title: Synthesis, conformation and T-helper cell stimulation of an O-linked glycopeptide epitope containing extended carbohydrate side-chains.
abstract: 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.
authors.affiliation: The Wistar Institute, Philadelphia, PA 19104, USA.
authors.forename: Mare
authors.initials: M
authors.lastname: Cudic
authors.forename: Hildegund C J
authors.initials: HC
authors.lastname: Ertl
authors.forename: Laszlo
authors.initials: L
authors.lastname: Otvos
mesh_terms: D000818:Animals; D002236:Carbohydrate Conformation; D018984:Epitopes, T-Lymphocyte / Q000737:chemistry*; D006020:Glycopeptides / Q000138:chemical synthesis* / Q000737:chemistry / Q000276:immunology*; D006031:Glycosylation; D006825:Hybridomas / Q000276:immunology; D007111:Immunity, Cellular / Q000187:drug effects; D016056:Immunodominant Epitopes / Q000737:chemistry; D008213:Lymphocyte Activation / Q000187:drug effects / Q000276:immunology; D051379:Mice; D008809:Mice, Inbred C3H; D008969:Molecular Sequence Data; D009698:Nucleoproteins / Q000737:chemistry; D009844:Oligosaccharides / Q000737:chemistry; D013329:Structure-Activity Relationship; D006377:T-Lymphocytes, Helper-Inducer / Q000187:drug effects* / Q000276:immunology
chemical_list: D018984:Epitopes, T-Lymphocyte; D006020:Glycopeptides; D016056:Immunodominant Epitopes; D009698:Nucleoproteins; D009844:Oligosaccharides; C090319:peptide 31D