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 1-4GlcNAc-R 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+ 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 (Vmax 140 nmol.h-1.mg protein-1; Km 1.02 mM), GalNAc beta 1-4GlcNAc (Vmax 105 nmol.h-1.mg protein-1; Km 0.99 mM), and GalNAc beta 1-4GlcNAc beta 1-OMe (Vmax 108 nmol.h-1.mg protein-1; Km 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 1H-NMR spectroscopy to be Gal beta 1-3GalNAc beta 1-4GlcNAc 1-OMe, respectively. The enzyme was inactive towards GlcNAc, GalNac beta 1-3 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.