Soluble human core 2 β6- N-acetylglucosaminyltransferase C2GnT1 requires its conserved cysteine residues for full activity

• Published in: Biochimica et biophysica acta. Proteins and proteomics 2003-05, Vol.1648 (1), p.62-74
• Main Authors: Yang, Xiaojing, Qin, Wensheng, Lehotay, Michael, Toki, Dale, Dennis, Philip, Schutzbach, John S., Brockhausen, Inka
• Format: Article
• Language: English
• Subjects: C2GnT1 and C2GNT3, core 2 β6-GlcNAc-transferase, UDP-GlcNAc: Galβ1–3GalNAc- (GlcNAc to GalNAc) β1,6-GlcNAc-transferase (EC 2.4.1.102); C2GnT2, UDP-GlcNAc: GlcNAcβ1–3GalNAc-(GlcNAc to GalNAc) β1,6-GlcNAc-transferase (EC 2.4.1.148); Core 2; Cysteine; Enzymes; GlcNAc-transferase; IGnT, I antigen β6-GlcNAc-transferase, UDP-GlcNAc: R-GlcNAcβ1–3Gal-(GlcNAc to Gal) β1,6-GlcNAc-transferase (EC 2.4.1.150); Mutant; O-glycan branching
• ISSN: 1570-9639; 1878-1454
• DOI: 10.1016/S1570-9639(03)00105-5

Human UDP-GlcNAc: Galβ1–3GalNAc- (GlcNAc to GalNAc) β1,6-GlcNAc-transferase (C2GnT1) is a member of a group of β6-GlcNAc-transferases that belongs to CAZy family 14. One of the striking features of these β6-GlcNAc-transferases is the occurrence of nine completely conserved cysteine residues that are located throughout the catalytic domain. We have expressed the soluble catalytic domain of human C2GnT1 in insect cells, and isolated active enzyme as a secreted protein. β-Mercaptoethanol (β-ME) and dithiothreitol (DTT) were found to stimulate the enzyme activity up to 20-fold, indicating a requirement for a reduced sulfhydryl for activity. When the enzyme was subjected to nonreducing PAGE, the migration of the protein was identical to the migration in reducing gels, demonstrating the absence of intermolecular disulfide bonds. This suggested that the monomer is the active form of the enzyme. Sulfhydryl reagents such as 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) and N-ethylmaleimide (NEM) inactivated the enzyme, and the inactivation was partially prevented by prior addition of donor or acceptor substrate and by sulfhydryl reducing agents. We therefore investigated the role of all nine conserved cysteine residues in enzyme stability and activity by site-directed mutagenesis where individual cysteine residues were changed to serine. All of the mutants were expressed as soluble proteins. Seven of the Cys mutants were found to be inactive, while C100S and C217S mutants had 10% and 41% activity, respectively, when compared to the wild-type enzyme. Wild-type and C217S enzymes had similar K M and V max values for acceptor substrate Galβ1–3GalNAcα- p-nitrophenyl (GGApnp), but the K M value for UDP-GlcNAc was higher for C217S than for the wild-type enzyme. In contrast to wild-type enzyme, C217S was not stimulated by reducing agents and was not inhibited by sulfhydryl specific reagents. These results suggest that Cys-217 is a free sulfhydryl in active wild-type enzyme and that Cys-217, although not required for activity, is in or near the active site of the protein. Since seven of the mutations were totally inactive, it is likely that these seven Cys residues play a role in maintaining an active conformation of soluble C2GnT1 by forming disulfide bonds. These bonds are only broken at high concentrations of disulfide reducing agents.