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Glycobiology Advance Access published online on September 29, 2004
Glycobiology, doi:10.1093/glycob/cwh158
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Received June 9, 2004
Revised September 21, 2004
Accepted September 22, 2004

ORIGINAL ARTICLES

A MUC1 tandem repeat reporter protein produced in CHO-K1 cells has sialylated core 1 O-glycans and becomes more densely glycosylated if co-expressed with polypeptide-GalNAc-T4 transferase

Fredrik J. Olson 1, Malin Bäckström 1, Hasse Karlsson 1, Joy Burchell 2, and Gunnar C. Hansson 1*

1 Department of Medical Biochemistry, Göteborg University, 413 90 Gothenburg, Sweden
2 Cancer Research UK Breast Cancer Biology, Guy's Hospital, London SE1 9RT, UK

* To whom correspondence should be addressed. E-mail: gunnar.hansson{at}medkem.gu.se.


  Abstract

A recombinant mucin O-glycosylation reporter protein, containing 1.7 tandem repeats (TR) from the transmembrane mucin MUC1, was constructed. The reporter protein, MUC1(1.7TR)-IgG2a, was produced in CHO-K1 cells to study the glycosylation of the MUC1 TR and the in vivo role of polypeptide-GalNAc-T4 glycosyltransferase. N-terminal sequencing of MUC1(1.7TR)-IgG2a showed that all five potential O-glycosylation sites within the TR were used, with an average density of 4.5 glycans per repeat. The least occupied site was Thr in the PDTR motif where 75 % of the molecules were glycosylated, compared to 88 - 97 % at the other sites. This glycan density was confirmed by an alternative LC-MS based approach. The O-linked oligosaccharides were released from MUC1(1.7TR)-IgG2a and analyzed by nano-LC-MS and LC-MS/MS. Four oligosaccharides were present, NeuAc{alpha}2-3Gal{beta}1-3GalNAcol, NeuAc{alpha}2-3Gal{beta}1-3(NeuAc{alpha}2-6)GalNAcol, Gal{beta}1-3(NeuAc{alpha}2-6)GalNAcol and Gal{beta}1-3GalNAcol, the two first being most abundant. Co-expression of the human polypeptide-GalNAc-T4 transferase with MUC1(1.7TR)-IgG2a increased the glycan occupancy at Thr in PDTR, Ser in VTSA and Ser in GSTA, supporting the function of GalNAc-T4 proposed from previous in vitro studies. The expression of GalNAc-T4 with a mutation in the first lectin domain ({alpha}) had no glycosylation effect on PDTR and GSTA, but surprisingly gave a dominant negative effect with a decreased glycosylation to around 50% at the Ser in VTSA. The results show that introduction of glycosyltransferases can specifically alter the sites for O-glycosylation in vivo.

Keywords: Mucin; O-glycosylation; Glycosyltransferase; Mass spectrometry; Edman sequencing.
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