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 beta-Gal residues was investigated by screening sulfotransferase activity present in 37 human tissue specimens toward the following synthesized acceptor moieties: Gal beta 1,3GalNAc alpha-O-Al, Gal beta 1,4GlcNAc beta-O-Al, Gal beta 1,3GlcNAc beta-O-Al, and mucin-type Gal beta 1,4GlcNAc beta 1,6(Gal beta 1,3)GalNAc alpha-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 Gal beta 1, 3GalNAc alpha-linkage and the ether (Group B) was directed toward the Gal beta 1,4GlcNAc branch beta 1,6 linked to the blood group T hapten, Enzyme activities found in breast tissues mere unique in showing a strict specificity for the T-hapten, Gal beta-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 sis human sera revealed a specificity of the serum enzyme toward beta 1,3 linked Gal, particularly, the T-hapten without beta 1,6 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 Gal beta 1,3GalNAc alpha-O-Al and Gal beta 1,3GlcNAc beta 1,6(Gal beta 1,3)GalNAc alpha-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-O-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 Gal beta 1,3GalNAc alpha-O-Al to act as an acceptor for Group A, (2) An alpha 1,3-fucosyl residue on the beta 1,6 branch in the mucin core structure did not affect the activity of Group A toward Gal linked beta 1,3 to GalNAc alpha-. (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 beta 1,6 branch owing to the presence of a 3-fucosyl or 6-sulfo group on GlcNAc did not hinder any action toward Gal linked beta 1,3 to GalNAc alpha, (5) Group A activity on Gal linked beta 1,3 to GalNAc remained unaffected by 3'-sulfation of the beta 1,6 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 Gal beta 1,4GlcNAc beta-O-Al and Gal beta 1,3GlcNAc beta-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.