title: Porcine, mouse and human galactose 3-O-sulphotransferase-2 enzymes have different substrate specificities; the porcine enzyme requires basic compounds for its catalytic activity.
abstract: Sulphation of galactose at the C-3 position is one of the major post-translational modifications of colorectal mucin. Thus we partially purified a Gal 3-O-sulphotransferase from porcine colonic mucosa (pGal3ST) and studied its enzymatic characteristics. The enzyme was purified 48500-fold by sequential chromatographies on hydroxyapatite, Con A (concanavalin A)-Sepharose, porcine colonic mucin-Sepharose, Cu2+-chelating Sepharose and AMP-agarose. Interestingly, the purified pGal3ST required submillimolar concentrations of spermine or basic lipids, such as D-sphingosine and N,N-dimethylsphingosine, for enzymatic activity. pGal3ST recognized Galbeta1-->3GalNAc (core 1) as an optimal substrate, and had weaker activity for Galbeta1-->3GlcNAc (type 1) and Galbeta1-->4GlcNAc (type 2). Substrate competition experiments proved that a single enzyme catalyses sulphation of all three oligosaccharides. Among the four human Gal3STs cloned to date, the substrate specificity of pGal3ST is most similar to that of human Gal3ST-2, which is also strongly expressed in colonic mucosa, although the kinetics of pGal3ST and human Gal3ST-2 were rather different. To determine whether pGal3ST is the orthologue of human Gal3ST-2, a cDNA encoding porcine Gal3ST-2 was isolated and the enzyme was expressed in COS-7 cells for analysis of substrate specificity. This revealed that porcine Gal3ST-2 has the same specificity as pGal3ST, indicating that pGal3ST is indeed the porcine equivalent of Gal3ST-2. The substrate specificity of mouse Gal3ST-2 was also different from those of human and porcine Gal3ST-2 enzymes. Mouse Gal3ST-2 preferred core 1 and type 2 glycans to type 1, and the K(m) values were much higher than those of human Gal3ST-2. These results suggest that porcine Gal3ST-2 requires basic compounds for catalytic activity and that human, mouse and porcine Gal3ST-2 orthologues have diverse substrate specificities.
authors.affiliation: Department of Biochemistry, Sasaki Institute, 2-2, Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Seko
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Sumiya
authors.forename: Katsuko
authors.initials: K
authors.lastname: Yamashita
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002384:Catalysis / Q000187:drug effects; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D019143:Evolution, Molecular; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007413:Intestinal Mucosa / Q000201:enzymology; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000378:metabolism*; D013466:Sulfurtransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013552:Swine
chemical_list: D008670:Metals; D013466:Sulfurtransferases; C432419:GAL3ST2 protein, human; C506605:Gal3St2 protein, mouse; D015238:Sulfotransferases