The effects of post-translational side-chain modifications on the stimulatory activity, serum stability and conformation of synthetic peptides carrying T helper cell epitopes

Abstract

Peptides 31D and VF 13, corresponding to the rabies virus nucleo- and glycoproteins, respectively, vigorously stimulate T helper cells of the appropriate specificity. Earlier we showed how internal and external glycosylation affects the major histocompatibility complex molecule (MHC)-binding ability and conformation of these T-cell epitopes (Otvos et al. (1994) Biochim. Biophys. Acta 1224, 68–76; Otvos et al. (1995) Biochim. Biophys. Acta 1267, 55–64). In the current report, we examined the T-helper cell stimulatory ability after introduction of a new set of post-translational modifications. To obtain general information concerning the effects of amino acid side-chain modifications on other biochemical properties of protein fragments, we studied the serum stability and the conformation of the 31D and VF13 peptides. We found that the extent of the reduction of the T-cell stimulatory activity depends upon the location in the sequence of the host amino acid residue. Generally, (β-linked sugars in mid-chain positions had a greater inhibitory effect than a-linked sugars attached to identical amino acids. In a case where mid-chain glycosylation just marginally reduced the T-cell stimulatory activity, the β-linked glycopeptide was significantly more resistant to serum proteases. This finding suggests that addition of β-linked carbohydrates might be superior to the addition of a-linked sugars for vaccine development, and generally for peptide agonist drug design. In addition, data presented here provide the first documentation that phosphorylation and sulfation of tyrosine residues may retain the MHC-binding ability and T-cell stimulatory activity of class II epitopes. The sulfated and the phosphorylated 31D peptides exhibited considerably increased serum stability compared to the unmodified parent peptide. Finally, all post-translational modifications destabilized the dominant α-helical or turn structures of the peptides presented in aqueous trifluoroethanol mixtures. While the circular dichroism spectra of the α- and β-linked VF13 glycopeptides with monosaccharides were almost indistinguishable, the structure of the glycopeptides depended upon the length of the sugar moiety. Significantly, incorporation of sulfate or phosphate groups resulted in identical peptide conformations.