O-glycosylated human MUC1 repeats are processed in vitro by immunoproteasomes.

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.