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O-Glycosylated Human MUC1 Repeats Are Processed In Vitro by Immunoproteasomes¹

Tanja Ninkovic^* and Franz-Georg Hanisch^2,*,

^* Center of Biochemistry, Medical Faculty, and Center for Molecular Medicine Cologne, University of Cologne, Köln, Germany

The targeting of epitopes on tumor-associated glycoforms of human MUC1 represents a primary goal in immunotherapeutic anticancer strategies. Effective immune responses to cancer cells certainly require the activation of specific cytotoxic T cell repertoires by cross-priming of dendritic cells either via immunoproteasomal or by endosomal processing of ectodomain epitopes on MUC1-positive carcinomas. Because no evidence is currently available on the capacities of human immunoproteasomes to cleave mucin-type O-glycosylated peptides, we performed in vitro studies to address the questions of whether glycosylated MUC1 repeats are cleaved by immunoproteasomes and in which way O-linked glycans control the site specificity of peptide cleavage via their localization and structures. We show for the first time that mucin-type O-glycosylated peptides are effective substrates of immunoproteasomes, however, the patterns of cleavage are qualitatively and quantitatively influenced by O-glycosylation. The nonglycosylated MUC1 repeat peptide (clusters of oligorepeats AHGVTSAPDTRPAPGSTAPP or AHGVTSAPESRPAPGSTAPA) is cleaved preferentially within or adjacent to the SAP and GST motifs with formation of a complex fragment pattern that includes major nona- and decapeptides. O-GalNAc modified peptides are largely resistant to proteolysis if these preferred cleavage sites are located adjacent to O-glycosylation, whereas peptides even with elongated glycans at more distant sites can form effective substrates yielding major glycopeptide fragments in the class I size range.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grant 1RO1 CA84106 (to F.-G.H.) and by the Köln-Fortune Programme (to F.-G.H.).

² Address correspondence and reprint requests to Dr. Franz-Georg Hanisch, Institute of Biochemistry II, Medical Faculty, University of Cologne, Joseph-Stelzmann-Strasse 52, 50931 Köln, Germany. E-mail address: franz.hanisch{at}uni-koeln.de

³ Abbreviations used in this paper: DC, dendritic cell; ER, endoplasmic reticulum; TFA, trifluoroacetic acid; ESI, electrospray ionization; Gal, galactose; GalNAc, N-acetylgalactosamine; LC, liquid chromatography; MS, mass spectrometry.