Key words: antigen presentation; class I MHC; glycopeptides; HLA; N-acetylglucosamine; post-translational modifications Acknowledgments: This work was supported by the Carlsberg Foundation, the Wellcome Trust, the Danish Medical Research Council, the Novo Nordisk Foundation, the Alfred Benzon Foundation, and the Danish Cancer Society. Received 21 September 1999, revised, accepted for publication 16 May 2000 Copyright c Munksgaard 2000 Tissue Antigens . ISSN 0001-2815 Tissue Antigens 2000: 56: 129–135 Printed in Denmark . All rights reserved 129 I.B. Kastrup Lectin purified human class I MHC-derived S. Stevanovic peptides: evidence for presentation of G. Arsequell G. Valencia glycopeptides in vivo J. Zeuthen H.-G. Rammensee T. Elliott J.S. Haurum Abstract: Previously, using synthetic glycopeptides carrying a natural cytosolic type of monosaccharide O-b-linked N-acetylglucosamine (GlcNAc) glycosylation of serine residues, we have shown that glycopeptides act as suitable substrates for TAP-mediated transport into the endoplasmic retic- ulum (ER), and that 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 amount of peptides which seem to be carrying O-b- linked monosaccharide GlcNAc. In the present report we provide further evidence that glycosylated peptides are indeed presented by class I MHC molecules in vivo. Thus, peptides derived from HLA-A*0201 were purified by wheat germ agglutinin (WGA) lectin affinity chromatography as pre- viously described. Subsequently, the peptides contained in the WGA-eluate were subjected to sequence analysis by Edman degradation. It was found that the peptides derived from HLA-A*0201 which had 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 evi- dence that the HLA-A*0201-derived peptides which bind to the WGA lectin are glycosylated with terminal GlcNAc residues. Together, these data provide further evidence for the natural presentation by human class I MHC of glycopeptides carrying terminal O-GlcNAc residues in vivo. Previously, we have demonstrated that peptides carrying a natural cytosolic type of O-linked monosaccharide glycosylation (O-GlcNAc) can be transported by TAP into the endoplasmic reticulum (ER) (1). Furthermore, such peptides bind efficiently to class I major histo- compatibility complex (MHC) molecules (2, 3), and elicit glycopep- tide-specific cytotoxic T-lymphocytes (CTL) in mice (2). The crystal structures of two H-2D b -glycopeptide complexes revealed how the glycan is exposed for recognition by glycopeptide-specific CTL (4). Finally, peptides isolated from human spleen class I MHC contain a small subset of peptides which carry the natural cytosolic O-b- GlcNAc glycosylation in vivo, as evidenced by their ability to act as Authors’ affiliations: I.B. Kastrup 1 , S. Stevanovic 2 , G. Arsequell 3 , G. Valencia 3 , J. Zeuthen 1 , H.-G. Rammensee 2 , T. Elliott 4 , J.S. Haurum 1,4 1 Institute of Cancer Biology, Danish Cancer Society, Copenhagen, Denmark, 2 Institute for Cell Biology, Department of Immunology, University of Tübingen, Germany, 3 Unit of Glycoconjugate, Chemistry, CIP-CSIC, Barcelona, Spain, 4 Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom Correspondence to: John S. Haurum Danish Cancer Society Strandboulevarden 49 2100 Copenhagen Denmark Fax: π45 3525 7721 e-mail: haurum/dadlnet.dk