Expression of heparan sulfate D-glucosaminyl 3-O-sulfotransferase isoformsreveals novel substrate specificities

The 3-O-sulfation of glucosamine residues is an important modification duri ng the biosynthesis of heparan sulfate (HS), Our previous studies have led us to purify and molecularly clone the heparan sulfate D-glucosaminyl 5-O-s ulfotransferase (5-OST-1), which is the key enzyme converting nonanticoagul ant heparan sulfate (HSinact) to anticoagulant heparan sulfate (HSact). In this study, we expressed and characterized the full-length cDNAs of 3-OST-1 homologous genes, designated as 3-OST-2, 3-OST-3(A), and 3-OST-3(B) as des cribed in the accompanying paper (Shworak, N. W., Liu, J., Petros, L. M., Z hang, L., Kobayashi, M., Copeland, N. G., Jenkins, N. A., and Rosenberg, R. D. (1999) J. Biol. Chem, 274, 5170-5184), All these cDNAs were successfull y expressed in COS-7 cells, and heparan sulfate sulfotransferase activities were found in the cell extracts, We demonstrated that 3-OST-2, 3-OST-3(A), and 3-OST-3(B) are heparan sulfate D-glucosaminyl 3-O-sulfotransferases be cause the enzymes transfer sulfate from adenosine 3'-phosphophate 5'-phosph o-[S-35]sulfate ([S-35]PAPS) to the 5-OH position of glucosamine, 3-OST-3(A ) and 3-OST-3(B) sulfate an identical disaccharide. HSact conversion activi ty in the cell extract transfected by 3-OST-1 was shown to be 300-fold grea ter than that in the cell extracts transfected by 5-OST-2 and 3-OST-3(A), s uggesting that 3-OST-2 and 3-OST-3(A) do not make HSact. The results of the disaccharide analysis of the nitrous acid-degraded [35S]HS suggested that 3-OST-2 transfers sulfate to GlcA2S-GlcNS and IdoA2S-GlcNS; 3-OST-3(A) tran sfers sulfate to IdoA2S-GlcNS. Our results demonstrate that the 3-O-sulfati on of glucosamine is generated by different isoforms depending on the sacch aride structures around the modified glucosamine residue. This discovery ha s provided evidence for a new cellular mechanism for generating a defined s accharide sequence in structurally complex HS polysaccharide.