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.
Center of Biochemistry, Medical Faculty, University of Cologne, Köln, Germany.
Tanja
T
Ninkovic
Franz-Georg
FG
Hanisch
D020816:Amino Acid Motifs / Q000276:immunology; D017951:Antigen Presentation / Q000276:immunology; D000951:Antigens, Neoplasm / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D002474:Cell-Free System / Q000201:enzymology / Q000276:immunology / Q000378:metabolism; D003713:Dendritic Cells / Q000201:enzymology / Q000276:immunology; D011992:Endosomes / Q000201:enzymology / Q000276:immunology; D018984:Epitopes, T-Lymphocyte / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007167:Immunotherapy; D018396:Mucin-1; D009077:Mucins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D009369:Neoplasms / Q000201:enzymology / Q000276:immunology / Q000628:therapy; D010455:Peptides / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D013602:T-Lymphocytes, Cytotoxic / Q000201:enzymology / Q000276:immunology
D000951:Antigens, Neoplasm; D018984:Epitopes, T-Lymphocyte; C060192:MUC1 protein, human; D018396:Mucin-1; D009077:Mucins; D010455:Peptides; D011134:Polysaccharides; D046988:Proteasome Endopeptidase Complex