MEMBRANE-INTERCALATED PROTEOGLYCAN OF A STROMA-INDUCING CLONE FROM LEWIS LUNG-CARCINOMA BINDS TO FIBRONECTIN VIA ITS HEPARAN-SULFATE CHAINS

A Lewis lung carcinoma-derived low metastatic clone, P29, with a capac ity to induce a fibrotic stromal response of host tissue, exhibits tum origenesis depending on an interstitial matrix formed by the induced s tromal cells. Using this clone, in the present study we isolated and c haracterized a membrane-intercalated proteoglycan that mediates intera ction between the tumor cells and interstitial matrix. The tumor cells were cultured in the presence of [H-3]glucosamine and [S-35]sulfate o r [S-35]methionine, and hydrophobic proteoglycans were isolated by chr omatography on DEAE-Sephacel and then Octyl-Sepharose CL-4B. Proteogly cans with high affinity to the octylresidue were obtained from the cel l layer but not to any significant extent from the medium. By CsCl den sity gradient centrifugation, they were separated into bottom, middle, and top subfractions, which were shown to consist of homogeneous spec ies with estimated M(r) values of 270,000 (named CPGIIIB), 200,000 (CP GIIIM), and 195,000 (CPGIIIT), respectively, by gel filtration on Seph arose CL-4B. These proteoglycans were intercalated into phosphatidylch oline liposomes, suggesting that they are all membrane-intercalated pr oteoglycans. Analyses of their glycosaminoglycans with chondroitinase ABC and heparitinase I plus II demonstrated that they all contain hepa ran sulfate as a major glycosaminoglycan (58-85%) and chondroitin 4-su lfate as a minor one (15-42%). Of these three proteoglycans, only CPGI IIB proteoglycan bound specifically to fibronectin-Sepharose 4B under physiological conditions. Molecular analyses of this proteoglycan by S epharose CL-4B or SDS-PAGE before and after treatments with glycosamin oglycan degradation enzymes or trifluoromethanesulfonic acid demonstra ted that CPGIIIB proteoglycan is a hybrid proteoglycan having heparan sulfate and chondroitin 4-sulfate chains on the same core protein with an M(r) of 40,000. Affinity chromatographies of the CPGIIIB proteogly can on fibronectin-Sepharose 4B after treatments with these enzymes de monstrated that it bound to fibronectin via its heparan sulfate chains . On the basis of the above results, we propose that the CPGIIIB prote oglycan mediates the interaction between the tumor cells and interstit ial matrix.