Characterization of a novel galactose beta1,3-N-acetylglucosaminyltransferase (beta3Gn-T8): the complex formation of beta3Gn-T2 and beta3Gn-T8 enhances enzymatic activity.
We characterized a novel member of the beta1,3-N-acetylglucosaminyltransferase (beta3Gn-T) gene family, beta3Gn-T8. A recombinant soluble form of beta3Gn-T8 was expressed in Pichia pastoris (P. pastoris), and its substrate specificity was compared with that of beta3Gn-T2. The two enzymes had similar substrate specificities and recognized tetraantennary N-glycans and 2,6-branched triantennary glycans in preference to 2,4-branched triantennary glycans, biantennary glycans, and lacto-N-neotetraose (LNnT), indicating their specificity for 2,6-branched structures such as [Galbeta1-->4GlcNAcbeta1-->2(Galbeta1-->4GlcNAcbeta1-->6)Manalpha1--> 6Man]. Interestingly, when soluble recombinant beta3Gn-T2 and beta3Gn-T8 were mixed, the Vmax/Km value of the mixture was 9.3- and 160-fold higher than those of individual beta3Gn-T2 and -T8, respectively. Sephacryl S-300 gel filtration of the enzymes revealed that apparent molecular weights of each beta3Gn-T2, beta3Gn-T8, and the mixture were 90-160, 45-65, and 110-210 kDa, respectively, suggesting that beta3Gn-T2 and -T8 can form a complex with enhanced enzymatic activity. This is the first report demonstrating that in vitro mixed glycosyltransferases show enhanced enzymatic activity through the formation of a heterocomplex. These results suggested that beta3Gn-T8 and beta3Gn-T2 are cooperatively involved in the elongation of specific branch structures of multiantennary N-glycans.
Seko
Akira
A
Department of Biochemistry, Sasaki Institute, 2-2, Kanda-Surugadai, Tokyo 101-0062, Japan.
Yamashita
Katsuko
K
D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry* / Q000302:isolation & purification; D010843:Pichia / Q000201:enzymology; D011134:Polysaccharides / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility; D013379:Substrate Specificity
D011134:Polysaccharides; D011994:Recombinant Proteins; C496333:B3GNT8 protein, human; D017351:N-Acetylglucosaminyltransferases; C428132:B3GNT2 protein, human