High density O-glycosylation of the MUC2 tandem repeat unit by N-acetylgalactosaminyltransferase-3 in colonic adenocarcinoma extracts

A synthetic peptide corresponding to the human MUC2 tandem repeat unit was glycosylated in vitro using UDP-GalNAc and extracts of colonic adenocarcino ma and paired normal mucosa, followed by fractionation of the products by r everse phase high-performance Liquid chromatography. Several peaks of glyco peptides 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 mRN A for N-acetylgalactosaminyltransferases-1, -2, and -3 were determined by r everse transcription-PCR, Only N-acetylgalactosaminyltransferase-3 mRNA was expressed at a higher level in the adenocarcinoma than in the normal tissu e. When the MUC2 tandem repeat peptide was glycosylated with a mixture of t he 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 an d characterized, Substitution at each Thr residue, as revealed by Edman deg radation sequencing, in conjunction with evidence obtained on mass spectrom etry indicated a heterogeneous pattern of site-specific glycosylation withi n 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 sugges t that O-glycosylation of the clustered Thr residues is a selective process controlled by N-acetylgalactosaminyltransferase-3 in the synthesis of clus tered carbohydrate antigens.