Presentation of cytosolic glycosylated peptides by human class I major histocompatibility complex molecules in vivo

Antigens presented by class I major histocompatibility complex (MHC) molecu les for recognition by cytotoxic T lymphocytes consist of 8-10-amino-acid-l ong cytosolic peptides. It is not known whether posttranslationally modifie d peptides are also presented by class I MHC molecules in vivo. Many differ ent posttranslational modifications occur on cytoplasmic proteins, includin g a cytosolic O-beta-linked glycosylation of serine and threonine residues with N-acetylglucosamine (GlcNAc). Using synthetic glycopeptides carrying t he monosaccharide O-beta-GlcNAc substitution on serine residues, we have sh own;that glycopeptides bind efficiently to class I MHC molecules and elicit a glycopeptide-specific cytotoxic T lymphocyte response in mice. In this s tudy, we provide evidence that peptides presented by human class I MHC mole cules in vivo encompass a small, significant amount of glycopeptides, const ituting up to 0.1% of total peptide. Further more, we find that carbohydrat e structures present on glycopeptides isolated from class I MHC molecules a re dominated by the cytosolic O-beta-GlcNAc substitution, and synthetic pep tides carrying this substitution are efficiently transported by TAP (transp orter associated with antigen presentation) into the endoplasmic reticulum. Thus, in addition to unmodified peptides, posttranslationally modified cyt osolic peptides carrying O-beta-linked GlcNAc can be presented by class I M HC molecules to the immune system.