A catalytically inactive beta 1,4-N-acetylglucosaminyltransferase III (GnT-III) behaves as a dominant negative GnT-III inhibitor.
beta 1,4-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 beta 1,4GlcNAc transferase and beta1,4Gal transferase-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.
Ihara
Hideyuki
H
Department of Biochemistry, Osaka University Medical School, Suita, Osaka, Japan.
Ikeda
Yoshitaka
Y
Koyota
Souichi
S
Endo
Takeshi
T
Honke
Koichi
K
Taniguchi
Naoyuki
N
D000595:Amino Acid Sequence; D002384:Catalysis; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017351:N-Acetylglucosaminyltransferases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D017351:N-Acetylglucosaminyltransferases; C054599:beta-1,4-mannosyl-glycoprotein beta-1,4-N-acetylglucosaminyltransferase