ATTACHMENT OF OLIGOSACCHARIDES TO PEPTIDE ANTIGEN PROFOUNDLY AFFECTS BINDING TO MAJOR HISTOCOMPATIBILITY COMPLEX CLASS-II MOLECULES AND PEPTIDE IMMUNOGENICITY

To investigate the immunogenicity of glycopeptides, a peptide fragment from hen egg lysozyme, HEL(81-96)-Y (here named 1) which is immunogenic in H-2(k) mice and known to bind to the murine major histocompatibility complex (MHC) class II molecule E(k), was synthesized in five different glycosylated forms. The N-terminal serine of HEL(81-96)-Y was derivatized with D-glucose (2), maltotriose (3), and a branched D-glucose pentasaccharide (3). Furthermore, 1 was prepared with a central serine or asparagine derivatized with the branched D-glucose pentasaccharide (5) and GlcNAc (6), respectively. The ability of the five glycopeptides and the non-glycosylated peptide, labeled with I-125, to bind to the two MMC class II molecules, A(k) and E(k), was Studied using a gel filtration assay. None of them could bind to Ak. Neither 5 nor 6 were able to bind to E(k) Surprisingly 2, 3 and 4 bound better to E(k) than did the non-glycosylated peptide 1. The increased binding varied depending on the type of oligosaccharide attached to the N terminus of the peptide. The better binding to E(k) Of glycopeptide 3 was found to be due to an increased association rate. The binding of 1 as well as 3 was optimal at pH 5.0. Functional studies showed that 4 was able to elicit a heteroclitic proliferative response from T cells of mice immunized with the native non-glycosylated peptide. Circular dichroism studies of 1 and 3 indicated a more unordered structure of 3 and a predominant alpha-helical conformation of 1, suggesting that the MHC class II molecule may bind to peptides which are in a non-alpha-helical conformation. These results demonstrate that glycosylation has considerable influence on peptide inzmunogenicity for T lymphocytes.