IN THE BIOSYNTHESIS OF N-GLYCANS IN CONNECTIVE-TISSUE OF THE SNAIL LYMNAEA-STAGNALIS OF INCORPORATION GLCNAC BY BETA-2GLCNAC-TRANSFERASE-I IS AN ESSENTIAL PREREQUISITE FOR THE ACTION OF BETA-2GLCNAC-TRANSFERASE-II AND BETA-2XYL-TRANSFERASE

Using a series of relevant substrates, connective tissue of the snail Lymnaea stagnalis was shown to contain beta 1-2 xylosyltransferase (be ta 2Xyl-T), beta 1-2 N-acetylglucosaminyltransferase I (beta 2GlcNAc-T I), and beta 1-2 N-acetylglucosaminyltransferase II (beta 2GlcNAc-T I l) activities. These enzymes are probably involved in the biosynthesis of the N-linked carbohydrate chains, like those present in hemocyanin . The products formed by incubation of GlcNAc beta 1-2Man alpha 1-6(Gl cNAc beta 1-2Man alpha 1-3)Man beta 1-R [where R = -4GlcNAc beta 1-4Gl cNAc or O-(CH2)(7)CH3] with UDP-Xyl and connective tissue microsomes h ave been purified and characterized by H-1-NMR spectroscopy in conjunc tion with methylation analysis to be GlcNAc beta 1-2Man alpha 1-6(GlcN Ac beta 1-2Man alpha 1-3)(Xyl beta 1-2)Man beta 1-R. Substrate specifi city studies focused on connective tissue beta 2Xyl-T show that the mi nimal structure requirements are fulfilled in GlcNAc beta 1-2Man alpha 1-3Man beta 1-O-(CH2)(7)CH3. The enzyme activity can therefore be cha racterized as UDP-Xyl:GlcNAc beta 1-2Man alpha 1-3Man beta-R (Xyl to M an beta) beta 1-2 xylosyltransferase. In substrate-specificity studies directed to connective tissue beta 2GlcNAc-T I, it could be demonstra ted that the enzyme is active towards accepters having at the minimum a Man alpha 1-3Man beta-R sequence, and that introduction of a beta Xy l residue at C2 of beta Man totally abolishes the enzyme activity. Xyl ose-containing oligosaccharides are not accepters for beta 2GlcNAc-T I . In combination with the substrate specificity of beta 2Xyl-T, this s hows that in snail connective tissue beta 2GlcNAc-T I must act before beta 2Xyl-T. The connective tissue beta 2GlcNAc-T II activity follows the earlier established biosynthetic routes. Based on the substrate sp ecificities of the various connective tissue glycosyltransferases know n so far, and the structures isolated from L. stagnalis hemocyanin, a partial biosynthetic scheme for N-glycosylation in snail connective ti ssue is proposed.