TAILIEUCHUNG - Báo cáo Y học: A catalytically inactive b1,4-N-acetylglucosaminyltransferase III (GnT-III) behaves as a dominant negative GnT-III inhibitor

b1,4-N-Acetylglucosaminyltransferase III (GnT-III) plays a regulatory role in the biosynthesis of N-glycans, and it has been suggested that its product, a bisecting GlcNAc, is involved in a variety of biological events as well as in regu-lating the biosynthesis of the oligosaccharides. In this study, it was found, on the basis of sequence homology, that GnT-III contains a small region that is signi®cantly homologous to both snail b1,4GlcNAc transferase andb1,4Gal trans-ferase-1. | Eur. J. Biochem. 269 193-201 2002 FEBS 2002 A catalytically inactive p1 4-W-acetylglucosaminyltransferase III GnT-III behaves as a dominant negative GnT-III inhibitor Hideyuki Ihara Yoshitaka Ikeda Souichi Koyota Takeshi Endo Koichi Honke and Naoyuki Taniguchi Department of Biochemistry Osaka University Medical School Suita Osaka Japan pi 4-N-Acetylglucosaminyltransferase III GnT-III plays a regulatory role in the biosynthesis of N-glycans and it has been suggested that its product a bisecting GlcNAc is involved in a variety of biological events as well as in regulating the biosynthesis of the oligosaccharides. In this study it was found on the basis of sequence homology that GnT-III contains a small region that is significantly homologous to both snail pi 4GlcNAc transferase and pi 4Gal trans-ferase-1. Subsequent mutational analysis demonstrated an absolute requirement for two conserved Asp residues Asp321 and Asp323 which are located in the most homologous region of rat GnT-III for enzymatic activity. The overexpression of Asp323-substituted catalytically inactive GnT-III in Huh6 cells led to the suppression of the activity of endogenous GnT-III but no significant decrease in its expression and led to a specific inhibition of the formation of bisected sugar chains as shown by structural analysis of the total N-glycans from the cells. These findings indicate that the mutant serves a dominant negative effect on a specific step in N-glycan biosynthesis. This type of dominant negative glycosyltransferase identified has potential value as a powerful tool for defining the precise biological roles of the bisecting GlcNAc structure. Keywords GnT-III glycosyltransferase bisecting GlcNAc N-glycan synthesis dominant negative effect. pi 4-N-Acetylglucosaminyltransferase III GnT-III catalyzes the transfer of GlcNAc from UDP-GlcNAc a glycosyl donor to a core p-mannose residue in N-linked oligosaccharides via a pi 4 linkage resulting in the formation of a bisected sugar chain i

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