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

Abstract

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 malignant 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

Inhibition of heparanase activity and heparanase‐induced angiogenesis by suramin analogues

“…3,29,30 Radioactivity was quantified on a liquid scintillation counter. Each experiment was performed 3 times, and the variation of elution positions (K av values) did not exceed Ϯ 15%.…”

“…HS degradation products were tested for their susceptibility to heparinase, chondroitinases ABC/AC and Flavobacterium heparitinases digestion (Seikagaku America, St. Petersburg, FL). 30 Preparation of heparanase-containing cellular extracts and heparanase purification Subconfluent 70W cells (2 ϫ 10 8 ) were harvested and solubilized in 50 mM Tris-HCl, pH 7.5, containing 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma Chemical Company, St. Louis, MO), 5 mM N-ethylmaleimide (Sigma), 0.05% (w/v) sodium azide, 0.5% (w/v) Triton X-100 at 4°C for 30 min. The cell extract was centrifuged at 12,000g for 30 min at 4°C, and protein concentration was determined using the BCA protein assay protocol (Pierce, Rockford, IL).…”

“…Heparanase was assayed as previously described 7,28,30 with some modifications. Heparanase activity was determined by degradation of [ 35 S]HS using high-speed gel permeation column chromatography (HPLC).…”

“…A cell survival index (SI) was subsequently derived and defined as the fluorescence in the experimental wells compared to that in control wells, with the blank values subtracted. IC 30 values were determined as the concentration of compound giving an SI of 70%.…”

“…[25][26][27] The ability of 70W cells to degrade HS in the ECM was studied by allowing the cells to interact with naturally produced sulfate-labeled ECM followed by gel filtration analysis of degradation products released into a culture medium. 3,30 Degradation of HS in the ECM was studied by incubation (72 hr at 37°C, pH 6.7) of high-heparanasecontaining 70W previously exposed to suramin or, alternatively, to NF 127, NF 145 and NF 171 with a metabolically sulfate-labeled ECM produced by cultured endothelial cells (MBE). Sulfate-labeled degradation products released into the incubation medium were subjected to gel filtration on Sepharose-6B columns.…”