The P1 histo-blood group antigen is present on human red blood cell glycoproteins.

BACKGROUND: The P1 antigen was first described in 1927 and belongs to the P1PK histo-blood group system, together with Pk and NOR. The A4GALT-encoded 4-α-galactosyltransferase synthesizes these antigens and has been considered to extend glycolipids exclusively. However, contradicting studies have been published regarding the presence of P1 on human glycoproteins. In other species, P1 occurs on glycoproteins. Furthermore, human ABH antigens occur on both glycolipids and glycoproteins and are biochemically related to P1. Thus, we hypothesized that P1 is present on RBC glycoproteins in humans. STUDY DESIGN AND METHODS: RBCs of known P1 /P2 status (phenotype and rs8138197 genotype) were used. The RBC surface glycans were modified with α-galactosidases, papain, and/or peptide-N-glycosidase F. RBC membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/immunoblot. A new P 1 /P 2 -allelic discrimination assay based on rs5751348 was validated. RESULTS: P1 occurs on various glycoproteins, seen as smearlike patterns in anti-P1-stained immunoblots with RBC membranes of P1 but not P2 or p phenotype. There was a significant difference between the staining of P 1 -homozygous and P 1 -heterozygous RBCs (P 1 P 1 > P 1 P 2 ), as well as intragenotypic variation. Immunoblotting banding patterns show major carriers at approximately 50 and 100 kDa. P1 staining was lost after treatment of RBCs with α-galactosidase of broad Galα-1,3/4/6-specificity. Peptide-N-glycosidase F treatment reduced the P1 signal, while papain or α-1,3-specific galactosidase did not. P 1 /P 2 status was confirmed by a new rs5751348 assay. CONCLUSION: Our data indicate that the P1 antigen can reside on human RBC glycoproteins. Glycosidase studies suggest that at least part of the epitopes occur on N-glycans.

A systematic study of single-nucleotide polymorphisms in the A4GALT gene suggests a molecular genetic basis for the P1/P2 blood groups.

BACKGROUND: The molecular mechanism for the formation of the P1/P2 blood groups remains unsolved. It has been shown that the P1/P2 polymorphism is connected to the different A4GALT gene expression levels in P1 and P2 red blood cells. STUDY DESIGN AND METHODS: The present investigation conducted a pilot investigation that involved the detailed and stepwise screening of single-nucleotide polymorphisms (SNPs) in the A4GALT gene, followed by a larger-scale association study. The transcription-inducing activity by the different genotypes of SNPs was analyzed using reporter assays. RESULTS: A total of 416 different SNP sites in the A4GALT genes from four P1 and four P2 individuals were analyzed in the pilot investigation, and 11 SNP sites, distributed in the A4GALT Intron 1 region, exhibited an association with the P1/P2 phenotypes. In the follow-up association study, the genotypes at the 11 SNPs of a total of 338 individuals across four different ethnic populations were determined, and the results show that two SNPs, rs2143918 and rs5751348, are consistently associated with the P1/P2 phenotypes. Reporter assays demonstrated significantly higher transcription-inducing activity by the SNPs bearing the P(1)-allele genotype than by the SNPs bearing the P(2)-allele genotype and that the difference in transcriptional activity was determined by the different genotypes at SNP rs5751348. CONCLUSION: The results of this investigation demonstrate a consistent association of A4GALT SNPs rs2143918 and rs5751348 with the P1/P2 phenotypes and suggest that SNP rs5751348 may lead to allelic variations in A4GALT gene expression and consequently leads to the formation of the P1/P2 phenotypes.

The P histo-blood group-related glycosphingolipid sialosyl galactosyl globoside as a preferred binding receptor for uropathogenic Escherichia coli: isolation and structural characterization from human kidney.

The P histo-blood group-related glycosphingolipid, sialosyl galactosyl globoside (SGG), has recently been implicated as a preferred binding receptor for uropathogenic Escherichia coli [Stapleton, A. E., Stroud, M. R., Hakomori, S., and Stamm, W. E. (1998) Infect. Immun. 66, 3856-3861]. We report here the purification and complete structural characterization of SGG from normal human kidney. Using metabolically [35S]-labeled E. coli as a probe, a monosialylated glycosphingolipid was isolated to homogeneity. The glycosphingolipid was purified by a combination of high-performance liquid chromatography and preparative high-performance thin-layer chromatography and its structure unambiguously elucidated by 1H NMR, electrospray ionization mass spectrometry, and methylation analysis. Its primary structure was shown to be identical to a previously characterized, developmentally regulated, globo-series glycolipid thought to be unique to human teratocarcinoma. The significance of this structure as a unique receptor in human kidney for uropathogenic E. coli and its role in the pathogenesis of urinary tract infections are discussed.

PA-I and PA-II lectin interactions with the ABO(H) and P blood group glycosphingolipid antigens may contribute to the broad spectrum adherence of Pseudomonas aeruginosa to human tissues in secondary infections.

Pseudomonas aeruginosa may cause serious infections in most human tissues/organs. Its adherence to them is mediated by a battery of adhesins including the PA-I and PA-II lectins, which are produced in this bacterium in high quantities. PA-I binds to the D-galactose of the erythrocyte glycosphingolipids exhibiting highest affinities for B and Pk (followed by P1) antigens, while PA-II preferentially binds to the L-fucose of H, A and B antigens. Intact P. aeruginosa cells also exhibit a clear Pk and P1 over p preference. Such affinities for the most common human ABH and P system antigens may underlie the widespread tissue infectivity and pathogenicity of this bacterium.

Evidence that verotoxins (Shiga-like toxins) from Escherichia coli bind to P blood group antigens of human erythrocytes in vitro.

The interaction of verotoxins (VTs) with human erythrocytes (RBCs) in vitro was investigated, with particular reference to the role of P blood group glycolipids that are structurally related to the known VT receptors. RBC binding of purified VT1, VT2, VT2c, and VT2e was detected by direct and indirect immunofluorescence. Glycolipids were extracted from defined RBCs, separated by thin-layer chromatography, and assessed for VT binding in an overlay assay by adding toxin and specific antibodies. All VTs bound to P1 phenotype (Pk, P, and P1 antigens) and P2 phenotype (Pk and P antigens) RBCs but not to p phenotype (lacking the Pk, P, and P1 antigens) RBCs. Binding of VT1 and VT2 was approximately 10-fold greater to P1 and the rare Pk2 (Pk antigen but no P1 or P antigen) phenotype cells than to P2 phenotype RBCs, whereas VT2e bound equally well to P1 and P2 phenotype cells. The VT1 and VT2 immunofluorescence results correlated with the detection of P1 and/or increased amounts of Pk (globotriaosylceramide) antigen; VT2e immunofluorescence correlated with the detection of P (globotetraosylceramide) antigen. The Pk band pattern and VT binding observed in the thin-layer chromatogram of human P1 and P phenotype RBC extracts varied from that of human kidney and Pk1 phenotype (Pk and P1 antigens) RBCs. We conclude that each VT binds to human RBCs in vitro by utilizing specific P blood group glycolipids as receptors. On P1 and P phenotype RBCs, the accessibility of the Pk antigen for VTs appeared to be restricted. The occurrence of VT-RBC binding in natural VT-producing Escherichia coli disease and its relevance for the pathophysiology of hemolytic uremic syndrome remain to be established.

Glycoproteins with Gal alpha 4Gal are absent from human erythrocyte membranes, indicating that glycolipids are the sole carriers of blood group P activities.

The antigenic determinants of the blood group P family (P1, P, Pk, and LKE antigens) are chemically based on Gal alpha 4Gal. For human erythrocytes it has been claimed that the majority of P1 determinants are expressed in glycoproteins, mainly band 4.5 (Haselberger, C. G., and Schenkel-Brunner, H. (1982) FEBS Lett. 149, 126-128). In the present work, the existence of Gal alpha 4Gal in glycoproteins of erythrocyte membranes (ghosts) of P1 positive and negative human individuals was carefully analyzed on replicas of sodium dodecyl sulfate-polyacrylamide electrophoresis gels using specific reagents (Escherichia coli HB101/pDC1 expressing the Pap gene and monoclonal antibodies with specificities for P1 and Pk antigens). No binding to glycoproteins was detected with any of these ligands when the ghosts had been pretreated with butanol to remove glycolipids. Therefore, all antigenic determinants of the P blood group family on human red cells are exclusively expressed in glycolipids and are absent from glycoproteins.

Erythrocyte P antigen: cellular receptor for B19 parvovirus.

The pathogenic human parvovirus B19 replicates only in erythroid progenitor cells. This virus was shown to bind to blood-group P antigen, as measured by hemagglutination. Erythrocytes lacking P antigen were not agglutinated with B19. Purified P antigen (globoside) blocked the binding of the virus to erythroid cells and the infectivity of the virus in a hematopoietic colony assay. Target cells were protected from infection by preincubation with monoclonal antibody to globoside. Knowledge of a parvovirus receptor has implications for understanding the pathogenesis of parvovirus infections and for the use of parvoviruses in gene therapy.

Characterization of a novel bacterial adhesion specificity of Streptococcus suis recognizing blood group P receptor oligosaccharides.

Streptococcus suis causes sepsis, meningitis, and other serious infections in piglets, and meningitis in humans. Hemagglutination inhibition experiments with mono- and oligosaccharides and glycoproteins indicated that galactose-binding strains of S. suis recognized the Gal alpha 1-4Gal sequence present in the P1 and Pk blood group antigen structures. In thin-layer chromatography overlay assays the bacteria bound to trihexosylceramide (GbO3) but not to globoside (GbO4) or Forssman glycolipid (GbO5), in contrast to P-fimbriated Escherichia coli, which bound only to the latter two. The S. suis adhesin also differed from that of E. coli in that some of the hydrogen bonds formed with the receptor, as determined with chemically modified receptor analogues, were different. In agreement with the binding specificity, the S. suis bacteria agglutinated best among P blood group erythrocytes those of the P1k and P2k type, and from different animal erythrocytes those from rabbit, which express GbO3 as the predominant glycolipid. Binding to frozen sections of pig pharyngeal tissue was decreased by the free GbO3 oligosaccharide and its protein conjugate, which indicated that the corresponding glycolipid may function as receptor for galactose-binding strains of S. suis in pig pharyngeal epithelium.