Sequential degradation of heparan sulfate in the subendothelial extracellular matrix by highly metastatic lymphoma cells.
A highly metastatic variant (ESb) of a methylcholanthrene-induced T lymphoma elaborates a heparan sulfate (HS) degrading endoglycosidase (heparanase) to a much higher extent than its non-metastatic parental subline (Eb). Whereas a serum-free medium conditioned by either subline contained a trypsin-like serine protease, heparanase activity was detected only in the ESb-conditioned medium (CM). ESb CM was incubated with a naturally produced, sulfate-labelled subendothelial extracellular matrix (ECM) or with a soluble, high-MW labelled proteoglycan first released from the ECM by incubation with Eb CM or with the partially purified ESb protease. Sulfate labelled degradation products were analyzed by gel filtration on Sephrose 6B. The optimal pH for degradation of ECM-bound HS was 6.2 as compared to pH 5.2 for degradation of the soluble proteoglycan. Heparanase-mediated degradation of both ECM-bound and soluble HS was inhibited by heparin. Addition of either trypsin, plasmin or to a lower extent, the purified ESb protease, stimulated between 5- and 20-fold the ESb CM-mediated degradation of ECM-bound HS but had no effect on heparanase-mediated degradation of the soluble proteoglycan. This stimulation was inhibited in the presence of heparin or protease inhibitors. These results indicate that both a protease and heparanase are involved in the ESb-mediated degradation of ECM-bound HS and that one enzyme produces a more accessible substrate for the next enzyme. This sequential cleavage is characteristic of degradation of a multimolecular structure such as the subendothelial ECM and hence cannot be detected in studies with its isolated constituents.
Bar-Ner
M
M
Kramer
M D
MD
Schirrmacher
V
V
Ishai-Michaeli
R
R
Fuks
Z
Z
Vlodavsky
I
I
D000818:Animals; D002460:Cell Line; D002850:Chromatography, Gel; D003470:Culture Media; D010450:Endopeptidases / Q000378:metabolism; D004727:Endothelium / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism*; D005966:Glucuronidase; D006025:Glycosaminoglycans / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D006497:Heparan Sulfate / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008223:Lymphoma / Q000139:chemically induced / Q000201:enzymology / Q000556:secondary*; D051379:Mice; D008970:Molecular Weight; D012697:Serine Endopeptidases; D013462:Sulfur Radioisotopes; D000099175:Heparanase
D003470:Culture Media; D010450:Endopeptidases; D005966:Glucuronidase; D006025:Glycosaminoglycans; D006026:Glycoside Hydrolases; D006493:Heparin; D006497:Heparan Sulfate; D012697:Serine Endopeptidases; D013462:Sulfur Radioisotopes; D000099175:Heparanase