MOLECULAR CHARACTERIZATION AND EXPRESSION OF HEPARAN-SULFATE 6-SULFOTRANSFERASE - COMPLETE CDNA CLONING IN HUMAN AND PARTIAL CLONING IN CHINESE-HAMSTER OVARY CELLS

Heparan-sulfate 6-sulfotransferase (HS6ST) catalyzes the transfer of s ulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of the N-sulfoglucosamine residue of heparan sulfate. The enzyme was purifie d to apparent homogeneity from the serum-free culture medium of Chines e hamster ovary (CHO) cells (Habuchi, H., Habuchi, O., and Kimata, K. (1995) J. Biol Chem. 270, 4172-4179). From the amino acid sequence dat a of the purified enzyme, degenerate oligonucleotides were designed an d used as primers for the reverse transcriptase polymerase chain react ion using poly(A)(+) RNA from CHO cells as a template. The amplified c DNA 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 si x peptides obtained after endoproteinase digestion of the purified enz yme. This cDNA clone was applied to the screening of a human fetal bra in cDNA library by crosshybridization. The isolated cDNA clones contai ned a whole open reading frame that predicts a type II transmembrane p rotein composed of 401 amino acid residues. No significant amino acid sequence identity to any other proteins, including heparan-sulfate 2-s ulfotransferases, was observed. When the cDNA for the entire coding se quence 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 affin ity chromatography showed the HS6ST activity alone. Northern blot anal ysis revealed the occurrence of a single transcript of 3.9 kilobases i n both human fetal brain and CHO cells. The results, together with the ones from our recent cDNA analysis of heparan sulfate 2-sulfotransfer ase (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 h eparan sulfate.