Molecular cloning and characterization of a human uronyl 2-sulfotransferase that sulfates iduronyl and glucuronyl residues in dermatan/chondroitin sulfate.
A partial-length human cDNA with a predicted amino acid sequence homologous to a previously described heparan sulfate iduronyl 2-sulfotransferase (Kobayashi, M., Habuchi, H., Yoneda, M., Habuchi, O., and Kimata, K. (1997) J. Biol. Chem. 272, 13980-13985) was obtained by searching the expressed sequence-tagged data bank. Northern blot analysis was performed using this homologous cDNA as a probe, which demonstrated ubiquitous expression of messages of 5.1 and 2.0 kilobases in a number of human tissues and in several human cancer cell lines. Since the human lymphoma Raji cell line had the highest level of expression, it was used to isolate a full-length cDNA clone. The full-length cDNA was found to contain an open reading frame that predicted a type II transmembrane protein composed of 406 amino acid residues. The cDNA in a baculovirus expression vector was expressed in Sf9 insect cells, and cell extracts were then incubated together with 3'-phosphoadenosine 5'-phospho[35S]sulfate and potential glycosaminoglycan acceptors. This demonstrated substantial sulfotransferase activity with dermatan sulfate, a small degree of activity with chondroitin sulfate, but no sulfotransferase activity with desulfated N-resulfated heparin. Analysis of [35S]sulfate-labeled disaccharide products of chondroitin ABC, chondroitin AC, and chondroitin B lyase treatment demonstrated that the enzyme only transferred sulfate to the 2-position of uronyl residues, which were preponderantly iduronyl residues in dermatan sulfate, but some lesser transfer to glucuronyl residues of chondroitin sulfate.
Kobayashi
M
M
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Sugumaran
G
G
Liu
J
J
Shworak
N W
NW
Silbert
J E
JE
Rosenberg
R D
RD
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D002809:Chondroitin Sulfates / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D003871:Dermatan Sulfate / Q000378:metabolism*; D020224:Expressed Sequence Tags; D005965:Glucuronates / Q000378:metabolism*; D020723:Glucuronic Acid; D006801:Humans; D007067:Iduronic Acid / Q000378:metabolism*; D008969:Molecular Sequence Data; D018411:Spodoptera; D015238:Sulfotransferases / Q000235:genetics* / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D005965:Glucuronates; D003871:Dermatan Sulfate; D007067:Iduronic Acid; D020723:Glucuronic Acid; D002809:Chondroitin Sulfates; D015238:Sulfotransferases; C118596:UST protein, human