Lectin purified human class I MHC-derived peptides: evidence for presentation of glycopeptides in vivo

Previously, using synthetic glycopeptides carrying a natural cytosolic type of monosaccharide O-beta-linked N-acetylglucosamine (GlcNAc) glycosylation of serine residues, we have shown that glycopeptides act as suitable subst rates for TAP-mediated transport into the endoplasmic reticulum (ER), and t hat they bind efficiently to class I major histocompatibility complex (MHC) molecules and can elicit glycopeptide-specific cytotoxic T-lymphocyte (CTL ) responses in mice. Recently, we have reported that peptides presented by human class I MHC molecules in vivo encompass a small but significant amoun t of peptides which seem to be carrying O-beta-linked monosaccharide GlcNAc . In the present report we provide further evidence that glycosylated pepti des are indeed presented by class I MHC molecules in vivo. Thus, peptides d erived from HLA-A*0201 were purified by wheat germ agglutinin (WGA) lectin affinity chromatography as previously described. Subsequently, the peptides contained in the WGA-eluate were subjected to sequence analysis by Edman d egradation. It was found that the peptides derived from HLA-A*0201 which ha d been retained by the O-GlcNAc-binding lectin WGA did indeed carry a HLA-A *0201 binding motif. Furthermore, using an enzymatic labeling procedure we present evidence that the HLA-A*0201-derived peptides which bind to the WGA lectin are glycosylated with terminal GlcNAc residues. Together, these dat a provide further evidence for the natural presentation by human class I MH C of glycopeptides carrying terminal O-GlcNAc residues in vivo.