title: Sequence-variant repeats of MUC1 show higher conformational flexibility, are less densely O-glycosylated and induce differential B lymphocyte responses.
abstract: The human epithelial cancer mucin MUC1 is able to break tolerance and to induce humoral immune responses in healthy subjects and in cancer patients. We recently showed that clusters of sequence-variant repeats are interspersed in the repeat domain of MUC1 at high frequency, which should contribute to the structural and immunological features of the mucin. Here we elucidated the potential effects exerted by sequence-variant repeats on their O-glycosylation. Evidence from in vitro glycosylation with polypeptide N-acetylgalactosaminyltransferases GalNAc-T1 and GalNAc-T2 in concert with mass spectrometric analyses of in vivo glycosylated MUC1 probes from transiently transfected HEK293 cells indicated reduced glycosylation densities of repeats with three concerted replacements: AHGVTSAPESRPAPGSTAPA. The Pro to Ala replacement in STAPA exerts not only proximal effects on the ppGalNAc-T2 preferred site at -3 and -4, but also more distant effects on the ppGalNAc-T1 preferred site at -15 (TSAPESRPAPGSTAPA). We also examined the conformational changes of MUC1 glycopeptides induced by the concerted DT to ES replacements and revealed a higher conformational flexibility of ES/P peptides compared to DT/P peptides. Differences in conformational flexibilities and in O-glycosylation densities could underlie the observed differential humoral responses in humans. We were able to show that the natural immunoglobulin G (IgG) responses to the repeat domain of MUC1 in sera from nonmalignant control subjects are preferentially directed to variant repeat clusters. In contrast, the IgG response in patients with adenocarcinoma shifted to higher frequencies of preferential DTR peptide binding.
authors.affiliation: Department of Obstetrics and Gynaecology, Vrije Universiteit Medical Center, De Boelelaan 1117, 1081 HV, Amsterdam, Netherlands.
authors.forename: Silvia
authors.initials: S
authors.lastname: von Mensdorff-Pouilly
authors.forename: Leo
authors.initials: L
authors.lastname: Kinarsky
authors.forename: Katja
authors.initials: K
authors.lastname: Engelmann
authors.forename: Stephan E
authors.initials: SE
authors.lastname: Baldus
authors.forename: René H
authors.initials: RH
authors.lastname: Verheijen
authors.forename: Michael A
authors.initials: MA
authors.lastname: Hollingsworth
authors.forename: Vladimir
authors.initials: V
authors.lastname: Pisarev
authors.forename: Simon
authors.initials: S
authors.lastname: Sherman
authors.forename: Franz-Georg
authors.initials: FG
authors.lastname: Hanisch
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology*; D001943:Breast Neoplasms / Q000276:immunology / Q000473:pathology; D005260:Female; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007075:Immunoglobulin M / Q000276:immunology; D008297:Male; D051379:Mice; D018396:Mucin-1 / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D017350:N-Acetylgalactosaminyltransferases / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D010051:Ovarian Neoplasms / Q000276:immunology / Q000473:pathology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013274:Stomach Neoplasms / Q000276:immunology / Q000473:pathology; D000097763:Polypeptide N-acetylgalactosaminyltransferase
chemical_list: D007074:Immunoglobulin G; D007075:Immunoglobulin M; D018396:Mucin-1; D017350:N-Acetylgalactosaminyltransferases; D011994:Recombinant Proteins; D000097763:Polypeptide N-acetylgalactosaminyltransferase