The lectin domain of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 1 is involved in O-glycosylation of a polypeptide with multiple acceptor sites.
Mucin type O-glycosylation begins with the transfer of GalNAc to serine and threonine residues on proteins by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminlytransferases. These enzymes all contain a lectin-like (QXW)(3) repeat sequence at the C terminus that consists of three tandem repeats (alpha, beta, and gamma). The putative lectin domain of one of the most ubiquitous isozymes, GalNAc-T1, is reportedly not functional. In this report, we have reevaluated the role of the GalNAc-T1 lectin domain. Deletion of the lectin domain resulted in a complete loss of enzymatic activity. We also found that GalNAc-T1 has two activities distinguished by their sensitivities to inhibition with free GalNAc; one activity is sensitive, and the other is resistant. In our experiments, the former activity is represented by the O-glycosylation of apomucin, an acceptor that contains multiple glycosylation sites, and the latter is represented by synthetic peptides that contain a single glycosylation site. Site-directed mutagenesis of the lectin domain selectively reduced the former activity and identified Asp(444) in the alpha repeat as the most important site for GalNAc recognition. A further reduction of the GalNAc-inhibitable activity was observed when both Asp(444) and the corresponding aspartate residues in the beta and the gamma repeats were mutated. This suggests a cooperative involvement of each repeat unit in the glycosylation of polypeptides with multiple acceptor sites.
D000588:Amines / Q000737:chemistry; D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D017353:Gene Deletion; D006031:Glycosylation; D007700:Kinetics; D037102:Lectins / Q000737:chemistry*; D008956:Models, Chemical; D009005:Monosaccharides / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017350:N-Acetylgalactosaminyltransferases / Q000737:chemistry* / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012276:Ricin / Q000737:chemistry; D012685:Sepharose / Q000378:metabolism; D013379:Substrate Specificity; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D000588:Amines; D003545:Cysteine; D004220:Disulfides; D037102:Lectins; D009005:Monosaccharides; D017350:N-Acetylgalactosaminyltransferases; D011994:Recombinant Proteins; D012276:Ricin; D012685:Sepharose; D000097763:Polypeptide N-acetylgalactosaminyltransferase