Glycobiology Advance Access originally published online on September 29, 2004
Glycobiology 2005 15(2):177-191; doi:10.1093/glycob/cwh158
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Glycobiology vol. 15 no. 2 © Oxford University Press 2005; all rights reserved.
A MUC1 tandem repeat reporter protein produced in CHO-K1 cells has sialylated core 1 O-glycans and becomes more densely glycosylated if coexpressed with polypeptide-GalNAc-T4 transferase
2 Department of Medical Biochemistry, Göteborg University, 413 90 Gothenburg, Sweden, and 3 Cancer Research UK Breast Cancer Biology, Guy's Hospital, London SE1 9RT, United Kingdom
1 To whom correspondence should be addressed; e-mail: gunnar.hansson{at}medkem.gu.se
Received on June 9, 2004; revised on September 21, 2004; accepted on September 22, 2004
A recombinant mucin O-glycosylation reporter protein, containing 1.7 tandem repeats (TRs) 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 liquid chromatography–mass spectrometry (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
2-3Galß1-3GalNAcol, NeuAc2-3Galß1-3(NeuAc2-6)GalNAcol, Galß1-3(NeuAc2-6)GalNAcol, and Galß1-3GalNAcol, the two first being most abundant. Coexpression 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 () 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.
Key words: Edman sequencing / glycosyltransferase / mass spectrometry / mucin / O-glycosylation
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