Heparan sulfate D-glucosaminyl 3-O-sulfotransferase-3A sulfates N- unsubstituted glucosamine residues

Abstract

3-O-Sulfation of glucosamine by heparan sulfate D-glucosaminyl 3-O- sulfotransferase (3-OST-1) is the key modification in anticoagulant heparan sulfate synthesis. However, the heparan sulfates modified by 3-OST-2 and 3- OST-3A, isoforms of 3-OST-1, do not have anticoagulant activity, although these isoforms transfer sulfate to the 3-OH position of glucosamine residues. In this study, we characterize the substrate specificity of purified 3-OST-3A at the tetrasaccharide level. The 3-OST-3A enzyme was purified from Sf9 cells infected with recombinant baculovirus containing 3-OST-3A cDNA. Two 3-OST-3A- modified tetrasaccharides were purified from the 3-O-35S-sulfated heparan sulfate that was digested by heparin lyases. These tetrasaccharides were analyzed using nitrous acid and enzymatic degradation combined with matrix- assisted laser desorption/ionization-mass spectrometry. Two novel tetrasaccharides were discovered with proposed structures of AUA2S-GlcNS- IdoUA2S-[35S]GlcNH23S and ΔUA2S-GlcNS-IdoUA2S-[3-35S]GlcNH23S6S. The results demonstrate that 3-OST-3A sulfates N-unsubstituted glucosamine residues, and the 3-OST-3A modification sites are probably located in defined oligosaccharide sequences. Our study suggests that oligosaccharides with N- unsubstituted glucosamine are precursors for sulfation by 3-OST-3A. The intriguing linkage between N-unsubstituted glucosamine and the 3-O-sulfation by 3-OST-3A may provide a clue to the potential biological functions of 3- OST-3A-modified heparan sulfate.