Characterization of a novel galactose β1,3-N-acetylglucosaminyltransferase (β3Gn-T8): the complex formation of β3Gn-T2 and β3Gn-T8 enhances enzymatic activity

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Abstract
We characterized a novel member of the β1,3-N-acetylglucosaminyltransferase (β3Gn-T) gene family, β3Gn-T8. A recombinant soluble form of β3Gn-T8 was expressed in Pichia pastoris (P. pastoris), and its substrate specificity was compared with that of β3Gn-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 [Galβ1→4GlcNAcβ1→2(Galβ1→4GlcNAcβ1→6)Manα1→ 6Man]. Interestingly, when soluble recombinant β3Gn-T2 and β3Gn-T8 were mixed, the Vmax/Km value of the mixture was 9.3- and 160-fold higher than those of individual β3Gn-T2 and -T8, respectively. Sephacryl S-300 gel filtration of the enzymes revealed that apparent molecular weights of each β3Gn-T2, β3Gn-T8, and the mixture were 90–160, 45–65, and 110–210 kDa, respectively, suggesting that β3Gn-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 β3Gn-T8 and β3Gn-T2 are cooperatively involved in the elongation of specific branch structures of multiantennary N-glycans.

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