*Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine;
Institute of Biological Anthropology, University of Oxford
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Abstract |
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Introduction |
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Many pathogens use sugar groups of surface glycoproteins, in particular sialic acid residues, as receptors for invasion (Karlsson 1995
). It has therefore been suggested that erythrocyte glycoproteins may function as decoy receptors (Gagneux and Varki 1999
) attracting pathogens to the erythrocyte, which as a mature cell lacks a nucleus and DNA replication, and away from target tissues (Gagneux and Varki 1999
). This represents a plausible function for GYPA which carries many complex sugar groups (Tomita and Marchesi 1975
; Pisano et al. 1993
) and is supported by studies in which GYPA has been shown to bind numerous viruses (Paul and Lee 1987
; Nishimura et al. 1988
; Tavakkol and Burness 1990
; Wybenga et al. 1996
) and bacteria (Baseman, Banai, and Kahane 1982
; Brooks et al. 1989
; Saada et al. 1991
). GYPA is, however, also known to be a principal receptor for the malarial parasite Plasmodium falciparum, which infects erythrocytes (Pasvol, Wainscoat, and Weatherall 1982
; Sim et al. 1994
). If GYPA generally functions as a decoy receptor, then it is likely to be under selection for targeting the diversity of potential pathogens. It is expected that this would cause diversifying selection on the gypa gene sequence.
To investigate the selective forces affecting the evolution of the GYPA receptor, a survey of sequence variation in the gypa gene was carried out. Tests for departures from neutrality were performed on pairwise comparisons of synonymous and nonsynonymous differences in the region encoding the mature GYPA product among six primate species and on genetic diversity in the region encoding the extracellular portion of GYPA in a human population.
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Materials and Methods |
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Allele Sequence of gypa Exons 24 Sampled from a Human Population
Purified genomic DNA from 33 unrelated individuals (Yoruba ethnicity, southwestern Nigeria) and a single chimpanzee (sample kindly provided by N. Mundy) were used as templates for amplification of a 3.6-kb region of gypa, from exon 2 to the boundary of exon 5 (see fig. 1
). gypa-specific amplification primers GYPAIfwd5'-GCTTAGCTCAGGGACTGGAGG-3' and GYPAIIIrev5'-CACCTTGCCTTTTAATAGAAAGC-3' were designed to ensure no amplification of a paralogous sequence in gypb or gype, which share strong homology with gypa. PCR products were ligated into pGem®-T Easy Vector (Promega) and transformed by heat shock into JM 109 E. Coli High Efficiency Competent Cells. Isolated plasmids were prepared using a Plasmid Miniprep column (QIAGEN). A single clone representing one allele was isolated and sequenced from each individual to avoid problems associated with determination of haplotypes in heterozygotes. Each template was sequenced using internal sequencing primers (available on request) with 3' BIG DYETM dye terminator cycle sequencing premix kit (Applied Biosystems). Sequencing was carried out on a Perkin-Elmer ABI PrismTM 377 DNA Sequencer (Applied Biosystems) and sequences were checked and assembled using Sequence Navigator Version 1.0.1 (Applied Biosystems). Polymorphic sites were identified, and all singletons in the data set were confirmed by reamplification from genomic DNA and sequencing to exclude any errors introduced by PCR or cloning (deposited human sequences: GenBank accession numbers: AJ30982845, AJ31131832; chimpanzee AJ309708).
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Prediction of O-linked Glycosylation Sites in Primate GYPA Sequences
Published primate gypa sequences (as above) were translated into amino acid sequences and entered into NetOGlyc Version 2.0 (Hansen et al. 1998
) (www.cbs.dtu.dk/services/NetOGlyc). This predicts O-glycosylation sites on serine and threonine residues in peptides based on the amino acid sequence context.
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Results |
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Analysis of Nine Other Nuclear Genes in Primates
A comparable analysis of the nine other nuclear genes, for which coding sequences in the same six primates were available (CCR5, fut1, A, G
, lyz, prm2, rhag, tyr, and zfy), indicated the exceptional nature of GYPA diversity in primates. None of these nine genes have an estimated excess of nonsynonymous changes in all lineages. All have average pairwise dN/dS ratios of less than 1.0 (except for rhag for which dN/dS = 1.00) (fig. 3a
). As indicated in figure 3b
the average dN pairwise difference for gypa (0.113) is exceptionally high (mean for the other genes = 0.018). Hence, the high dN/dS ratio is caused by a high rate of nonsynonymous substituion and not by a low rate of synonymous substitution (the average dS pairwise difference for gypa (0.055) is typical; mean for the other genes = 0.059). Together these data highlight the distinctive signature of diversifying selection on gypa across the evolution of higher primates. Comparable sequences are not yet available for other mammalian orders, except for the mouse homologue that is so divergent from primate sequences that a proper alignment for analysis is not possible (Matsui, Natori, and Obinata 1989
).
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Discussion |
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The joint signatures of between-species and within-species diversifying selection can, however, be explained by the ability of GYPA to bind nonerythrocytic pathogens to the erythrocyte surface (Baseman, Banai, and Kahane 1982
; Paul and Lee 1987
; Nishimura et al. 1988
; Brooks et al. 1989
; Tavakkol and Burness 1990
; Saada et al. 1991
; Wybenga et al. 1996
) by acting as a decoy receptor. Surface glycoproteins containing sialic acids, are principal receptors used by pathogens to invade their target tissues (Karlsson 1995
). Given its abundance on the erythrocyte surface and its heavy glycosylation (including sialic acid groups), GYPA could function as a decoy receptor attracting pathogens that bind cell surface sugar groups to the anucleated erythrocyte and away from more vital target tissues. Such a flypaper strategy would explain why the GYPA sequences have diverged so greatly among species, with GYPA in each species adapting specifically to its own pathogens.
This hypothesis also explains the maintenance of GYPA variation within humans by balancing selection, because heterozygotes that express two structural forms of GYPA would be potentially able to target more pathogens. In addition to the primate sequence data given here, there is also general evidence of variation in the glycosylation found on the erythrocyte surface both within humans (Gardner et al. 1989
) and among different mammals (Gagneux and Varki 1999
). This is supported by comparing the predicted O-linked glycosylation sites of primate GYPA sequences, which demonstrate how changes in amino acid sequence elsewhere in the protein can influence the predicted glycosylation of conserved serine or threonine residues through indirect effects such as protein folding (fig. 5
). These differences in amino acid sequence that affect the folding and tertiary structure of surface proteins may also affect pathogen binding. Consistent with this, it has been observed that human erythrocytes with the GYPA allelic blood group M antigen are preferentially bound by certain strains of Escherichia coli, compared with cells with only N (Vaisanen et al. 1982
; Brooks et al. 1989
), where the difference between the two blood groups is defined by amino acid differences and not by glycosylation differences. Functional studies on the interaction between GYPA and nonerythrocytic pathogens and the effect that sequence variation has on glycosylation and pathogen binding will be important for testing the pathogen decoy hypothesis further. The strong non-neutral patterns in the gypa gene identified in this study highlight the erythrocyte surface as an important area for evolutionary studies.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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Abbreviations: GYPA, Glycophorin A; dN, number of nonsynonymous substitutions per nonsynonymous site; dS, number of synonymous substitutions per synonymous site.
Keywords: natural selection
erythrocyte
glycophorin A
infectious disease
Address for correspondence and reprints: Jake Baum, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom. jacob.baum{at}lshtm.ac.uk
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References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aminoff D., 1988 The role of sialoglycoconjugates in the aging and sequestration of red cells from circulation Blood Cells 14:229-257[ISI][Medline]
Baseman J. B., M. Banai, I. Kahane, 1982 Sialic acid residues mediate Mycoplasma pneumoniae attachment to human and sheep erythrocytes Infect. Immun 38:389-391[ISI][Medline]
Binks R. H., J. Baum, A. M. J. Oduola, D. E. Arnot, H. A. Babiker, G. Kremsner, C. Roper, B. M. Greenwood, D. J. Conway, 2001 Population genetic analysis of the Plasmodium falciparum erythrocyte binding antigen-175 (eba-175) gene Mol. Biochem. Parasitol 114:63-70[ISI][Medline]
Brooks D. E., J. Cavanagh, D. Jayroe, J. Janzen, R. Snoek, T. J. Trust, 1989 Involvement of the MN blood group antigen in shear-enhanced hemagglutination induced by the Escherichia coli F41 adhesin Infect. Immun 57:377-383[ISI][Medline]
Cartron J., J. London, 1992 The protein and gene structure of red cell glycophorins Pp. 101151 in P. Agre and J. Cartron, eds. Protein blood group antigens of the human red cell: structure, function, and clinical significance. Johns Hopkins University Press, Baltimore
Fu Y. X., W. H. Li, 1993 Statistical tests of neutrality of mutations Genetics 133:693-709
Gagneux P., A. Varki, 1999 Evolutionary considerations in relating oligosaccharide diversity to biological function Glycobiology 9:747-755
Gardner B., S. F. Parsons, A. H. Merry, D. J. Anstee, 1989 Epitopes on sialoglycoprotein alpha: evidence for heterogeneity in the molecule Immunology 68:283-289[ISI][Medline]
Goldman N., Z. Yang, 1994 A codon-based model of nucleotide substitution for protein-coding DNA sequences Mol. Biol. Evol 11:725-736
Hansen J. E., O. Lund, N. Tolstrup, A. A. Gooley, K. L. Williams, S. Brunak, 1998 NetOGlyc: prediction of mucin type O-glycosylation sites based on sequence context and surface accessibility Glycoconj. J 15:115-130[ISI][Medline]
Huang C. H., O. O. Blumenfeld, 1995 MNSs blood groups and major glycophorins: molecular basis for allelic variation Pp. 153188 in J. P. Cartron and P. Rouger, eds. Blood cell biochemistry: molecular basis of major human blood group antigens. Plenum Press, New York
Hudson R. R., M. Kreitman, M. Aguade, 1987 A test of neutral molecular evolution based on nucleotide data Genetics 116:153-159
Kaessmann H., V. Wiebe, G. Weiss, S. Paabo, 2001 Great ape DNA sequences reveal a reduced diversity and an expansion in humans Nat. Genet 27:155-156[ISI][Medline]
Karlsson K. A., 1995 Microbial recognition of target-cell glycoconjugates Curr. Opin. Struct. Biol 5:622-635[ISI][Medline]
Kudo S., M. Fukuda, 1989 Structural organization of glycophorin A and B genes: glycophorin B gene evolved by homologous recombination at Alu repeat sequences Proc. Natl. Acad. Sci. USA 86:4619-4623[Abstract]
Kumar S., K. Tamura, M. Nei, 1994 MEGA: Molecular Evolutionary Genetics Analysis software for microcomputers Comput. Appl. Biosci 10:189-91[Abstract]
Matsui Y., S. Natori, M. Obinata, 1989 Isolation of the cDNA clone for mouse glycophorin, erythroid-specific membrane protein Gene 77:325-332[ISI][Medline]
Miller L. H., J. D. Haynes, F. M. McAuliffe, T. Shiroishi, J. R. Durocher, M. H. McGinniss, 1977 Evidence for differences in erythrocyte surface receptors for the malarial parasites, Plasmodium falciparum and Plasmodium knowlesi J. Exp. Med 146:277-281[Abstract]
Mourant A. E., A. C. Kopec, K. Domaniewska-Sobczak, 1978 Blood groups and diseases Oxford University Press, Oxford
Nei M., T. Gojobori, 1986 Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions Mol. Biol. Evol 3:418-426[Abstract]
Nielsen R., Z. Yang, 1998 Likelihood models for detecting positively selected amino acid sites and applications to the HIV-1 envelope gene Genetics 148:929-936
Nishimura H., K. Sugawara, F. Kitame, K. Nakamura, 1988 Attachment of influenza C virus to human erythrocytes J. Gen. Virol 69:2545-2553[Abstract]
Pasvol G., J. S. Wainscoat, D. J. Weatherall, 1982 Erythrocytes deficiency in glycophorin resist invasion by the malarial parasite Plasmodium falciparum Nature 297:64-66[ISI][Medline]
Paul R. W., P. W. Lee, 1987 Glycophorin is the reovirus receptor on human erythrocytes Virology 159:94-101[ISI][Medline]
Pisano A., J. W. Redmond, K. L. Williams, A. A. Gooley, 1993 Glycosylation sites identified by solid-phase Edman degradation: O-linked glycosylation motifs on human glycophorin A Glycobiology 3:429-435[Abstract]
Pohlmann S., F. Baribaud, B. Lee, G. J. Leslie, M. D. Sanchez, K. Hiebenthal-Millow, J. Munch, F. Kirchhoff, R. W. Doms, 2001a. DC-SIGN interactions with human immunodeficiency virus type 1 and 2 and simian immunodeficiency virus J. Virol 75:4664-4672
Pohlmann S., E. J. Soilleux, F. Baribaud, G. J. Leslie, L. S. Morris, J. Trowsdale, B. Lee, N. Coleman, R. W. Doms, 2001b. DC-SIGNR, a DC-SIGN homologue expressed in endothelial cells, binds to human and simian immunodeficiency viruses and activates infection in trans Proc. Natl. Acad. Sci. USA 98:2670-2675
Rearden A., A. Magnet, S. Kudo, M. Fukuda, 1993 Glycophorin B and glycophorin E genes arose from the glycophorin A ancestral gene via two duplications during primate evolution J. Biol. Chem 268:2260-2267
Rozas J., R. Rozas, 1999 DnaSP version 3: an integrated program for molecular population genetics and molecular evolution analysis Bioinformatics 15:174175.
Saada A. B., Y. Terespolski, A. Adoni, I. Kahane, 1991 Adherence of Ureaplasma urealyticum to human erythrocytes Infect. Immun 59:467-469[ISI][Medline]
Sim B. K. L., C. E. Chitnis, K. Wasniowska, T. J. Hadley, L. H. Miller, 1994 Receptor and ligand domains for invasion of erythrocytes by Plasmodium falciparum Science 264:1941-1944[ISI][Medline]
Tajima F., 1989 Statistical method for testing the neutral mutation hypothesis by DNA polymorphism Genetics 123:585-595
Tavakkol A., A. T. Burness, 1990 Evidence for a direct role for sialic acid in the attachment of encephalomyocarditis virus to human erythrocytes Biochemistry 29:10684-10690[ISI][Medline]
Tomita M., V. T. Marchesi, 1975 Amino-acid sequence and oligosaccharide attachment sites of human erythrocyte glycophorin Proc. Natl. Acad. Sci. USA 72:2964-2968[Abstract]
Vaisanen V., T. K. Korhonen, M. Jokinen, C. G. Gahmberg, C. Ehnholm, 1982 Blood group M specific haemagglutinin in pyelonephritogenic Escherichia coli Lancet 1:1192.
Wybenga L. E., R. F. Epand, S. Nir, J. W. Chu, F. J. Sharom, T. D. Flanagan, R. M. Epand, 1996 Glycophorin as a receptor for Sendai virus Biochemistry 35:9513-9518[ISI][Medline]
Yang Z., 1998 Likelihood ratio tests for detecting positive selection and application to primate lysozyme evolution Mol. Biol. Evol 15:568-573[Abstract]
Yang Z., R. Nielsen, N. Goldman, A. M. Pedersen, 2000 Codon-substitution models for heterogeneous selection pressure at amino acid sites Genetics 155:431-449