Characterization of a Heparan Sulfate 3-O-Sulfotransferase-5, an Enzyme Synthesizing a Tetrasulfated Disaccharide

Hideo Mochizuki, Keiichi Yoshida, Masanori Gotoh, Shigemi Sugioka, Norihiro Kikuchi, Yeon-Dae Kwon, Akira Tawada, Kennichi Maeyama, Niro Inaba, Toru Hiruma, Koji Kimata, Hisashi Narimatsu
2003 Journal of Biological Chemistry  
Heparan sulfate D-glucosaminyl 3-O-sulfotransferases (3-OSTs) catalyze the transfer of sulfate from 3-phos- phoadenosine 5-phosphosulfate (PAPS) to position 3 of the glucosamine residue of heparan sulfate and heparin. A sixth member of the human 3-OST family, named 3-OST-5, was recently reported ((2002) J. Biol. Chem. 277, 37912-37919). In the present study, we cloned putative catalytic domain of the human 3-OST-5 and expressed it in insect cells as a soluble enzyme. Recombinant 3-OST-5 only
more » ... ant 3-OST-5 only exhibited sulfotransferase activity toward heparan sulfate and heparin. When incubated heparan sulfate with [ 35 S]PAPS, the highest incorporation of 35 S was observed, and digestion of the product with a mixture of heparin lyases yielded two major 35 S-labeled disaccharides, which were determined as ⌬HexA-GlcN(NS,3S,6S) and ⌬HexA(2S)-GlcN(NS,3S ) by further digestion with 2-sulfatase and degradation with mercuric acetate. However, when used heparin as acceptor, we identified a highly sulfated disaccharide unit as a major product. This had a structure of ⌬HexA(2S)-GlcN(NS,3S,6S). Quantitative real-time PCR analysis revealed that 3-OST-5 was highly expressed in fetal brain, followed by adult brain and spinal cord, and at very low or undetectable levels in the other tissues. Finally, we detected a tetrasulfated disaccharide unit in bovine intestinal heparan sulfate. To our knowledge, this is the first report to describe not only the natural occurrence of tetrasulfated disaccharide unit but also the enzymatic formation of this novel structure.
doi:10.1074/jbc.m301861200 pmid:12740361 fatcat:5wlpxhxjfbcuzao5hv4ol7oyam