Nuclear Magnetic Resonance-Based Dissection of a Glycosyltransferase Specificity for the Mucin MUC1 Tandem Repeat
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- 作者：Richard D. Brokx ; Leigh Revers ; Qinghong Zhang ; Shaoxian Yang ; Tapas K. Mal ; Mitsuhiko Ikura ; and Jean Garié ; py
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：December 2, 2003
- 年：2003
- 卷：42
- 期：47
- 页码：13817 - 13825
- 全文大小：149K
- 年卷期：v.42,no.47(December 2, 2003)
- ISSN：1520-4995
文摘
The human glycoprotein MUC1 mucin plays a critical role in cancer progression. Breast, ovarian,and colon cancer cells often display unique cell-surface antigens corresponding to aberrantly glycosylatedforms of the MUC1 tandem repeat. In this report, 15N- and 13C-labeled forms of a recombinant MUC1construct containing five tandem repeats were used as substrates to define the order and kinetics of additionof N-acetylgalactosamine (GalNAc) moieties by a recombinant active form of the human enzyme UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase I (ppGalNAc-T1; residues 40-559). HeteronuclearNMR experiments were performed to assign resonances associated with the two serines (Ser5 and Ser15)and three threonines (Thr6, Thr14, and Thr19) present in the 20-residue long MUC1 repeat. The kineticsand order of addition of GalNAc moieties (Tn antigen) on the MUC1 construct by human ppGalNAc-T1were subsequently dissected by NMR spectroscopy. Threonine 14 was shown to be rapidly glycosylatedby ppGalNAc-T1 with an initial rate of 25 
M/min, followed by Thr6 (8.6 M/min). The enzyme alsomodified Ser5 at a slower rate (1.7 M/min), an event that started only after the glycosylation of Thr14and Thr6 side chains was mostly completed. Ser15 and Thr19 remained unglycosylated by ppGalNAc-T1. Corresponding O-glycosylation sites within all five tandem repeats were simultaneously modified byppGalNAc-T1, suggesting that each repeat behaves as an independent substrate unit. This studydemonstrated that the hydroxyl oxygens of Thr14 and to a lesser extent Thr 6 represent the two dominantsubstrates modified by ppGalNAc-T1 within the context of a complex MUC1 peptide substrate. Moreimportantly, the availability of defined isotopically labelled MUC1 glycopeptide substrates and the relativesimplicity of their NMR spectra will facilitate the analysis of other transferases within the O-glycosylationpathways and the rational design of tumor-associated MUC1 antigens.
M/min, followed by Thr6 (8.6 M/min). The enzyme alsomodified Ser5 at a slower rate (1.7 M/min), an event that started only after the glycosylation of Thr14and Thr6 side chains was mostly completed. Ser15 and Thr19 remained unglycosylated by ppGalNAc-T1. Corresponding O-glycosylation sites within all five tandem repeats were simultaneously modified byppGalNAc-T1, suggesting that each repeat behaves as an independent substrate unit. This studydemonstrated that the hydroxyl oxygens of Thr14 and to a lesser extent Thr 6 represent the two dominantsubstrates modified by ppGalNAc-T1 within the context of a complex MUC1 peptide substrate. Moreimportantly, the availability of defined isotopically labelled MUC1 glycopeptide substrates and the relativesimplicity of their NMR spectra will facilitate the analysis of other transferases within the O-glycosylationpathways and the rational design of tumor-associated MUC1 antigens.