IDENTIFICATION OF A NOVEL UDP-GAL-GALNAC-BETA-1-4GLCNAC-R BETA-1-3-GALACTOSYLTRANSFERASE IN THE CONNECTIVE-TISSUE OF THE SNAIL LYMNAEA-STAGNALIS

Connective tissue of the freshwater pulmonate Lymnaea stagnalis was shown to contain galactosyltransferase activity capable of transferring Gal from UDP-Gal in beta-1-3 linkage to terminal GalNAc of GalNAc-beta-1-4GlcNAcR [R = beta-1-2Man-alpha-1-O(CH2)8COOMe, beta-1-OMe, or alpha, beta-1-OH]. Using GalNAc-beta-1-4GlcNAc-beta-1-2Man-alpha-1-O(CH2)8COOMe as substrate, the enzyme showed an absolute requirement for Mn2+ with an optimum Mn2+ with an optimum Mn2+ concentration between 12.5 mM and 25 mM. The divalent cations Mg2+, Ca2+, Ba2+ and Cd2+ at 12.5 mM could not substitute for Mn2+. The galactosyltransferase activity was independent of the concentration of Triton X-100, and no activation effect was found. The enzyme was active with GalNAc-beta-1-4GlcNAc-beta-1-2Man-alpha-1-O(CH2)8COOMe (V(max) 140 nmol . h-1 . mg protein- 1; K(m) 1.02 mM), GalNAc-beta-1-4GlcNAc (V(max) 105 nmol . h-1 . mg protein-1; K(m) 0.99 mM), and GalNAc-beta-1-4GlcNAc-beta-1-OMe (V(max) 108 nmol . h-1 . mg protein-1; K(m) 1.33 mM). The products formed from GalNAc-beta-1-4GlcNAc-beta-1-2Man-alpha-1-O(CH2)8COOMe and GalNAc-beta-1-4GlcNAc-beta-1-OMe were purified by high performance liquid chromatography, and identified by 500-MHz H-1-NMR spectroscopy to be Gal-beta-1-3GalNAc-beta-1-4GlcNAc-beta-1-2Man-alpha-1-O(CH2)8COOMe and Gal-beta-1-3GalNAc-beta-1-4GlcNAc-beta-1-OMe, respectively. The enzyme was inactive towards GlcNAc, GalNAc-beta-1-3Gal-alpha-1-OMe, GalNAc-alpha-1-OC6H5, GalNAc-alpha-1-ovine-submaxillary-mucin, lactose and N-acetyllactosamine. This novel UDP-Gal:GalNAc-beta-1-4GlcNAc-R beta-1-3-galactosyltransferase is believed to be involved in the biosynthesis of the hemocyanin glycans of L. stagnalis.