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 Galβ1,4GlcNacβ units and 6-sulfoGalβ1,4GlcNAcβ, and Gal3ST-4 for the mucin core-2 trisaccharide

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 Galβ1,4GlcNAcβ1,6(3-O-MeGalβ1,3)GalNAcα-O-Bn (where Bn is benzyl) and the disaccharide Galβ1,4GlcNAc served as high affinity acceptors for Gal3ST-2 and Gal3ST-3, 3-O-MeGalβ1,4GlcNAcβ 1,-6(Galβ1,3)GalNAcα-O-Bn and Galβ1,3GalNAcα-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 (Galβ1,3GalNAcβ1,3Galα-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 Galβ1,4GlcNAcβ unit on mucin core-2 as well as the Globo H precursor could be inhibited competitively by Galβ1,4GlcNAcβ 1,6(3-O-sulfoGalβ1,3)GalNAcα-O-Bn but not 3-O-sulfoGalβ 1,-4GlcNAcβ1,6(Galβ1,3)GalNAcα-O-Bn. Remarkably these sulfotransferases were uniquely specific for sul. fated substrates: Gal3ST-3 utilized Galβ1,4(6-O-sulfo)-GlcNAcβ-O-Al as acceptor, Gal3ST-2 acted efficiently on Galβ1,3(6-O-sulfo) GlcNAcβ-O-Al, and Gal3ST-4 acted efficiently on Galβ1,3(6-O-sulfo)GalNAcα-O-Al. Mg²⁺, Mn²⁺, and Ca²⁺ stimulated the activities of Gal3ST-2, whereas only Mg²⁺ augmented Gal3ST-3 activity. Divalent cations did not stimulate Gal3ST-4, although inhibition was noted at high Mn²⁺ concentrations. The fine substrate specificities of Gal3STs indicate a distinct physiological role for each enzyme.