N-Acetylglucosaminyltransferases Involved in N-Glycan Biosynthesis
We have succeeded in the purification and cDNA cloning of several enzymes involved in the biosynthesis of biologically important N-glycans of glycoproteins. These enzymes are responsible for the formation of branching structures of the core of N-glycans. To detect the activities with ease and sensitivity, we developed assay methods for these glycosyltransferases including N-acetylglucosaminyltransferases (GnTs) III, IV, V, VI, and IX using fluorescence-labeled oligosaccharides as acceptor substrates. The branching structures of N-glycans such as β1,6 GlcNAc branching and bisecting GlcNAc are formed by the actions of GnT-V and GnT-III, respectively. It has been reported that these N-glycans may play important roles in cell growth, cancer invasion, and metastasis. The assay methods for glycosyltransferases, including these enzymes involved in the branching formation of the core structure of N-glycan, are described (Fig. 1). The procedure described here is based on the methodology originally developed by Hase et al. (1978) and reported by our group earlier (Taniguchi et al. 1989). Each enzymatic product can be separated from the reaction mixture using reversed-phase HPLC. These methods allowed us to assay the activities with high sensitivity and substrate specificities of these glycosyltransferases.
KeywordsEnzymatic Product Acceptor Substrate Crude Enzyme Preparation Typical Elution 80TM Column
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