title: High density O-glycosylation of the MUC2 tandem repeat unit by N-acetylgalactosaminyltransferase-3 in colonic adenocarcinoma extracts.
abstract: A synthetic peptide corresponding to the human MUC2 tandem repeat unit was glycosylated in vitro using UDP-GalNAc and extracts of colonic adenocarcinoma and paired normal mucosa, followed by fractionation of the products by reverse phase high-performance liquid chromatography. Several peaks of glycopeptides with different numbers of GalNAc residues attached were detected. It is notable that the adenocarcinoma extract was capable of glycosylating peptides to a much greater extent than was normal mucosa. The levels of mRNA for N-acetylgalactosaminyltransferases-1, -2, and -3 were determined by reverse transcription-PCR. Only N-acetylgalactosaminyltransferase-3 mRNA was expressed at a higher level in the adenocarcinoma than in the normal tissue. When the MUC2 tandem repeat peptide was glycosylated with a mixture of the normal mucosa extract and recombinant N-acetylgalactosaminyltransferase-3, larger amounts of glycopeptides with higher contents of GalNAc residues were produced. The MUC2 tandem repeat peptides glycosylated extensively by recombinant N-acetylgalactosaminyltransferase-1, -2, or -3 were prepared and characterized. Substitution at each Thr residue, as revealed by Edman degradation sequencing, in conjunction with evidence obtained on mass spectrometry indicated a heterogeneous pattern of site-specific glycosylation within the MUC2 tandem repeat. It was found that maximum numbers of 6, 8, and 11 GalNAc residues were incorporated by N-acetylgalactosaminyltransferases-1, -2, and -3, respectively, and that only N-acetylgalactosaminyltransferase-3 could completely glycosylate both consecutive sequences composed of three and five Thr residues in the MUC2 tandem repeat unit. These results suggest that O-glycosylation of the clustered Thr residues is a selective process controlled by N-acetylgalactosaminyltransferase-3 in the synthesis of clustered carbohydrate antigens.
authors.affiliation: Department of Biotechnology, Faculty of Engineering, Kyoto Sangyo University, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Inoue
authors.forename: S
authors.initials: S
authors.lastname: Takahashi
authors.forename: I
authors.initials: I
authors.lastname: Yamashina
authors.forename: M
authors.initials: M
authors.lastname: Kaibori
authors.forename: T
authors.initials: T
authors.lastname: Okumura
authors.forename: Y
authors.initials: Y
authors.lastname: Kamiyama
authors.forename: S
authors.initials: S
authors.lastname: Vichier-Guerre
authors.forename: D
authors.initials: D
authors.lastname: Cantacuzène
authors.forename: H
authors.initials: H
authors.lastname: Nakada
mesh_terms: D000116:Acetylgalactosamine / Q000378:metabolism; D000230:Adenocarcinoma / Q000201:enzymology* / Q000276:immunology / Q000378:metabolism; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000096:biosynthesis* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003110:Colonic Neoplasms / Q000201:enzymology* / Q000276:immunology / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007202:Indicators and Reagents; D007413:Intestinal Mucosa / Q000201:enzymology / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D017879:Isothiocyanates; D055262:Mucin-2; D009077:Mucins / Q000378:metabolism*; D017350:N-Acetylgalactosaminyltransferases / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020080:Tandem Repeat Sequences; D014020:Tissue Extracts / Q000378:metabolism; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives
chemical_list: D015295:Antigens, Tumor-Associated, Carbohydrate; D007202:Indicators and Reagents; D007527:Isoenzymes; D017879:Isothiocyanates; C524625:MUC2 protein, human; D055262:Mucin-2; D009077:Mucins; D012333:RNA, Messenger; D014020:Tissue Extracts; C012926:4-(N,N-dimethylaminoazobenzene)-4'-isothiocyanate; D004124:p-Dimethylaminoazobenzene; D017350:N-Acetylgalactosaminyltransferases; D000116:Acetylgalactosamine