Molecular cloning and expression of Chinese hamster ovary cell heparan-sulfate 2-sulfotransferase.
Heparan-sulfate 2-sulfotransferase (HS2ST), which catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to L-iduronic acid at position 2 in heparan sulfate, was purified from cultured Chinese hamster ovary (CHO) cells to apparent homogeneity (Kobayashi, M., Habuchi, H., Habuchi, O., Saito, M., and Kimata, K. (1996) J. Biol. Chem. 271, 7645-7653). The internal amino acid sequences were obtained from the peptides after digestion of the purified protein with a combination of endoproteinases. Mixed oligonucleotides based on the peptide sequences were used as primers to obtain a probe fragment by reverse transcriptase-polymerase chain reaction using CHO cell poly(A)+ RNA as template. The clone obtained from a CHO cDNA library by screening with the probe is 2.2 kilobases in size and contains an open reading frame of 1068 bases encoding a new protein composed of 356 amino acid residues. The protein predicts a type II transmembrane topology similar to other Golgi membrane proteins. Messages of 5.0 and 3.0 kilobases were observed in Northern analysis. Evidence that the cDNA clone corresponds to the purified HS2ST protein is as follows. (a) The predicted amino acid sequence contains all five peptides obtained after endoproteinase digestion of the purified protein; (b) the characteristics of the predicted protein fit those of the purified protein in terms of molecular mass, membrane localization, and N-glycosylation; and (c) when the cDNA containing the entire coding sequence of the enzyme in a eukaryotic expression vector was transfected into COS-7 cells, the HS2ST activity increased 2.6-fold over controls, and the FLAG-HS2ST fusion protein purified by affinity chromatography showed the HS2ST activity alone.
Kobayashi
M
M
Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi 480-11, Japan.
Habuchi
H
H
Yoneda
M
M
Habuchi
O
O
Kimata
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D019556:COS Cells; D002384:Catalysis; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary / Q000737:chemistry; D005260:Female; D015723:Gene Library; D008969:Molecular Sequence Data; D010053:Ovary / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D015238:Sulfotransferases / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D018076:DNA, Complementary; D015238:Sulfotransferases; C106358:heparan-sulfate 2-sulfotransferase