Attachment of oligosaccharides to peptide antigen profoundly affects binding to major histocompatibility complex class II molecules and peptide immunogenicity

Abstract

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 (4). 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 ¹²⁵I, to bind to the two MHC class II molecules, A^k and E^k, was studied using a gel filtration assay. None of them could bind to A^k. 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 4 was found to be due to an increased association rate. The binding of 1 as well as 4 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 4 indicated a more unordered structure of 4 and a predominant α-helical conformation of 1, suggesting that the MHC class II molecule may bind to peptides which are in a non-α-helical conformation. These results demonstrate that glycosylation has considerable influence on peptide immunogenicity for T lymphocytes.