Heparanase and a Synthetic Peptide of Heparan Sulfate-interacting Protein Recognize Common Sites on Cell Surface and Extracellular Matrix Heparan Sulfate

Dario Marchetti, Shouchun Liu, William C. Spohn, Daniel D. Carson
1997 Journal of Biological Chemistry  
Heparanase is an endo-␤-D-glucuronidase that degrades the glycosaminoglycan chains of heparan sulfate (HS) proteoglycans at specific sites. Elevated levels of heparanase are associated with the metastatic potential of melanoma and other types of tumor cells. We previously reported heparanase degradation of cell surface HS subpopulations of the human adenocarcinoma cell line RL95. In the present study, heparanase activity was examined on RL95 cell surface HS subpopulations in the presence of a
more » ... nthetic peptide (CRPKAKAKAKAK-DQTK) of heparin/heparan sulfate-interacting protein (HIP; Liu, S., Smith, ). Heparanase digestion generated HS fragments from cell surface-or extracellular matrix-derived HS of approximately 25 and 9 kDa, respectively. In contrast, HS of various size classes isolated from proteoglycans secreted or released by RL95 and endothelial cells in culture were not susceptible to heparanase digestion. Incubation of heparanase-containing melanoma cellular extracts or partially purified heparanase preparations with cell surface-or ECM-derived HS and HIP peptide, but not a scrambled sequence of this peptide or other HS-binding proteins present in ECM, completely inhibited heparanase action. Conversely, predigestion of cell surface HS with either heparanase-containing cellular extracts or with secreted or partially purified heparanase destroyed binding to HIP peptide. Preincubation of HS with HIP peptide prevented subsequent heparanase digestion. Collectively, these data demonstrate that HIP peptide and heparanase recognize specific, common motifs within HS chains at cell surfaces and in ECM and may mutually modulate HS-dependent activities. Metastasis occurs via a sequential and complex series of interactions between tumor cells and normal host cells and tissues (1, 2). During the formation of metastases, migrating cells are confronted by natural tissue barriers, such as connective tissue stroma and basal lamina (2, 3). The ability of ma-lignant cells to penetrate these barriers depends upon the presence of enzymes capable of degrading extracellular matrix (ECM) 1 components (1-5). For these reasons, considerable effort has been focused on the study of tissue-degradative enzymes produced and secreted by metastatic tumor cells as well as normal cells of the tissue being invaded. Important ECM targets for degradation by invading melanoma cells are the heparan sulfate (HS) chains found on proteoglycans (3, 6, 7). HS are highly negatively charged linear polysaccharides consisting of alternating residues of uronic acids and glucosamine. Proteins containing one or more covalently attached HS chains are called HS proteoglycans (HSPGs). The dynamic role of HSPGs in biology has become increasingly apparent (8 -23). As a result of characterizing heparin (HP) and HS binding sites related to the initial attachment of trophoblast cells to uterine epithelial cells of murine and human origin, we recently reported the cell surface expression and molecular cloning of a novel HP/HS-interacting protein (HIP) of human epithelial and endothelial cells (24 -26). HIP not only recognizes HS and HP in a highly specific fashion, but it also binds a subset of HP and forms of HS enriched at cell surfaces and in ECM. In contrast, HIP does not bind intracellular or secreted forms of HS. Furthermore, HIP also appears to bind the anticoagulantly active species of HP efficiently and with high affinity. 2 HP octasaccharides, but not hexasaccharides, are large enough to bind HIP with high affinity. Thus, HIP appears to recognize a motif that at least overlaps the anticoagulant motif in HP and HS chains. In light of the above findings, activities that mediate HSPG degradation are expected to have significant regulatory consequences. Indeed, HSPG catabolism is observed in inflammation, wound repair, diabetes, and cancer metastasis, suggesting that enzymes that degrade the HS chains play important roles in pathologic processes (3, 4). Furthermore, malignant cells are capable of modulating cellular interactions with HSPGs by producing and releasing a HS-degrading enzyme, heparanase (3, 7, 27, 28) . Recently, we reported that purified, high M r subpopulations of cell surface HS were more sensitive to heparanase action than secreted HS (29). In the present study, we have investigated the relationship between tumor (melanoma) heparanase activity and HIP binding in HS subpopulations whether on the cell surface, secreted, or deposited in the ECM. By use of sensitive heparanase assays that separate
doi:10.1074/jbc.272.25.15891 pmid:9188488 fatcat:yd3vjz7d2rdrldatjlerg3lr2m