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Glycobiology Advance Access published online on December 18, 2008
Glycobiology, doi:10.1093/glycob/cwn144
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© The Author 2008. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Localisation of O-glycans in MUC1 glycoproteins using electron-capture dissociation fragmentation mass spectrometry

Carina Sihlbom1, Iris van Dijk Härd1, Martin E Lidell1, Thomas Noll2,3, Gunnar C Hansson1 and Malin Bäckström1,4

1 Institute of Biomedicine, Department of Medical Biochemistry, University of Gothenburg, 405 30 Gothenburg, Sweden
2 Research Centre Jülich, Institute for Biotechnology 2, 52425 Jülich, Germany
3 Present address: Technical Faculty, University of Bielefeld, 33614 Bielefeld, Germany

To whom correspondence should be addressed; Tel. +46 31 786 3496, Fax +46 31 416108, e-mail: malin.backstrom{at}medkem.gu.se

MUC1 is a mucin glycoprotein containing multiple tandem repeats of 20 amino acids, with five serines and threonines that can be O-glycosylated. Here, we investigated the O-glycosylation site occupancy in MUC1 glycoproteins produced in two mutant CHO cell lines, Lec3.2.8.1 and ldlD. We found that the average site occupancy was higher in MUC1 from Lec3.2.8.1 than from ldlD and that the occupancy increased with the number of tandem repeats in the protein and also depended on the culture conditions used for production. Moreover, we describe the successful use of electron-capture dissociation (ECD) fragmentation, coupled to on-line liquid chromatography mass spectrometry, to determine the glycosylation of individual sites in recombinant MUC1 proteins with 16 tandem repeats. We analysed MUC1 tandem repeat peptides with 1–5 GalNAc residues by ECD fragmentation and we found that the first site to be glycosylated was either Ser-5 or Thr-6, with a second addition of GalNAc at Thr-14. For peptides with three GalNAc residues, several different variants of glycopeptides were found, indicating a heterogeneous order of glycosylation at this stage. In contrast, only one variant was found for peptides with four GalNAc residues, where Thr-19 in the PDTR motif was left unglycosylated, indicating that this site is glycosylated last. The results gave novel insight into the order of GalNAc substitution in MUC1 in vivo.

Key words: Chinese hamster ovary cells / mucin / O-glycosylation / perfusion culture / recombinant protein


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