IDENTIFICATION OF AMINO-ACIDS AT THE JUNCTION OF EXON-3 AND EXON-7 THAT ARE USED FOR THE GENERATION OF GLYCOSYLATION-RELATED HUMAN CD45RO AND CD45RO-LIKE ANTIGEN SPECIFICITIES

The CD45 transmembrane protein tyrosine phosphatase plays an essential role in lymphocyte activation. In humans, CD45 is composed of five is oforms that are generated by alternative splicing of three exons of a common precursor mRNA. Expression of the smallest molecular mass 180-k D CD45 isoform (CD45-O) results from splicing out of exons 4(A), 5(B), and 6(C), which encode peptide regions near the NH2 terminus, and is regulated during T cell maturation and activation. Two monoclonal anti bodies (mAb), UCHL1 (anti-CD45RO) and A6 (anti-CD45RO-like), were stud ied that selectively bind to murine transfectant cells expressing the human CD45-0 isoform. The anti-CD45RO-like A6 mAb, but not the anti-CD 45RO UCHL1 mAb, also weakly reacted with transfectant cells expressing the human CD45 isoforms that contained exons 4 and 5(AB), or exon 5(B ) encoded sequences. The structural basis of the antigen specificities of these two different human anti-CD45RO mAbs was investigated at the molecular level by using potential glycosylation-defective CD45-0 iso form variants containing amino acid substitutions at the junction of e xons 3 and 7. Replacement of the threonine residue at position 8 (last amino acid encoded in exon 3 and a putative O-linked carbohydrate anc horage site) by an alanine, completely abrogated the reactivity of the UCHL1 mAb, but did not affect that of the A6 mAb. Conversely, replace ment of either the asparagine at position 174 or the serine at positio n 176 (the first two putative carbohydrate anchorage sites in exon 7) by alanine, abrogated the reactivity of the A6 mAb, but not that of th e UCHL1 mAb. Both the UCHL1 and A6 epitopes were dependent on the pres ence of O-linked carbohydrates; and the UCHL1, but not the A6 epitope, was dependent on the presence of sialic acid. These results demonstra te a pivotal role for the amino acids encoded at the junction of exons 3 and 7 for the generation of glycosylation-related CD45RO epitopes t hat are expressed in a cell lineage- and activation-regulated fashion.