Synthesis, conformation and T-helper cell stimulation of an O-linked glycopeptide epitope containing extended carbohydrate side-chains.

To answer the question whether or not T cells to immunodominant protein fragments recognize glycosylated antigens, we synthesized a series of glycopeptides corresponding to peptide 31D, a major T-helper cell epitope of the rabies virus nucleoprotein. Thr4 of the epitope is known to allow mono- or disaccharide side-chain substitutions in either alpha- or beta-anomeric configuration without interfering with MHC-binding. To model naturally occurring glycoprotein fragments that carry extended sugar chains, we prepared Fmoc-Ser/Thr-OPfp building blocks containing alpha- and beta-linked linear tri- and heptasaccharides. Peptide 31D was synthesized with the complex carbohydrates attached to Thr4, and the T-helper cell activity of the glycopeptides was determined. Addition of alpha-linked carbohydrates, that mimic most of the natural O-linked glycoproteins, resulted in a major drop in the T-cell stimulatory ability in a sugar length-dependent manner. In contrast, the cytosolic glycoprotein mimicking beta-linked glycopeptides retained their T-cell stimulatory activity, with the trisaccharide-containing analogue being almost as potent as the unglycosylated peptide. When the peptides were preincubated with diluted human serum, all peptides lost their ability to stimulate the 9C5.D8-H hybridoma. These findings indicated that (i) in contrast to cytosolic glycosylation, incorporation of long O-linked carbohydrates into T-helper cell epitopes abrogates the antigenicity of these protein fragments, and (ii) glycosylation is not a viable alternative to improve the immunogenic properties of subunit peptide vaccines. Glycosylation with all four carbohydrate moieties similarly destroyed the inducible alpha-helical structure of peptide 31D as detected by CD, indicating that the differences in the T-cell activity were not due to different peptide conformations.