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

Abstract

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. This suggests that GalNAc-T1 contains free cysteine residues interacting with the UDP moiety of the sugar donor. For the functional analysis of the cysteine residues, several conserved cysteine residues in GalNAc-T1 were mutated individually to alanine. All of the mutations except one resulted in complete inactivation or a drastic decrease in the activity, of the enzyme. We identified only Cys212 and Cys214, among the conserved cysteine residues in GalNAc-T1, as free cysteine residues, by cysteine-specific labeling of GalNAc-T1. To investigate the role of these two cysteine residues, we generated cysteine to serine mutants (C212S and C214S). The serine mutants were more active than the corresponding alanine mutants (C212A and C214A). Kinetic analysis demonstrated that the affinity of the serine-mutants for UDP-GalNAc was decreased, as compared to the wild type enzyme. The affinity for the acceptor apomucin, on the other hand, was essentially unaffected. The functional importance of the introduced serine residues was further demonstrated by the inhibition of all serine mutant enzymes with diisopropyl fluorophosphate. In addition, the serine mutants were more resistant to modification by PCMPS. Our results indicate that Cys212 and Cys214 are sites of PCMPS modification, and that these cysteine residues are involved in the interaction with the UDP moiety of UDP-GalNAc.

The beginnings of mucin biosynthesis: The crystal structure of UDP-GalNAc:polypeptide α- N -acetylgalactosaminyltransferase-T1

“…Two disulfide bonds, C106-C339 and C330-C408, are formed from four invariant cysteine residues. Mutating any of these Cys to Ala significantly reduces expression and abolishes enzyme activity (32). The three remaining catalytic domain Cys are moderately conserved (conservation was determined by comparison of 38 known enzymatically active ppGaNTases described to date) and do not form disulfide bonds.…”

“…Two of these (C212 and C214) have been proposed to interact with UDP-GalNAc based upon chemical modification and mutagenesis studies. Mutating either Cys to Ala reduces activity to 6% and 17%, respectively, of wild-type levels (32). The third Cys (C235) is in a hydrophobic pocket and forms a close contact with M291.…”

Cloning and Expression of a Brain-Specific Putative UDP-GalNAc: Polypeptide N-Acetylgalactosaminyltransferase Gene

“…gion. They had several characteristics commonly found in the GalNAc-transferase family: 1) a glycosyltransferase 1 (GT1) motif, a conserved sequence commonly found in glycosyltransferases, 23) 2) a DXH sequence in the GT1 motif, a putative binding site for a sugar donor and/or a metal ion, 24) 3) a Gal/GalNAc-T motif consisting of about 40 amino acid residues, 23) 4) conserved acidic, histidine, and cysteine residues, 23,25,26) and 5) (QXW) 3 repeats, a C-terminal lectinlike domain. 27) We, then, investigated the expression of pt-GalNAc-T. First, the expression of human pt-GalNAc-T in the brain was examined by Northern blot analysis.…”

Characterization of a Novel Polypeptide N-Acetylgalactosaminyltransferase (dGalNAc-T3) from Drosophila

“…It is reported that these are reduced Cys residues, and are involved in binding with UDPGalNAc. 25) Interestingly, dGalNAc-T3 contains a 39 amino acid residue insertion at the N-terminus of the (QXW) 3 a repeat, which is not found in the other GalNAc-transferases. Moreover, it also has a short insertion (approximately 5-10 amino acid residues) in each repeat of the lectin-like domain.…”

“…Tenno et al reported that the modification of mammalian GalNAc-T1 with a Cys-specific reagent, PCMPS, irreversibly inhibited the enzyme with a K i of 0.03 mM and that Cys212 and Cys214, which are C-terminal to the DXH motif, are the sites of the modification and are involved in the binding with UDP-GalNAc. 25) dGalNAc-T3, containing both Cys residues corresponding to mammalian Cys212 and Cys214, was also incubated with increasing concentrations of PCMPS. PCMPS effectively inhibited dGalNAc-T3 with an apparent K i of 0.05 mM (Fig.…”