ANTIGENIC DETERMINANTS ENCODED BY ALTERNATIVELY SPLICED EXONS OF CD45ARE DETERMINED BY THE POLYPEPTIDE BUT INFLUENCED BY GLYCOSYLATION

Antibodies recognising the products of alternatively spliced exons nea r the N-terminus of the leukocyte common antigen, CD45, have been wide ly used to distinguish populations of lymphocytes with different funct ional properties. These alternatively spliced regions contain a high c ontent 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 prot ein determinants. In this study the exons of CD45 were expressed in Es cherichia coli as non-glycosylated proteins fused to glutathione S-tra nsferase (GST). Fourteen out of 17 mAbs specific for human CD45R react ed with a fusion protein containing exons 4, 5 and 6 (ABC) of human CD 45, and four out of six mAbs specific for rat CD45R reacted with an eq uivalent rat protein, mAbs recognising the product of rat exon B are r eported for the first time. Kinetic analysis of MRC OX22 antibody bind ing to spleen CD45 and to GST fusion proteins showed that the carbohyd rate affected the kinetics of binding of antibodies to the protein bac kbone. In conclusion, heterogeneity in the glycosylation of heavily O- glycosylated cell surface proteins can affect interactions of these pr oteins both directly through the carbohydrate and indirectly through e ffects on the protein backbone.