COMPARISON OF THE EFFECTS OF AMINO-ACID SUBSTITUTIONS AND BETA-N-GLYCOSYLATION VS ALPHA-O-GLYCOSYLATION ON THE T-CELL STIMULATORY ACTIVITY AND CONFORMATION OF AN EPITOPE ON THE RABIES VIRUS GLYCOPROTEIN

The first potential N-glycosylation site of the rabies virus glycoprot ein, the antigen that carries epitopes for glycoprotein-specific T-cel ls and virus neutralizing antibodies, is glycosylated inefficiently. R ecently, we showed that addition of a beta-N-acetyl-glucosamine moiety to the asparagine residue in the corresponding synthetic fragment V V E D E G C T N L S G F (amino acids 29-41), significantly diminished t he T-cell stimulatory activity and reduced the characteristic alpha-he licity of the peptide. The amino acid sequence of the glycoprotein in this region exhibits some degree of variability among different rabies virus and rabies virus related strains, including the replacement of the asparagine residue with aspartic acid or threonine. In the current study, stimulation of a specific T-cell clone by various viral strain s and appropriate tridecapeptide sequences and their analogs was inves tigated. The T-cell recognition pattern of the rabies and rabies-relat ed viruses was identical to that of the synthetic peptides representin g the respective epitope sequences. While the asparagine could be repl aced without complete loss of T-cell stimulatory activity, amino acid modifications at the C-terminus of the peptide were not tolerated. In contrast to glycosylation of the asparagine, coupling of an N-acetyl-g alactosamine moiety at the serine, or galactosyl-N-acetyl-galactosamin e moieties at the threonines preceding or replacing the asparagine (al l O-linked sugars in the natural alpha-anomeric configuration) resulte d in epitopes that lowered rather than abolished the T-cell stimulator y activity. All non-glycosylated peptides assumed a low-to-medium heli city in trifluoroethanol. O-glycosylation was more efficient than N-gl ycosylation in breaking the helical conformation of the peptides to re sult in the formation of reverse-turns or unordered structure.