Antigenic determinants encoded by alternatively spliced exons of CD45 are determined by the polypeptide but influenced by glycosylation.
Antibodies recognising the products of alternatively spliced exons near the N-terminus of the leukocyte common antigen, CD45, have been widely used to distinguish populations of lymphocytes with different functional properties. These alternatively spliced regions contain a high content of serine and threonine residues (average 35%) and are heavily O-glycosylated. Despite evidence that the O-glycosylation contributes significantly to the antigenic character of this region of CD45, work with leukosialin and mucin glycoproteins leads to the prediction that the majority of epitopes in the N-terminal exons should be linear protein determinants. In this study the exons of CD45 were expressed in Escherichia coli as non-glycosylated proteins fused to glutathione S-transferase (GST). Fourteen out of 17 mAbs specific for human CD45R reacted with a fusion protein containing exons 4, 5 and 6 (ABC) of human CD45, and four out of six mAbs specific for rat CD45R reacted with an equivalent rat protein. mAbs recognising the product of rat exon B are reported for the first time. Kinetic analysis of MRC OX22 antibody binding to spleen CD45 and to GST fusion proteins showed that the carbohydrate affected the kinetics of binding of antibodies to the protein backbone. In conclusion, heterogeneity in the glycosylation of heavily O-glycosylated cell surface proteins can affect interactions of these proteins both directly through the carbohydrate and indirectly through effects on the protein backbone.
Cyster
J G
JG
MRC Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, UK.
Fowell
D
D
Barclay
A N
AN
D017398:Alternative Splicing / Q000276:immunology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001483:Base Sequence; D000939:Epitopes / Q000276:immunology*; D005982:Glutathione Transferase / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D017493:Leukocyte Common Antigens / Q000096:biosynthesis / Q000276:immunology*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational / Q000276:immunology*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D017493:Leukocyte Common Antigens