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-A1, 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 accepters for Group A but were very active with Group B. An examination 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-hapten/acrylamide 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-A1 and Galβ1,,4GlcNAcβ1,6(Galβ1,3,3)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 (<100,000 Da) as observed by Sephacryl S-100 HR column chromatography. The following effects upon Gal: 3-0-sulfotransferase activities by fucose, sulfate, and other substituents on the carbohydrate chains were noted. (1) A methyl or GlcNAc substituent on C-6 of GalNAc diminished the ability of Galβ1,3GalNAcα-O-Al to act as an acceptor for Group A. (2) An α,1,3-fucosyl residue on the β1,6 branch in the mucin core structure did not affect the activity of Group A toward Gal linked β1,3 to GalNAcα-. (3) Lewis x and Lewis a terminals did not serve as accepters for either Group A or B enzymes. (4) Elimination of Group B activity on Gal in the β1,6 branch owing to the presence of a 3-fucosyl or 6-sulfo group on GlcNAc did not hinder any action toward Gal linked β1,3 to GalNAcα. (5) Group A activity on Gal linked β1,3 to GalNAc remained unaffected by 3'-sulfation of the β1,6 branch. The reverse was true for Group B. (6) The acceptor activity of the T-hapten was increased somewhat upon C-6 sulfation of GalNAc, whereas, C-6 sialylation resulted in an 85% loss of activity. (7) A novel finding was that Galβ1,4GlcNAcβ-O-Al and Galβ1,3GlcNAcβ-O-Al, upon C-6 sulfation of the GlcNAc moiety, became 100% inactive and 5- to 7-fold active, respectively, in their ability to serve as accepters for Group B.