The enzymatic sulfation of glycoprotein carbohydrate units: blood group T-hapten specific and two other distinct Gal:3-O-sulfotransferases as evident from specificities and kinetics and the influence of sulfate and fucose residues occurring in the carbohydrate chain on C-3 sulfation of terminal Gal

Enzymatic 3-O-sulfation of terminal β-Gal residues was investigated by screening sulfotransferase activity present in 37 human tissue specimens toward the following synthesized acceptor moieties: Galβ1,3GalNAcα-O-Al, Galβ1,4GlcNAcβ-O-Al, Galβ1,3GlcNAcβ-O-Al, and mucin-type Galβ1,4GlcNAcβ1,6(Galβ1,3)GalNAcα-O-Bn structures containing a C-3 methyl substituent on either Gal. Two distinct types of Gal: 3-O-sulfotransferases were revealed. One (Group A) was specific for the Galβ1, 3GalNAcα- linkage and the other (Group B) was directed toward the Galβ1,4GlcNAc branch β1,6 linked to the blood group T hapten. Enzyme activities found in breast tissues were unique in showing a strict specificity for the T-hapten. Galβ-O-allyl or benzyl did not serve as acceptors for Group A but were very active with Group B. An exainination of activity present in six human sera revealed a specificity of the serum enzyme toward β1,3 linked Gal, particularly, the T-hapten without β1,6 branching. Group A was highly active toward T-haptenlacrylamide copolymer, anti-freeze glycoprotein, and fetuin O-glycosidic asialo glycopeptide; less active toward fetuin triantennary asialo glycopeptide; and least active toward bovine IgG diantennary glycopeptide. Group B was moderately and highly active, respectively, with the latter two glycopeptides noted and least active with the first two. Competition experiments performed with Galβ1,3GaLNAcα-O-Al and Galβ1,4GlcNAcβ1,6(Galβ1,3)GalNAcα-O-Bn having a C-3 substituent (methyl or sulfate) on either Gal reinforced earlier findings on the specificity characteristics of Group A and Group B. Group A displayed a wider range of optimal activity (pH 6.0–7.4), whereas Group B possessed a peak of activity at pH 7.2. Mg2+ stimulated Group A 55% and Group B 150%, whereas Mn+2 stimulated Group B 130% but inhibited Group A 75%. Ca2+ stimulated Group B 100% but inhibited Group A 35%. Group A and Group B enzymes appeared to be of the same molecular size (