Glycobiology Advance Access originally published online on April 6, 2005
Glycobiology 2005 15(8):735-746; doi:10.1093/glycob/cwi058
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Sequence-variant repeats of MUC1 show higher conformational flexibility, are less densely O-glycosylated and induce differential B lymphocyte responses
2 Department of Obstetrics and Gynaecology, Vrije Universiteit Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, Netherlands; 3 Eppley Cancer Institute, UNMC, 986805 Nebraska Medical Center, Omaha, NE 68198-6805; 4 Institute of Biochemistry II, Medical Faculty, University of Cologne, Joseph-Stelzmann-Str. 52, 50931 Köln, Germany; 5 Institute of Pathology, University Clinic, University of Cologne, Joseph-Stelzmann-Str. 9, 50931 Köln, Germany; and 6 Central Bioanalytics, Center of Molecular Medicine, University of Cologne, Joseph-Stelzmann-Str. 52, 50931 Köln, Germany
1 To whom correspondence should be addressed; e-mail: franz.hanisch{at}uni-koeln.de
Received on September 24, 2004; revised on March 23, 2005; accepted on March 23, 2005
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.
Key words: B cell response / cancer/MUC1 / O-glycosylation / peptide conformation
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