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.
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 Thr(3), Thr(6), Thr(5), Thr(2), Thr(4) and Thr(1), respectively. An exception was the presence of a glycopeptide with three GalNAc residues at Thr(1), Thr(4) and Thr(5). 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.
Iida
S
S
Laboratory 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.
Takeuchi
H
H
Kato
K
K
Yamamoto
K
K
Irimura
T
T
D000116:Acetylgalactosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003110:Colonic Neoplasms / Q000378:metabolism* / Q000473:pathology; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D008861:Microsomes / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D055262:Mucin-2; D009077:Mucins / Q000737:chemistry / Q000378:metabolism*; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D020449:Repetitive Sequences, Amino Acid; D020539:Sequence Analysis, Protein; D013912:Threonine / Q000378:metabolism*; D014407:Tumor Cells, Cultured
C524625:MUC2 protein, human; D055262:Mucin-2; D009077:Mucins; D009842:Oligopeptides; D010446:Peptide Fragments; D013912:Threonine; D000116:Acetylgalactosamine