Sequential degradation of heparan sulfate in the subendothelial extracellular matrix by highly metastatic lymphoma cells

Abstract

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.

The Impact of Heparanese and Heparin on Cancer Metastasis and Angiogenesis

“…Heparanase activity was further characterized as an endoglucuronidase and found to be inhibited by species of heparin [36,38]. This latter finding was confirmed by other laboratories in multitude experimental settings, providing the basis for the development of heparanase-inhibiting non-anticoagulant species of heparin (see below).…”

P‐selectin‐ and heparanase‐dependent antimetastatic activity of non‐anticoagulant heparins

“…This material provides a readily accessible soluble substrate for the heparanase enzyme and is produced by proteolytic enzymes degrading the proteoglycan core protein and residing in the ECM and cell lysates (16). We have previously demonstrated that degradation of heparan sulfate proteoglycans in the ECM involves the sequential action of proteases and heparanase and that inhibition of heparanase is associated with a marked accumulation of soluble, nearly intact proteoglycans (16,38). This was also the case for incubation of the ECM with B16-BL6 melanoma cells in the presence of RO.H or 100 NA-RO.H (data not shown).…”

Coordinate secretion and functional synergism of T cell‐associated serine proteinase‐1 (MTSP‐1) and endoglycosidase(s) of activated T cells

“…Previous investigations have shown that the invasive capacity of various cell types, including malignant T lymphoma cells, only correlated with their ability to release low-but not high-M, sulfated compounds from ECM-associated heparan sulfate proteoglycans [2]. These data indicate that under physiological conditions cleavage of proteoglycan core protein structures by proteolytic enzymes alone is not sufficient to promote cell invasion but that additional enzymatic degradation of the ECM-associated heparan sulfate proteoglycans is required for this process to occur.…”

“…The labeled ECM was then ready to be used in degradation experiments. Previous studies have shown that 70% -75% of the total ECM-bound radioactivity were incorporated into heparan sulfate side chains [2].…”