Molecular Cloning and Expression of Chinese Hamster Ovary Cell Heparan-sulfate 2-Sulfotransferase*
- From the Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi 480-11 and the §Department of Life Science, Aichi University of Education, Kariya 448, Japan
Abstract
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.
Footnotes
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↵* This work was supported in part by special coordination funds from the Science and Technology Agency of the Japanese Government and the New Energy and Industrial Technology Development Organization and by a special research fund from Seikagaku Corp.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the DNA Data Bank of Japan and the GenBank™/EMBL Data Bank with accession number(s) D88811 .
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↵‡ Contributed equally to this work.
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↵¶ To whom correspondence should be addressed. Tel.: 81-52-264-4811 (ext. 2088); Fax: 81-561-63-3532.
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↵1 The abbreviations used are: HS, heparan sulfate; HS2ST, heparan-sulfate 2-sulfotransferase; HS6ST, heparan-sulfate 6-sulfotransferase; CHO, Chinese hamster ovary; HPLC, high performance liquid chromatography; PCR, polymerase chain reaction; bp, base pair(s).
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- Received November 5, 1996.
- Revision received March 3, 1997.
