THE ENZYMATIC SULFATION OF GLYCOPROTEIN CARBOHYDRATE UNITS - BLOOD-GROUP T-HAPTEN SPECIFIC AND 2 OTHER DISTINCT GAL-3-O-SULFOTRANSFERASES AS EVIDENT FROM SPECIFICITIES AND KINETICS AND THE INFLUENCE OF SULFATEAND 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 spe cimens toward the following synthesized acceptor moieties: Gal beta 1, 3GalNAc alpha-O-Al, Gal beta 1,4GlcNAc beta-O-Al, Gal beta 1,3GlcNAc b eta-O-Al, and mucin-type Gal beta 1,4GlcNAc beta 1,6(Gal beta 1,3)GalN Ac 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 a nd the ether (Group B) was directed toward the Gal beta 1,4GlcNAc bran ch beta 1,6 linked to the blood group T hapten, Enzyme activities foun d in breast tissues mere unique in showing a strict specificity for th e 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 t oward beta 1,3 linked Gal, particularly, the T-hapten without beta 1,6 branching, Group A was highly active toward T-hapten/acrylamide copol ymer, anti-freeze glycoprotein, and fetuin O-glycosidic asialo glycope ptide; 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 glycop eptides noted and least active with the first two. Competition experim ents performed with Gal beta 1,3GalNAc alpha-O-Al and Gal beta 1,3GlcN Ac beta 1,6(Gal beta 1,3)GalNAc alpha-O-Bn having a C-3 substituent (m ethyl or sulfate) on either Gal reinforced earlier findings on the spe cificity characteristics of Group A and Group B, Group A displayed a w ider 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 G roup B enzymes appeared to be of the same molecular size (<100,000 Da) as observed by Sephacryl S-100 HR column chromatography, The followin g 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 o f 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 be ta 1,3 to GalNAc alpha-. (3) Lewis x and Lewis a terminals did not ser ve as accepters for either Group A or B enzymes, (4) Elimination of Gr oup 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 towar d Gal linked beta 1,3 to GalNAc alpha, (5) Group A activity on Gal lin ked 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 activi ty 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 1,3Gl cNAc beta-O-Al, upon C-6 sulfation of the GlcNAc moiety, became 100% i nactive and 5-to 7-fold active, respectively, in their ability to serv e as accepters for Group B.