Function of the lectin domain of polypeptide N-acetylgalactosaminyltransferase 1.

All UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases cloned to date contain a lectin domain at the C-terminus, consisting of three tandem repeat sequences (alpha,beta, and gamma). We previously reported that the alpha repeat of one of the most ubiquitous isozymes, GalNAc-T1, is a functional lectin that recognizes O-linked GalNAc residues on the acceptor polypeptides with multiple acceptor sites; the domain appears not to be involved in the glycosylation of acceptors with a single acceptor site. In this report, we studied the function of the beta and gamma repeats in the GalNAc-T1 lectin domain, by site-directed mutagenesis and analysis of the catalytic properties of mutant enzymes.

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.

An efficient assay for dolichyl phosphate-mannose: protein O-mannosyltransferase.

A novel method for quantifying the reaction product from dolichyl phosphoryl mannose:polypeptide mannosyltransferase (protein mannosyl transferase; PMT), was developed. The assay quantifies the amount of radioactivity incorporated into the acceptor peptide YNPTSV from dolichyl phosphoryl [3H]mannose (Dol-P-Man). A novel delivery system, large unilamellar vesicles (LUV), composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), is used to keep the poorly soluble donor substrate, Dol-P-Man, in solution.

Identification of two cysteine residues involved in the binding of UDP-GalNAc to UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 1 (GalNAc-T1).

Biosynthesis of mucin-type O-glycans is initiated by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases, which contain several conserved cysteine residues among the isozymes. We found that a cysteine-specific reagent, p-chloromercuriphenylsulfonic acid (PCMPS), irreversibly inhibited one of the isozymes (GalNAc-T1). Presence of either UDP-GalNAc or UDP during PCMPS treatment protected GalNAc-T1 from inactivation, to the same extent.

The acceptor specificity of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases.

The in vitro and in vivo specificity of the family of peptide:N-acetylgalactosaminyltransferases (GalNAcT) is analyzed on the basis of the reactivity and/or inhibitory activity of peptides and protein segments. The transferases appear to be multi-substrate enzymes with extended active sites containing a least nine subsites that interact cooperatively with a linear segment of at least nine amino acid residues on the acceptor polypeptide. Functional acceptor sites are located on the surface of the protein and extended conformations (beta-strand conformation) are preferred.