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 Gal1,4GlcNAc1,6(3-O-MeGal1,3)GalNAc␣-O-Bn (where Bn is benzyl) and the disaccharide Gal1,4GlcNAc served as high affinity acceptors for Gal3ST-2 and Gal3ST-3, 3-O-MeGal1,4GlcNAc1,-6(Gal1,3)GalNAc␣-O-Bn and Gal1,3GalNAc␣-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 (Gal1,3GalNAc1,3Gal␣-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 Gal1,4GlcNAc unit on mucin core-2 as well as the Globo H precursor could be inhibited competitively by Gal1,4GlcNAc1,6(3-OsulfoGal1,3)GalNAc␣-O-Bn but not 3-O-sulfoGal1,-4GlcNAc1,6(Gal1,3)GalNAc␣-O-Bn. Remarkably these sulfotransferases were uniquely specific for sulfated substrates: Gal3ST-3 utilized Gal1,4(6-O-sulfo)-GlcNAc-O-Al as acceptor, Gal3ST-2 acted efficiently on Gal1,3(6-O-sulfo)GlcNAc-O-Al, and Gal3ST-4 acted efficiently on Gal1,3(6-O-sulfo)GalNAc␣-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.
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