptide was produced by CNBr


cleavage, a methionine was included at the N terminus to increase the length of the peptide to 24 residues. Two degenerated primers for amino acids 245-252 (primer A) at the N terminus and amino acids 261-268 (primer B) at the C terminus were chemically synthesized and used to amplify the coding sequence of Pe-5 peptide from pooled human bone marrow mRNA of Fy(a-b+) individuals. The expected PCRamplified product of 72 oligonucleotides was cloned and sequenced; it had an open reading frame of the exact sequence of the Pe-5 peptide (data not shown). The 24-mer oligonucleotide probe having codon usage for amino acids 251-258 successfully identified true gpD protein cDNA clones. Nucleotide Sequence of the gpD Protein cDNA Clones. A nonampkified human bone marrow cDNA library constructed from pooled mRNA of Fy(a-b+) individuals was screened with the 24-mer probe. Of 1.9 x 106 recombinant AZAP II phage, four positive clones were selected and sequenced. All clones had overlapping sequences but did not fully extend gpD cDNA. Clone Fyb8l of 1085 bp, the only clone that included the ultimate 5' end, and clone Fyb7l of 1083 bp, which extended from nt 185 to the poly(A)+ tail, were the longest. Clone Fyb3l of 989 bp and clone Fyb82 of 726 bp extended from nt 275 and 527, respectively, to the poly(A) tail. Combination of clone Fyb8l with any other clone generated the full-length cDNA of gpD protein. Fig. 1A shows the overlapping and combination of the two longest clones. The joined Fyb7l-81 clone predicted an open reading frame that started at position 176 and stopped at position 1192, encoding a polypeptide of 338 amino acid residues (Fig. 1B). A GenBank sequence search (release 77) at the National Center for Biotechnology Information using the BLAST network service yielded a significant protein sequence homology to human and rabbit interleukin-8 receptors and quasi-total nucleotide sequence homology with a human hippocampus cDNA clone HHCMF86 (see below). Verification of the 5' end of clone Fyb8l was done by primer extension. The extended product of an antisense primer (from position 57 to 80, Fig. 1B) yielded a sequence of 80 nt that matched exactly with the predicted size at the 5' end of the Fyb8l clone (data not shown). At positions 176-178, the initiation codon is not embedded within a sequence context most frequently associated with mammalian translation initiation (13). We assumed, however, that it is the true initiation codon for the following reasons: (i) it is the only ATG codon at the 5' end; and (ii) from the first methionine residue, the polypeptide encoded by the combined clones has the same molecular mass as that of deglycosylated gpD protein (6). At the 3' end, clone Fyb7l-81 included the consensus poly(A) addition signal AATTAAA (Fig. 1B). Both clones had a perfect nucleotide sequence match, except at the 5' end, where several base substitutions yielded six different amino acid predictions. These discrepancies were not a sequencing error because both DNA strands were sequenced several times. They were a consequence of protein heterogeneity because the cDNA library was constructed from mRNA of several Fy(a-b+) individuals. To establish that clone Fyb7l-81 had a coding sequence specific to gpD protein, we compared the translated sequence with the partial amino acid sequence data obtained from the six peptides described in the legend of Fig. 1B. Portions ofthe predicted amino acid sequence matched with Pe-1 peptide sequenced by the o-phthalaldehyde reagent, with four peptides isolated by reverse-phase HPLC (Pe-2, Pe-3, Pe-4, and Pe-6 peptides), and with the Pe-5 peptide isolated by SDS/ PAGE. However, two of a total of 62 residues did not match. Thus, the residues at position 92 and 327 were tryptophan by codon sequence analysis, but they were isoleucine and arginine, respectively, by amino acid sequence determination.

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10795

Because tryptophan is a very unstable residue, the discrepancies may be a technical problem in amino acid sequence analysis. On the other hand, they may be due to the heterogeneity of gpD protein. Additional evidence that clone Fyb7l-81 encoded gpD protein was provided by RNA blot and ELISA analysis. Probe Fyb8l did not detect any bone marrow mRNA in Duffy-negative individuals, but it detected an =1.27-kb transcript representing the full-length of gpD mRNA in Duffypositive individuals (see Fig. 3A). Anti-Fy6 antibody reacted with a 35-mer synthetic peptide (residues 9-44, see Fig. 1B), predicted by the Fyb7l-81 clone (data not shown). The absence of gpD protein-specific mRNA in Fy(a-b-) phenotypes (see below) and the reaction of anti-Fy6 with a peptide derived from a gpD cDNA are strong indications that the clones we isolated are true Duffy clones. Amino Acid Sequence and Membrane Topology of gpD Protein. The predicted translation product of the Fyb7l-81 clone is an acidic protein of isoelectric point 5.65 and M, 35,733. The protein carries at the N terminus only two potential canonical sequences for N-glycosylation to asparagine residues (14). This result agrees with previous investigations that N-glycosidase F digestion increases gpD protein mobility on SDS/PAGE and with the chemical detection of N-acetylglucosamine (6, 15, 16). Predictions of transmembrane helices locations from sequence data using the hydropathy map of Engelman et al. (17) and a scanning window of 20 residues show that the bulk of the protein is embedded in the membrane (Fig. 2). Nine transmembrane a-helices, a hydrophilic domain of 66 residues at the N terminus, a hydrophilic domain of 25 residues at the C terminus, and short protruding hydrophilic connecting segments were predicted. The pair of helices, D and E, is so closely spaced that the pair may be arranged as coupled -&

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10796

anti-parallel helices. A schematic illustrationi of gpD protein topology is shown in Fig. 2. The charge-difference rule proposed by lHartmann et al. (18) predicts that the N terminus is on the exo cellular side and the C terminus of the protein is on the cytoplaLsmic side of the membrane. The N-terminal prediction is v alidated by the finding of the two potential N-glycosylation sites on the N terminus. Moreover, the reaction of anti-Fy6 with a synthetic peptide deduced from this domain establishe :s its exocellular location experimentally because the antibod)y binds to erythrocytes. The signal-anchor sequence (19, 201) for membrane insertion probably lies in the first transmembrrane a-helix that follows the N-terminal domain. From there oon, the protein is deeply buried in the membrane and exits at re sidue 314 on the cytoplasmic side of the membrane (Fig. 2). The topological predictions of helices, hydrophilic connectinjg segments, and the location of the C-terminal fragment shouild be substantiated by direct biochemical and immunochennical analysis. Duffy gpD protein is deeply buried in the membrane like the membrane-associated fragment of band 3 (21), the human blood group Rh polypeptide (22, 23), bacteric)rhodopsin (24), and lipophilin (25). The significant homology( of gpD protein with interleukin-8 receptors is very intriguing (26, 27). If gpD protein binds chemokines and has the abililty to activate a signal-transduction cascade, this gives rise tc) gpD protein as another class of proinflammatory mediatc )rs. Thus, gpD protein is not present in leukocytes becaus e a rabbit polyclonal antibody (anti-gpD) against purified and denatured gpD protein that reacts with erythrocytes alnd their precursors does not react with any leukocytes (unpu blished results). Northern and Southern Analyses. On RN A blot analysis Fyb71 or Fyb8l clone detected an -1.27-kb mRNA species in the bone marrow of the three Duffy-positLive phenotypes but not in individuals of Fy(a-b-) phenotypxe (Fig. 3A). The absence of gpD mRNA was consistent with the absence of gpD protein in Duffy-negative individuals. Anti-gpD antibody did not react with any erythrocyte memlbrane protein of Fy(a-b-) erythrocytes (data not shown). Duffy-negative A

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FIG. 3. RNA and Southern blots probed with either the Fyb7l or the Fyb81 insert. (A) Human bone marrow poly(A)+ RNA from the four phenotypes. Lanes: 1, 10 ug of Fy(a-b-) mRNA; 2, 5 pg of Fy(a+b-) mRNA; 3, 5 pg of Fy(a-b+) mRNA; and 4, 2 pg of Fy(a+b+) mRNA. RNAs were resolved on a 2% denaturing agarose gel, blotted, hybridized, and autoradiographed for 72 hr at -80°C. RNA size markers human 28S (5.1 kb) rRNA, 18S (2.0 kb) rRNA, and the 1.35-kb GIBCO/BRL marker (Life Technologies, Grand Island, NY) were used to calculate the size of gpD mRNA. The actin probe at bottom was used as control of sample loading. RNA integrity was indicated by the presence of the two rRNAs in the poly(A)+ fraction and the actin probe. (B) Human genomic DNAs from the four phenotypes. Each lane contained 10 pIg of digested DNA. Lanes: 1-4, Fy(a-b-); 5-8, Fy(a+b-); 9-12, Fy(a-b+) DNA. Enzyme digestions were as follows: lanes: 1, 5, and 9, BamHI; 2, 6, and 10, EcoRI; 3, 7, and 11, Hinfl; and 4, 8, and 12, Pst I. DNAs were resolved on 0.8% agarose gel, blotted, hybridized, and autoradiographed for 7 days at -80°C. Sizes were calculated from the positions of GIBCO/BRL DNA markers.

individuals do not express gpD protein because they do not synthesize Duffy-specific mRNA. On Southern blot analysis Fyb71 or Fyb81 probe hybridized with DNA of Duffy-positive and -negative individuals (Fig. 3B). They identified a single band of 6.5 kb in BamHI, two bands of 12 kb and 2 kb in EcoRl, and two bands of 3.5 kb and 1.4 kb in Pst I-digested DNA. These findings agree with the restriction map of the Fyb7l and Fyb81 clones and show a single-copy gene. Determination of the structural differences among the genes of Duffy-positive and -negative individuals should clarify the mechanism of gpD gene repression in negative individuals. A functional silencer element described in other systems may selectively repress transcription of gpD gene in the erythrocytes of Fy(a-b-) individuals (28). The Duffy system is different from the ABO (29) and Kell (Colvin Redman, personal communication) systems, where mRNA has been found in individuals who do not express the blood group determinants. As indicated in Fig. 4, a 1.27-kb mRNA species was found in adult spleen and kidney, fetal liver but not in adult liver, and K-562 erythroleukemia cells. Hybridization with the ,B-globin probe showed a strong signal in bone marrow and fetal liver; it showed a weak signal in adult spleen but showed no signal in adult liver, brain, and kidney (data not shown). The presence of gpD mRNA in fetal liver was expected because fetal liver is an erythropoietic organ. In human brain, a strong band of 8.5 kb and a faint band of 2.2 kb were detected. This finding is interesting, as it indicates there is a Duffy-related protein in brain. This idea is also supported by the quasi-total homology between Fyb7l-81 clone and a human hippocampus cDNA clone HHCMF86, which was recently identified (30). However, it is unlikely that the 8.5-kb brain mRNA codes for a Duffy protein with long 5' and 3' untranslated sequences. Perhaps the brain mRNA codes for a larger protein that has extensive homology with gpD protein. The homologies of these mRNA species with gpDspecific mRNA remain to be demonstrated by sequence analysis; however, the findings strongly indicate that gpD protein or a similar protein is produced in kidney, nonhemopoietic spleen cells, and probably in brain. In summary, we have isolated four cDNA clones that encode gpD protein, the major subunit of the Duffy blood group antigenic system. It is a highly hydrophobic intramembrane glycoprotein with nine putative transmembrane a-helices. The cognate gene is present in Duffy-positive and -negative individuals, but the bone marrow of Duffy-negative individuals does not synthesize gpD-specific mRNA. In adult kidney, spleen, and fetal liver, the mRNA has the same size as gpD mRNA; however, in brain the mRNA is much larger. kb

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FIG. 4. RNA blot analysis of poly(A)+ RNA from human tissues probed with the insert of Fyb8l clone. Lanes 1, 3, 5, and 7 contained 2 pg of Fy(a-b+) bone marrow, fetal liver, adult spleen, and erythroleukemia (K-562) mRNAs, respectively. Lanes 2, 4, and 6 contained 7 pg of total brain, adult liver, and adult kidney mRNA, respectively. RNAs were resolved on a 1.5% denaturing agarose gel and autoradiographed for 5 days at -80°C.

Medical Sciences: Chaudhuri

et

al.

The clones that we have characterized will provide the elements to investigate (i) the structural components of gpD genes, (ii) the biosynthesis and expression of gpD protein in human bone marrow and other tissues, (iii) the structurefunction of this erythrocyte-membrane protein that might exist in other cell types and may function as a chemokine receptor, and (iv) the role as the receptor for P. vivaxmerozoite invasion. Note Added in Proof. Since submission of this article, a family of chemotactic and proinflammatory soluble peptides, including interleukin 8 (IL-8), melanoma growth-stimulating activity (MGSA), monocyte chemotactic peptide 1 (MCP-1), and regulated on activation, normal T-cell expressed and secreted peptide (RANTES), has been shown to bind to Duffy-positive erythrocytes only. This observation strongly supports the idea that gpD protein is an additional class of chemokine receptor (31). We are grateful to Dr. J. Adamson for his support and encouragement. We thank Drs. M. Nichols and P. Rubinstein for the Fy6

murine monoclonal antibody, Drs. D. Engelman and R. Macnab for the FOAMPC program, and the Laboratory of Immunohematology for erythrocyte antigen typing. We are indebted to H. Hlawaty, M. Noble, and K. Stone for technical assistance and P. Lim for secretarial assistance. This research was partially supported by Grant HL 39021 from the National Institutes of Health and New York Blood Center Institutional Funds. 1. Marsh, W. L. (1975) Crit. Rev. Clin. Lab. Sci. 5, 387-412. 2. Nichols, M. E., Rubinstein, P., Barnwell, J., de Cordoba, S. R. & Rosenfield, R. E. (1987) J. Exp. Med. 166, 776-785. 3. Miller, L. H., Mason, S. J., Clyde, D. F. & McGinniss, M. H. (1976) N. Engl. J. Med. 295, 302-304. 4. Miller, L. H., Mason, S. J., Dvorak, J. A., McGinniss, M. H. & Rdthman, K. I. (1985) Science 189, 561-563. 5. Chaudhuri, A., Zbrzezna, V., Johnson, C., Nichols, M., Rubinstein, P., Marsh, W. L. & Pogo, A. 0. (1989) J. Biol. Chem. 264, 13770-13774. 6. Chaudhuri, A. & Pogo, A. 0. (1993) in Blood Cell Biochemistry, eds. Cartron, J.-P. & Rouger, P. (Plenum, New York), Vol. 6, in press. 7. LeGendre, N. & Matsudaira, P. T. (1989) in A Practical Guide to Protein and Peptide Purification for Microsequencing, ed. Matsudaira, P. T. (Academic, New York), pp. 49-69.


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