A
Lewis lung carcinoma-derived low metastatic clone, P29, with a capacity to induce a fibrotic stromal response of host tissue, exhibits
tumorigenesis depending on an interstitial matrix formed by the induced stromal cells. Using this clone, in the present study we isolated and characterized a membrane-intercalated
proteoglycan that mediates interaction between the
tumor cells and interstitial matrix. The
tumor cells were cultured in the presence of [3H]
glucosamine and [35S]
sulfate or [35S]
methionine, and hydrophobic
proteoglycans were isolated by chromatography on
DEAE-Sephacel and then
Octyl-Sepharose CL-4B.
Proteoglycans with high affinity to the octylresidue were obtained from the cell layer but not to any significant extent from the medium. By
CsCl density gradient centrifugation, they were separated into bottom, middle, and top subfractions, which were shown to consist of homogeneous species with estimated M(r) values of 270,000 (named CPGIIIB), 200,000 (CPGIIIM), and 195,000 (CPGIIIT), respectively, by gel filtration on
Sepharose CL-4B. These
proteoglycans were intercalated into
phosphatidylcholine liposomes, suggesting that they are all membrane-intercalated
proteoglycans. Analyses of their
glycosaminoglycans with
chondroitinase ABC and
heparitinase I plus II demonstrated that they all contain
heparan sulfate as a major
glycosaminoglycan (58-85%) and
chondroitin 4-sulfate as a minor one (15-42%). Of these three
proteoglycans, only CPGIIIB
proteoglycan bound specifically to
fibronectin-
Sepharose 4B under physiological conditions. Molecular analyses of this
proteoglycan by
Sepharose CL-4B or SDS-PAGE before and
after treatments with
glycosaminoglycan degradation
enzymes or
trifluoromethanesulfonic acid demonstrated 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
proteoglycan on
fibronectin-
Sepharose 4B after treatments with these
enzymes demonstrated that it bound to
fibronectin via its
heparan sulfate chains. On the basis of the above results, we propose that the CPGIIIB
proteoglycan mediates the interaction between the
tumor cells and interstitial matrix.