title: Identification of physiologically relevant substrates for cloned Gal: 3-O-sulfotransferases (Gal3STs): distinct high affinity of Gal3ST-2 and LS180 sulfotransferase for the globo H backbone, Gal3ST-3 for N-glycan multiterminal Galbeta1, 4GlcNAcbeta units and 6-sulfoGalbeta1, 4GlcNAcbeta, and Gal3ST-4 for the mucin core-2 trisaccharide.
abstract: Sulfated glycoconjugates regulate biological processes such as cell adhesion and cancer metastasis. We examined the acceptor specificities and kinetic properties of three cloned Gal:3-O-sulfotransferases (Gal3STs) ST-2, ST-3, and ST-4 along with a purified Gal3ST from colon carcinoma LS180 cells. Gal3ST-2 was the dominant Gal3ST in LS180. While the mucin core-2 structure Galbeta1,4GlcNAcbeta1,6(3-O-MeGalbeta1,3)GalNAcalpha-O-Bn (where Bn is benzyl) and the disaccharide Galbeta1,4GlcNAc served as high affinity acceptors for Gal3ST-2 and Gal3ST-3, 3-O-MeGalbeta1,4GlcNAcbeta1,-6(Galbeta1,3)GalNAcalpha-O-Bn and Galbeta1,3GalNAcalpha-O-Al (where Al is allyl) were efficient acceptors for Gal3ST-4. The activities of Gal3ST-2 and Gal3ST-3 could be distinguished with the Globo H precursor (Galbeta1,3GalNAcbeta1,3Galalpha-O-Me) and fetuin triantennary asialoglycopeptide. Gal3ST-2 acted efficiently on the former, while Gal3ST-3 showed preference for the latter. Gal3ST-4 also acted on the Globo H precursor but not the glycopeptide. In support of the specificity, Gal3ST-2 activity toward the Galbeta1,4GlcNAcbeta unit on mucin core-2 as well as the Globo H precursor could be inhibited competitively by Galbeta1,4GlcNAcbeta1,6(3-O-sulfoGalbeta1,3)GalNAcalpha-O-Bn but not 3-O-sulfoGalbeta1,-4GlcNAcbeta1,6(Galbeta1,3)GalNAcalpha-O-Bn. Remarkably these sulfotransferases were uniquely specific for sulfated substrates: Gal3ST-3 utilized Galbeta1,4(6-O-sulfo)-GlcNAcbeta-O-Al as acceptor, Gal3ST-2 acted efficiently on Galbeta1,3(6-O-sulfo)GlcNAcbeta-O-Al, and Gal3ST-4 acted efficiently on Galbeta1,3(6-O-sulfo)GalNAcalpha-O-Al. Mg(2+), Mn(2+), and Ca(2+) stimulated the activities of Gal3ST-2, whereas only Mg(2+) augmented Gal3ST-3 activity. Divalent cations did not stimulate Gal3ST-4, although inhibition was noted at high Mn(2+) concentrations. The fine substrate specificities of Gal3STs indicate a distinct physiological role for each enzyme.
authors.affiliation: Department of Molecular and Cellular Biophysics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
authors.forename: E V
authors.initials: EV
authors.lastname: Chandrasekaran
authors.forename: Sukhwinder S
authors.initials: SS
authors.lastname: Lakhaman
authors.forename: Ram
authors.initials: R
authors.lastname: Chawda
authors.forename: Conrad F
authors.initials: CF
authors.lastname: Piskorz
authors.forename: Sriram
authors.initials: S
authors.lastname: Neelamegham
authors.forename: Khushi L
authors.initials: KL
authors.lastname: Matta
mesh_terms: D000117:Acetylglucosamine / Q000737:chemistry*; D000818:Animals; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000737:chemistry; D001667:Binding, Competitive; D016466:CHO Cells; D045744:Cell Line, Tumor; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009077:Mucins / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D014312:Trisaccharides / Q000737:chemistry
chemical_list: D015295:Antigens, Tumor-Associated, Carbohydrate; C472765:Globo-H; D009077:Mucins; D014312:Trisaccharides; D012313:RNA; D015238:Sulfotransferases; C097036:galactose 3-O-sulfotransferase; C432916:galactose-3-O-sulfotransferase 3; D000117:Acetylglucosamine