Open AccessA catalytically inactive β1,4‐ N ‐acetylglucosaminyltransferase III (GnT‐III) behaves as a dominant negative GnT‐III inhibitor
Author(s) -
Ihara Hideyuki,
Ikeda Yoshitaka,
Koyota Souichi,
Endo Takeshi,
Honke Koichi,
Taniguchi Naoyuki
Publication year - 2002
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.0014-2956.2001.02640.x
Subject(s) - glycosyltransferase , biosynthesis , mutant , transferase , glycan , biochemistry , enzyme , chemistry , homology (biology) , homologous chromosome , biology , gene , glycoprotein
β1,4‐ 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 β1,4GlcNAc transferase and β1,4Gal 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.