MOLECULAR-CLONING AND EXPRESSION OF CHINESE-HAMSTER OVARY CELL HEPARAN-SULFATE 2-SULFOTRANSFERASE

Heparan-sulfate a-sulfotransferase (HS2ST), which catalyzes the transf er of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to L-iduronic acid at position 2 in heparan sulfate, was purified from cultured Chi nese hamster ovary (CHO) cells to apparent homogeneity (Kobayashi, M., Habuchi, H., Habuchi, O., Saito, M,, and Kimata, K, (1996) J, Biol. C hem, 271, 7645-7653), The internal amino acid sequences were obtained from the peptides after digestion of the purified protein with a combi nation 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 w ith the probe is 2.2 kilobases in size and contains an open reading fr ame of 1068 bases encoding a new protein composed of 356 amino acid re sidues, The protein predicts a type II transmembrane topology similar to other Gels membrane proteins, Messages of 5.0 and 3.0 kilobases wer e observed in Northern analysis, Evidence that the cDNA clone correspo nds to the purified HS2ST protein is as follows, (a) The predicted ami no acid sequence contains all five peptides obtained after endoprotein ase digestion of the purified protein; (b) the characteristics of the predicted protein fit those of the purified protein in terms of molecu lar mass, membrane localization, and N-glycosylation; and (c) when the cDNA containing the entire coding sequence of the enzyme in a eukaryo tic expression vector was transfected into COS-7 cells, the HS2ST acti vity increased 2.6-fold over controls, and the FLAG-HS2ST fusion prote in purified by affinity chromatography showed the HS2ST activity alone .