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

Stenfelt, L. LU ; Westman, Julia LU and Olsson, M.L. LU (2017) 27th Regional Congress of the International Society of Blood Transfusion, ISBT 2017 In Vox Sanguinis 112(S1). p.25-25
Abstract
Background: The P1 antigen was first described in 1927 but not categorized with the Pk antigen in the P1PK histo-blood group system until 2010. Individuals of P1 phenotype have both the Pk and P1 antigens on their red blood cell (RBC) surface, while P2 individuals lack P1 and have lower amounts of Pk. Like in the ABO system, the antigens are carbohydrate epitopes built up stepwise by glycosyltransferases. The antigens of the P1PK system are synthesized by 4-a-galactosyltransferase (a1,4GalT) encoded by A4GALT. Gala4Gal-terminating antigens like Pk and P1 can function as receptors for P-fimbriated Escherichia coli (E. coli). Moreover, the terminal and internal Gala4Galb sequence is a known Shiga toxin receptor. Antibodies against the... (More)
Background: The P1 antigen was first described in 1927 but not categorized with the Pk antigen in the P1PK histo-blood group system until 2010. Individuals of P1 phenotype have both the Pk and P1 antigens on their red blood cell (RBC) surface, while P2 individuals lack P1 and have lower amounts of Pk. Like in the ABO system, the antigens are carbohydrate epitopes built up stepwise by glycosyltransferases. The antigens of the P1PK system are synthesized by 4-a-galactosyltransferase (a1,4GalT) encoded by A4GALT. Gala4Gal-terminating antigens like Pk and P1 can function as receptors for P-fimbriated Escherichia coli (E. coli). Moreover, the terminal and internal Gala4Galb sequence is a known Shiga toxin receptor. Antibodies against the antigens of the P1PK system are naturally-occurring. Anti-P1 induced hemolytic transfusion reactions are rare but a few cases have been reported. The anti-P1PPk found in plasma from individuals of the p (P1PPk-) phenotype is also associated with recurrent miscarriages. In humans, a1,4GalT has been considered to extend glycans on glycosphingolipids exclusively. However, in certain other species, such as the pigeon, the P1 epitope resides on glycoproteins as well. Interestingly, the majority of the human A, B and H antigens are found on glycoproteins and they share the same precursor as P1, namely paragloboside. Aims: This work is based on a hypothesis stated years ago regarding the P1 glycoepitope. Haselberger et al. [FEBS Lett., 1982] published that P1 is carried on glycoproteins in humans. However, this was later contradicted firmly by Yang et al. [J Biol Chem., 1994]. The aim of this work was to find out if P1 is present on glycoproteins and if so, to characterize carrier candidates on human RBCs. Methods: RBCs from peripheral blood of apparently healthy volunteers were used for the study. P1 phenotyping and SNP genotyping was used to determine P1/P2 status. RBC glycans were digested with a-galactosidase from green coffee bean, a1,3- specific GH110 a-galactosidase of bacterial origin (B-zyme), papain, neuraminidase and/or peptide-N-glycosidase (PNGase) F. RBCs were lysed and the hemoglobin was washed away while the unsealed membranes were collected. Denatured membrane proteins were separated in SDS-PAGE and transferred to a low fluorescence PVDF membrane, immunoblotted with monoclonal anti-P1 and staining quantified in relation to total protein loaded. Results: We show clearly that P1 occurs on various glycoproteins, seen as a smearlike pattern in Western blots with cell membranes from P1 but not P2 or p RBCs. Furthermore, there was a significant difference between the staining of RBCs from P1-homozygous (stronger) and P1-heterozygous (weaker) individuals. The amount detected varied between samples of the same genotype, which is consistent with earlier studies of P1 expression on RBCs. A tendency toward a banding pattern suggested possible carriers at ∼50 and ∼100 kDa. P1 staining was lost after treatment of the RBCs with coffee bean a-galactosidase as the enzyme destroys the P1 epitope. PNGase F treatment reduced the P1 signal whilst B-zyme did not. Summary/Conclusions: This study provides data showing that P1 does indeed reside on human RBC glycoproteins and not only on glycosphingolipids. The epitope was detected in a gene-dose dependent manner on glycoproteins of P1-positive RBC membranes. The signal was reduced when using N-glycan-specific PNGase F, indicating that at least some of the epitopes are carried on N-glycans, which mimics the ABH antigens. Attempts to identify specific carrier proteins are in progress. (Less)
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keywords
antibody, blood group ABH antigen, carrier protein, endogenous compound, epitope, galactosidase, galactosyltransferase, globotriaosylceramide, glycan, glycopeptidase, glycoprotein, glycosphingolipid, membrane protein, papain, paragloboside, protein, sialidase, blood transfusion reaction, denaturation, erythrocyte membrane, Escherichia coli, fluorescence, genotype, heterozygosity, homozygosity, human, human tissue, lysis, phenotype, pigeon, polyacrylamide gel electrophoresis, precursor, species, Western blotting
in
Vox Sanguinis
volume
112
issue
S1
pages
1 pages
publisher
Federation of European Neuroscience Societies and Blackwell Publishing Ltd
conference name
27th Regional Congress of the International Society of Blood Transfusion, ISBT 2017
ISSN
1423-0410
DOI
10.1111/vox.12530
language
English
LU publication?
yes
id
af69cca1-f4bd-48f4-a7f6-d7ba223f09c3
date added to LUP
2017-09-01 13:30:15
date last changed
2017-09-08 14:53:59
@misc{af69cca1-f4bd-48f4-a7f6-d7ba223f09c3,
  abstract     = {Background: The P1 antigen was first described in 1927 but not categorized with the Pk antigen in the P1PK histo-blood group system until 2010. Individuals of P1 phenotype have both the Pk and P1 antigens on their red blood cell (RBC) surface, while P2 individuals lack P1 and have lower amounts of Pk. Like in the ABO system, the antigens are carbohydrate epitopes built up stepwise by glycosyltransferases. The antigens of the P1PK system are synthesized by 4-a-galactosyltransferase (a1,4GalT) encoded by A4GALT. Gala4Gal-terminating antigens like Pk and P1 can function as receptors for P-fimbriated Escherichia coli (E. coli). Moreover, the terminal and internal Gala4Galb sequence is a known Shiga toxin receptor. Antibodies against the antigens of the P1PK system are naturally-occurring. Anti-P1 induced hemolytic transfusion reactions are rare but a few cases have been reported. The anti-P1PPk found in plasma from individuals of the p (P1PPk-) phenotype is also associated with recurrent miscarriages. In humans, a1,4GalT has been considered to extend glycans on glycosphingolipids exclusively. However, in certain other species, such as the pigeon, the P1 epitope resides on glycoproteins as well. Interestingly, the majority of the human A, B and H antigens are found on glycoproteins and they share the same precursor as P1, namely paragloboside. Aims: This work is based on a hypothesis stated years ago regarding the P1 glycoepitope. Haselberger et al. [FEBS Lett., 1982] published that P1 is carried on glycoproteins in humans. However, this was later contradicted firmly by Yang et al. [J Biol Chem., 1994]. The aim of this work was to find out if P1 is present on glycoproteins and if so, to characterize carrier candidates on human RBCs. Methods: RBCs from peripheral blood of apparently healthy volunteers were used for the study. P1 phenotyping and SNP genotyping was used to determine P1/P2 status. RBC glycans were digested with a-galactosidase from green coffee bean, a1,3- specific GH110 a-galactosidase of bacterial origin (B-zyme), papain, neuraminidase and/or peptide-N-glycosidase (PNGase) F. RBCs were lysed and the hemoglobin was washed away while the unsealed membranes were collected. Denatured membrane proteins were separated in SDS-PAGE and transferred to a low fluorescence PVDF membrane, immunoblotted with monoclonal anti-P1 and staining quantified in relation to total protein loaded. Results: We show clearly that P1 occurs on various glycoproteins, seen as a smearlike pattern in Western blots with cell membranes from P1 but not P2 or p RBCs. Furthermore, there was a significant difference between the staining of RBCs from P1-homozygous (stronger) and P1-heterozygous (weaker) individuals. The amount detected varied between samples of the same genotype, which is consistent with earlier studies of P1 expression on RBCs. A tendency toward a banding pattern suggested possible carriers at ∼50 and ∼100 kDa. P1 staining was lost after treatment of the RBCs with coffee bean a-galactosidase as the enzyme destroys the P1 epitope. PNGase F treatment reduced the P1 signal whilst B-zyme did not. Summary/Conclusions: This study provides data showing that P1 does indeed reside on human RBC glycoproteins and not only on glycosphingolipids. The epitope was detected in a gene-dose dependent manner on glycoproteins of P1-positive RBC membranes. The signal was reduced when using N-glycan-specific PNGase F, indicating that at least some of the epitopes are carried on N-glycans, which mimics the ABH antigens. Attempts to identify specific carrier proteins are in progress.},
  articleno    = {3B-S06-01},
  author       = {Stenfelt, L. and Westman, Julia and Olsson, M.L.},
  issn         = {1423-0410},
  keyword      = {antibody,blood group ABH antigen,carrier protein,endogenous compound,epitope,galactosidase,galactosyltransferase,globotriaosylceramide,glycan,glycopeptidase,glycoprotein,glycosphingolipid,membrane protein,papain,paragloboside,protein,sialidase,blood transfusion reaction,denaturation,erythrocyte membrane,Escherichia coli,fluorescence,genotype,heterozygosity,homozygosity,human,human tissue,lysis,phenotype,pigeon,polyacrylamide gel electrophoresis,precursor,species,Western blotting},
  language     = {eng},
  month        = {06},
  note         = {Conference Abstract},
  number       = {S1},
  pages        = {25--25},
  publisher    = {Federation of European Neuroscience Societies and Blackwell Publishing Ltd},
  series       = {Vox Sanguinis},
  title        = {The P1 histo-blood group antigen is present on human red blood cell glycoproteins},
  url          = {http://dx.doi.org/10.1111/vox.12530},
  volume       = {112},
  year         = {2017},
}