Identification of a novel UDP-GalNAc:GlcNAcβ-R β1-4 N-acetylgalactosaminyltransferase from the albumen gland and connective tissue of the snail Lymnaea stagnalis

Abstract

Both the albumen gland, one of the female accessory sex glands, and connective tissue of the freshwater snail Lymnaea stagnalis contain N-acetylgalactosaminyltransferase activity, capable of transferring GalNAc from UDP-GalNAc in beta1-4 linkage to the terminal GlcNAc residue of GlcNAcbeta-R. The albumin gland enzyme was partially purified by affinity chromatography on UDP-hexanolamineSepharose 4B. Using GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-2Manalpha1-3)Manalpha1-4GlcNAcbeta1-4GlcNAc or GlcNAcbeta1-OMe as substrates, the enzyme showed an absolute requirement for Mn2+ with an optimum concentration of 12.550 mM. The optimal pH was approximately pH 7.0. The enzyme activity was independent of the Triton X-100 concentration in the range 0.252.5%, and no activation effect was found. The more labile connective tissue microsomal enzyme, subjected to the same optimization procedure, gave comparable results. Both enzyme activities have similar substrate specificities towards GlcNAc or GlcNAcbetal-OMe, and towards oligosaccharides or glycopeptides with a non-reducing terminal beta-GlcNAc unit, but cannot act on GlcNAcalpha1-OMe. Saccharides with non-reducing terminal Gal or GalNAc residues, and free GalNAc, Gal or Glc residues are not acceptors. Product analysis was carried out for albumen gland N-acetylgalactosaminyltransferase and four acceptors having GlcNAcbeta1-R as the terminal non-reducing unit, and for connective tissue N-acetylgalactosaminyltransferase with GlcNAcbeta1-OMe as acceptor. In all instances, products with GalNAc beta1-4-linked to GlcNAc were obtained, showing that the connective tissue and the albumen gland activities are probably from one enzyme. This enzyme activity can be identified as UDP-GalNAc:GlcNAcbeta-Rbeta1-4 N-acetylgalactosaminyltransferase, and is probably involved in the biosynthesis of N,N'-diacetyllactosediamine-containing glycoproteins, like hemocyanin, in the snail L. stagnalis.