Characteristics of proteoglycans associated with cell membrane of ascites Tawa sarcoma cells

We investigated the biochemical characteristics of cell membrane-associated proteoglycans extracted from ascites Tawa sarcoma cells. Proteoglycans were extracted with 4 M Gdn-HCl, and purified by DEAE-Sephacel. The extract sample was fractionated into two proteoglycan fractions, TC-I and TC-II, and eluted at salt concentrations of approximately 0.35 M and 0.45 M NaCl, respectively, by HPLC ion exchange chromatography using a Bio-Scale DEAE 5 column in 7 M urea. After HPLC gel filtration using a TSK gel G 6000 PW column, the fractions were further analysed by hydrophobic interaction chromatography on Octyl-Sepharose in 4 M Gdn-HCl. Since TC-I displayed hydrophobic properties while TC-II was non-hydrophobic, the former was regarded as the proteoglycan associated with the cell membrane. Cellulose acetate membrane electrophoresis confirmed that both TC-I and TC-II contained only heparan sulfate as a sugar chain, and that the degree of sulfation of TC-I and TC-II was lower than that for normal tissue. Immunoblotting with monoclonal antibody HepSS-1 showed that TC-I and TC-II contained two heparan sulfate proteoglycans with Mr of about 30 kDa and 45 kDa, respectively. These results indicate that the proteoglycan associated with the cell membrane of ascites Tawa sarcoma cells is a small and undersulfated-heparan sulfate proteoglycan.