N. Itano et al., MEMBRANE-INTERCALATED PROTEOGLYCAN OF A STROMA-INDUCING CLONE FROM LEWIS LUNG-CARCINOMA BINDS TO FIBRONECTIN VIA ITS HEPARAN-SULFATE CHAINS, Journal of Biochemistry, 114(6), 1993, pp. 862-873
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.