Characterization of a heparan sulfate 3-O-sulfotransferase-5, an enzyme synthesizing a tetrasulfated disaccharide.
Heparan sulfate d-glucosaminyl 3-O-sulfotransferases (3-OSTs) catalyze the transfer of sulfate from 3'-phosphoadenosine 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 (Xia, G., Chen, J., Tiwari, V., Ju, W., Li, J.-P., Malmstrom, A., Shukla, D., and Liu, J. (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 exhibited sulfotransferase activity toward heparan sulfate and heparin. When incubated heparan sulfate with [35S]PAPS, the highest incorporation of35S was observed, and digestion of the product with a mixture of heparin lyases yielded two major35S-labeled disaccharides, which were determined as DeltaHexA-GlcN(NS,3S,6S) and DeltaHexA(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 DeltaHexA(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.
Glycogene Function Team, Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Central-2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8586, Japan.
Hideo
H
Mochizuki
Keiichi
K
Yoshida
Masanori
M
Gotoh
Shigemi
S
Sugioka
Norihiro
N
Kikuchi
Yeon-Dae
YD
Kwon
Akira
A
Tawada
Kennichi
K
Maeyama
Niro
N
Inaba
Toru
T
Hiruma
Koji
K
Kimata
Hisashi
H
Narimatsu
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004187:Disaccharides / Q000096:biosynthesis* / Q000737:chemistry; D005260:Female; D006493:Heparin / Q000737:chemistry / Q000378:metabolism; D006497:Heparan Sulfate / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D008969:Molecular Sequence Data; D011247:Pregnancy; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014018:Tissue Distribution
D004247:DNA; D004187:Disaccharides; D006493:Heparin; D006497:Heparan Sulfate; D012333:RNA, Messenger; D011994:Recombinant Proteins; D015238:Sulfotransferases; C102180:heparan sulfate D-glucosaminyl 3-O-sulfotransferase