Abstract
Heparan-sulfate 6-sulfotransferase (HS6ST) catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of the N-sulfoglucosamine residue of heparan sulfate. The enzyme was purified to apparent homogeneity from the serum-free culture medium of Chinese hamster ovary (CHO) cells (Habuchi, H., Habuchi, O., and Kimata, K. (1995) J. Biol Chem. 270, 4172-4179). From the amino acid sequence data of the purified enzyme, degenerate oligonucleotides were designed and used as primers for the reverse transcriptase-polymerase chain reaction using poly(A)+ RNA from CHO cells as a template. The amplified cDNA fragment was then used as a probe to screen a cDNA library of CHO cells. The cDNA clone thus obtained encoded a partial peptide sequence composed of 236 amino acid residues that included the sequences of six peptides obtained after endoproteinase digestion of the purified enzyme. This cDNA clone was applied to the screening of a human fetal brain cDNA library by cross-hybridization. The isolated cDNA clones contained a whole open reading frame that predicts a type II transmembrane protein composed of 401 amino acid residues. No significant amino acid sequence identity to any other proteins, including heparan-sulfate 2-sulfotransferases, was observed. When the cDNA for the entire coding sequence of the protein was inserted into a eukaryotic expression vector and transfected into COS-7 cells, the HS6ST activity increased 7-fold over the control. The FLAG fusion protein purified by anti-FLAG affinity chromatography showed the HS6ST activity alone. Northern blot analysis revealed the occurrence of a single transcript of 3.9 kilobases in both human fetal brain and CHO cells. The results, together with the ones from our recent cDNA analysis of heparan-sulfate 2-sulfotransferase (Kobayashi, M., Habuchi, H., Yoneda, M., Habuchi, O., and Kimata, K. (1997) J. Biol. Chem. 272, 13980-13985), suggest that at least two different gene products are responsible for 6- and 2-O-sulfations of heparan sulfate.
Highlights
Heparan sulfate proteoglycans are ubiquitously present on the cell surface and in the extracellular matrix, including the basement membrane [1, 2]
Amino Acid Sequence of Chinese Hamster Heparan-sulfate 6-Sulfotransferase—Heparan-sulfate 6-sulfotransferase (HS6ST) purified from the serum-free culture medium of Chinese hamster ovary (CHO) cells was immobilized on a polyvinylidene difluoride membrane after SDS-polyacrylamide gel electrophoresis
The following lines of evidence suggest that the cloned cDNA encodes a human homologue of the hamster HS6ST protein that we previously purified from CHO cell culture medium [35]
Summary
Amino Acid Sequence of Peptides Derived from Purified Protein— HS6ST was purified from the serum-free culture medium of CHO cell as described previously [35]. The bands of the 45- and 52-kDa proteins, which had the HS6ST activity [35], were excised, and a part of the membrane was used for N-terminal amino acid sequencing. Hybond Nϩ nylon membrane (Amersham Pharmacia Biotech) replicas of the plaques from the gt cDNA library were fixed by the alkali fixation method recommended by the manufacturer and prehybridized in a solution containing 50% formamide, 5ϫ SSPE (SSPE ϭ sodium chloride/sodium phosphate/EDTA buffer), 5ϫ Denhardt’s solution, 0.5% SDS, and 0.05 mg/ml denatured salmon sperm DNA for 3 h ster ovary; HPLC, high performance liquid chromatography; PCR, polymerase chain reaction. The amino acid sequences of different peptides after sequential in situ digestion of purified CHO cell HS6ST with endoproteinases Asp-N and Lys-C and trypsin were obtained as described under “Experimental Procedures.”.
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