TAILIEUCHUNG - Báo cáo khoa học: Identification of two cysteine residues involved in the binding of UDP-GalNAc to UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 1 (GalNAc-T1)

Biosynthesis ofmucin-typeO-glycans is initiated by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyl-transferases, which contain several conserved cysteine resi-dues 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. | Eur. J. Biochem. 269 4308-4316 2002 FEBS 2002 doi Identification of two cysteine residues involved in the binding of UDP-GalNAc to UDP-GalNAc polypeptide V-acetylgalactosaminyltransferase 1 GalNAc-T1 Mari Tenno1 Shinya Toba1 Ferenc J Kezdy3 Ake P. Elhammer3 and Akira Kurosaka1 2 1 Department of Biotechnology Faculty of Engineering and 2Institute for Comprehensive Research Kyoto Sangyo University Kamigamo-motoyama Kyoto Japan 3Pharmacia Corporation Kalamazoo Michigan USA Biosynthesis of mucin-type O-glycans is initiated by a family of UDP-GalNAc polypeptide N-acetylgalactosaminyl-transferases 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 I uoclional 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 acceptorapomucin on the otherhand was essentially unaffected. The functional importance of the introduced serine residues was further demonstrated

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