Mucin 2 (MUC2) is the major intestinal mucin. O-glycans are attached to MUC2 in a potentially diverse arrangement, which is crucial for their interaction with endogeneous and exogeneous lectins. In the present report, five oligopeptides [PTTTPITTTT(K), ITTTTTVTPT(K), TVTPTPTPTG(K), PTPTGTQTPT(K) and TQTPTTTPIT(K)] corresponding to portions of the MUC2 tandem repeat domain were synthesized, and incubated with UDP-N-acetyl-D-galactosamine (UDP-GalNAc) and detergent-soluble microsomes, prepared from the human colon carcinoma cell line LS174T. The products were fractionated by reverse-phase HPLC and characterized by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry. Oligopeptides with GalNAc residues derived from PTTTPITTTT(K), containing consecutive threonine residues, were found to be glycosylated with 1-7 GalNAc residues per single peptide. The sequences of all glycopeptides were determined. The results indicated that the predominant sites of the first through to the sixth GalNAc incorporation were Thr3, Thr6, Thr5, Thr2, Thr4 and Thr1, respectively. An exception was the presence of a glycopeptide with three GalNAc residues at Thr1, Thr4 and Thr5. Oligopeptides containing alternating threonine residues [TVTPTPTPTG(K) and PTPTGTQTPT(K)] were not fully glycosylated under the same conditions or even after prolonged incubations. Thus, the preferential order and maximum number of GalNAc incorporation into threonine residues of MUC2 core peptides depends on the peptide sequence, when the microsome fraction of LS174T cells is used as a source of N-acetyl-D-galactosaminyltransferases.
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Research Article|
April 10 2000
Order and maximum incorporation of N-acetyl-d-galactosamine into threonine residues of MUC2 core peptide with microsome fraction of human-colon-carcinoma LS174T cells
Shin-ichiro IIDA;
Shin-ichiro IIDA
1Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
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Hideyuki TAKEUCHI;
Hideyuki TAKEUCHI
1Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
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Kentaro KATO;
Kentaro KATO
1Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
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Kazuo YAMAMOTO;
Kazuo YAMAMOTO
1Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
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Tatsuro IRIMURA
Tatsuro IRIMURA
1
1To whom correspondence should be addressed (e-mail irimura@;mol.f.u-tokyo.ac.jp).
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Publisher: Portland Press Ltd
Received:
September 06 1999
Revision Received:
December 07 1999
Accepted:
February 11 2000
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 2000
2000
Biochem J (2000) 347 (2): 535–542.
Article history
Received:
September 06 1999
Revision Received:
December 07 1999
Accepted:
February 11 2000
Citation
Shin-ichiro IIDA, Hideyuki TAKEUCHI, Kentaro KATO, Kazuo YAMAMOTO, Tatsuro IRIMURA; Order and maximum incorporation of N-acetyl-d-galactosamine into threonine residues of MUC2 core peptide with microsome fraction of human-colon-carcinoma LS174T cells. Biochem J 15 April 2000; 347 (2): 535–542. doi: https://doi.org/10.1042/bj3470535
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