Identification of a novel UDP‐Gal:GalNAcβ1‐4GlcNAc‐R

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Abstract
Connective tissue of the freshwater pulmonateLymnaea stagnalis was shown to contain galactosyltrans ferase activity capable of transferring Gal from UDP‐Gal in β1‐3 linkage to terminal GalNAc of GalNAcβ1‐4GlcNAc‐R [R =β1‐2Manα1‐O(OH2)8 COOMe, β1‐OMe, or α,β1‐OH]. Using GalNAcβ1‐4GlcNAcβ‐2Manα‐1‐O(CH2)8 COOMe 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 GalNAcβ1‐4GlcNAcβ1‐2Manα1‐O(CH2)8 COOMe (Vmax 140 nmol · h−1· mg protein−1; Km 1.02 mM), GalNAcβ‐4GlcNAc (Vmax 105 nmol · h−1· mg protein−1; km 0.99 mM), and GalNAcβ‐4GlcNAcβ1‐OMe (Vmax 108 nmol · h−1· mg protein−1; Km1.33 mM). The products formed from GalNAcβ1‐4GlcNAcβ1‐2Manα1‐O(CH2)8COOMe and GalNAcβ1‐4GlcNAcβ1‐OMe were purified by high performance liquid chromatography, and identified by 500‐MHz1H‐NMR spectroscopy to be Galβ1‐3GalNAcβ1‐4GlcNAcβ1‐2Manα1‐O(CH2)8COOMe and Galβ‐3GalNAcβ1‐4GlcNAcβ1‐OMe, respectively. The enzyme was inactive was inactive towards GlcNAc, GalNAcβ1‐3Galα‐OMe, GalNAcα1‐OC6H5, GalNAcα1 – ovine‐submaxillary‐mucin, lactose andN‐acetyllactosamine. This novel UDPGal:GalNAcβ1‐4GlcNAc‐R β1‐3‐galactosyltransferase is believed to be involved in the biosynthesis of the hemocyanin glycans of L. stagnalis.

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