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• W1971939635 abstract "The endo-β-glucuronidase, heparanase, is an enzyme that cleaves heparan sulfate at specific intra-chain sites, yielding heparan sulfate fragments with appreciable size and biological activities. Heparanase activity has been traditionally correlated with cell invasion associated with cancer metastasis, angiogenesis, and inflammation. In addition, heparanase up-regulation has been documented in a variety of primary human tumors, correlating with increased vascular density and poor postoperative survival, suggesting that heparanase may be considered as a target for anticancer drugs. In an attempt to identify the protein motif that would serve as a target for the development of heparanase inhibitors, we looked for protein domains that mediate the interaction of heparanase with its heparan sulfate substrate. We have identified three potential heparin binding domains and provided evidence that one of these is mapped at the N terminus of the 50-kDa active heparanase subunit. A peptide corresponding to this region (Lys158–Asp171) physically associates with heparin and heparan sulfate. Moreover, the peptide inhibited heparanase enzymatic activity in a dose-responsive manner, presumably through competition with the heparan sulfate substrate. Furthermore, antibodies directed to this region inhibited heparanase activity, and a deletion construct lacking this domain exhibited no enzymatic activity. NMR titration experiments confirmed residues Lys158–Asn162 as amino acids that firmly bound heparin. Deletion of a second heparin binding domain sequence (Gln270–Lys280) yielded an inactive enzyme that failed to interact with cell surface heparan sulfate and hence accumulated in the culture medium of transfected HEK 293 cells to exceptionally high levels. The two heparin/heparan sulfate recognition domains are potentially attractive targets for the development of heparanase inhibitors. The endo-β-glucuronidase, heparanase, is an enzyme that cleaves heparan sulfate at specific intra-chain sites, yielding heparan sulfate fragments with appreciable size and biological activities. Heparanase activity has been traditionally correlated with cell invasion associated with cancer metastasis, angiogenesis, and inflammation. In addition, heparanase up-regulation has been documented in a variety of primary human tumors, correlating with increased vascular density and poor postoperative survival, suggesting that heparanase may be considered as a target for anticancer drugs. In an attempt to identify the protein motif that would serve as a target for the development of heparanase inhibitors, we looked for protein domains that mediate the interaction of heparanase with its heparan sulfate substrate. We have identified three potential heparin binding domains and provided evidence that one of these is mapped at the N terminus of the 50-kDa active heparanase subunit. A peptide corresponding to this region (Lys158–Asp171) physically associates with heparin and heparan sulfate. Moreover, the peptide inhibited heparanase enzymatic activity in a dose-responsive manner, presumably through competition with the heparan sulfate substrate. Furthermore, antibodies directed to this region inhibited heparanase activity, and a deletion construct lacking this domain exhibited no enzymatic activity. NMR titration experiments confirmed residues Lys158–Asn162 as amino acids that firmly bound heparin. Deletion of a second heparin binding domain sequence (Gln270–Lys280) yielded an inactive enzyme that failed to interact with cell surface heparan sulfate and hence accumulated in the culture medium of transfected HEK 293 cells to exceptionally high levels. The two heparin/heparan sulfate recognition domains are potentially attractive targets for the development of heparanase inhibitors. Heparan-sulfate proteoglycans (HSPGs) 1The abbreviations used are: HSPGs, heparan sulfate proteoglycans; HS, heparan sulfate; ECM, extracellular matrix; WT, wild type; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; CHO, Chinese hamster ovary; CM, conditioned medium; PBS, phosphate-buffered saline; HA, hyaluronic acid; GS, glycol-split. are members of the glycosaminoglycan family, a class of molecules that consists of unbranched, repeated disaccharide units attached to a core protein. Proteoglycans are present essentially in every tissue compartment, localized in the extracellular matrix (ECM), on the cell surface, intracellularly in granules, and even in the nucleus (1Couchman J.R. Nature Rev. Mol. Cell. Biol. 2003; 4: 926-937Crossref PubMed Scopus (342) Google Scholar, 2Hiscock D.R. Yanagishita M. Hascall V.C. J. Biol. Chem. 1994; 269: 4539-4546Abstract Full Text PDF PubMed Google Scholar, 3Kramer K.L. Yost H.J. Annu. Rev. Genet. 2003; 37: 461-484Crossref PubMed Scopus (114) Google Scholar). Virtually all cells express at least one proteoglycan on their surface. Membrane-associated proteoglycans are mostly HS that can be either transmembrane (syndecan) or glycosylphosphatidylinositol-anchored (glypican). From mice to worms, embryos that lack HS die during gastrulation (3Kramer K.L. Yost H.J. Annu. Rev. Genet. 2003; 37: 461-484Crossref PubMed Scopus (114) Google Scholar), suggesting a critical developmental role for HSPGs. HSPGs play key roles in numerous biological settings, including cytoskeleton organization, cell/cell, and cell/ECM interactions (4Iozzo R.V. San Antonio J.D. J. Clin. Investig. 2001; 108: 349-355Crossref PubMed Scopus (402) Google Scholar, 5Sasisekharan R. Shriver Z. Venkataraman G. Narayanasami U. Nature Rev. Cancer. 2002; 2: 521-528Crossref PubMed Scopus (555) Google Scholar, 6Simons M. Horowitz A. Cell. Signal. 2001; 13: 855-862Crossref PubMed Scopus (122) Google Scholar). For biological function, HSPGs exert their multiple functional repertoire via several distinct mechanisms that combine structural, biochemical, and regulatory aspects. By interacting with other macromolecules such as laminin, fibronectin, and collagen IV, HSPGs contribute to the structural integrity, self-assembly, and insolubility of the ECM and basement membrane. ECM components are, however, only one class of HSPG-binding proteins. In fact, numerous enzymes, growth factors, cytokines, and chemokines are sequestered by HSPGs on the cell surface and ECM, most often as an inactive reservoir (7Capila I. Linhardt R.J. Angew. Chem. Int. Ed. Engl. 2002; 41: 391-397Crossref PubMed Scopus (1550) Google Scholar, 8Munoz E.M. Linhardt R.J. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1549-1557Crossref PubMed Scopus (169) Google Scholar). Cleavage of HSPGs would ultimately release these polypeptides and convert them into bioactive mediators, thus ensuring rapid tissue response to local environmental alterations. The protein core of HSPGs is susceptible to cleavage by several classes of proteases (9Fitzgerald M.L. Wang Z. Park P.W. Murphy G. Bernfield M. J. Cell Biol. 2000; 148: 811-824Crossref PubMed Scopus (347) Google Scholar, 10Schulz J.G. Annaert W. Vandekerckhove J. Zimmermann P. De Strooper B. David G. J. Biol. Chem. 2003; 278: 48651-48657Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 11Werb Z. Cell. 1997; 91: 439-442Abstract Full Text Full Text PDF PubMed Scopus (1132) Google Scholar). A more delicate way to modify HSPGs is provided by the endo-β-glucuronidase, heparanase, an enzyme that cleaves HS at specific intra-chain sites, yielding HS fragments with appreciable size and biological activities (12Freeman C. Parish C.R. Biochem. J. 1998; 330: 1341-1350Crossref PubMed Scopus (173) Google Scholar, 13Pikas D.S. Li J.-P. Vlodavsky I. Lindahl U. J. Biol. Chem. 1998; 273: 18770-18777Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 14Vlodavsky I. Friedmann Y. Elkin M. Aingorn H. Atzmon R. Ishai-Michaeli R. Bitan M. Pappo O. Peretz T. Michal I. Spector L. Pecker I. Nat. Med. 1999; 5: 793-802Crossref PubMed Scopus (725) Google Scholar, 15Vlodavsky I. Friedmann Y. J. Clin. Investig. 2001; 108: 341-347Crossref PubMed Scopus (548) Google Scholar). Heparanase activity has been correlated traditionally with cell invasion associated with cancer metastasis, angiogenesis, and inflammation (16Nakajima M. Irimura T. Di Ferrante N. Nicolson G.L. J. Biol. Chem. 1984; 259: 2283-2290Abstract Full Text PDF PubMed Google Scholar, 17Nakajima M. Irimura T. Nicolson G.L. J. Cell. Biochem. 1988; 36: 157-167Crossref PubMed Scopus (281) Google Scholar, 18Parish C.R. Coombe D.R. Jakobsen K.B. Bennett F.A. Underwood P.A. Int. J. Cancer. 1987; 40: 511-518Crossref PubMed Scopus (161) Google Scholar, 19Vlodavsky I. Eldor A. Haimovitz-Friedman A. Matzner Y. Ishai-Michaeli R. Lider O. Naparstek Y. Cohen I.R. Fuks Z. Invasion Metastasis. 1992; 12: 112-127PubMed Google Scholar). A proof of concept for this notion has been provided recently by applying short interfering RNA and ribozyme technologies (20Edovitsky E. Elkin M. Zcharia E. Peretz T. Vlodavsky I. J. Natl. Cancer Inst. 2004; 96: 1219-1230Crossref PubMed Scopus (227) Google Scholar). In addition, heparanase up-regulation has been documented in a variety of primary human tumors correlating with increased vascular density and poor postoperative survival, in some cases (21Gohji K. Hirano H. Okamoto M. Kitazawa S. Toyoshima M. Dong J. Katsuoka Y. Nakajima M. Int. J. Cancer. 2001; 95: 295-301Crossref PubMed Scopus (110) Google Scholar, 22Koliopanos A. Friess H. Kleeff J. Shi X. Liao Q. Pecker I. Vlodavsky I. Zimmermann A. Buchler M.W. Cancer Res. 2001; 61: 4655-4659PubMed Google Scholar, 23Rohloff J. Zinke J. Schoppmeyer K. Tannapfel A. Witzigmann H. Mossner J. Wittekind C. Caca K. Br. J. Cancer. 2002; 86: 1270-1275Crossref PubMed Scopus (107) Google Scholar, 24Ohkawa T. Naomoto Y. Takaoka M. Nobuhisa T. Noma K. Motoki T. Murata T. Uetsuka H. Kobayashi M. Shirakawa Y. Yamatsuji T. Matsubara N. Matsuoka J. Haisa M. Gunduz M. Tsujigiwa H. Nagatsuka H. Hosokawa M. Nakajima M. Tanaka N. Lab. Investig. 2004; 84: 1289-1304Crossref PubMed Scopus (69) Google Scholar, 25Sato T. Yamaguchi A. Goi T. Hirono Y. Takeuchi K. Katayama K. Matsukawa S. J. Surg. Oncol. 2004; 87: 174-181Crossref PubMed Scopus (74) Google Scholar). Heparanase overexpression has also been noted in several pathologies other than cancer and inflammation (26Dempsey L.A. Brunn G.J. Platt J.L. Trends Biochem. Sci. 2000; 25: 349-351Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 27Katz A. Van-Dijk D.J. Aingorn H. Erman A. Davies M. Darmon D. Hurvitz H. Vlodavsky I. Isr. Med. Assoc. J. 2002; 4: 996-1002PubMed Google Scholar, 28Levidiotis V. Freeman C. Tikellis C. Cooper M.E. Power D.A. J. Am. Soc. Nephrol. 2004; 15: 68-78Crossref PubMed Scopus (83) Google Scholar, 29Xiao Y. Kleeff J. Shi X. Buchler M.W. Friess H. Hepatol. Res. 2003; 26: 192-198Crossref PubMed Scopus (34) Google Scholar), suggesting a broader pathological repertoire than originally thought. The heparanase cDNA encodes for a protein of 543 amino acids that undergoes proteolytic processing at two potential cleavage sites, Glu109–Ser110 and Gln157–Lys158, yielding an 8-kDa polypeptide at the N terminus and a 50-kDa polypeptide at the C terminus that heterodimerize to form an active heparanase enzyme (30Fairbanks M.B. Mildner A.M. Leone J.W. Cavey G.S. Mathews W.R. Drong R.F. Slightom J.L. Bienkowski M.J. Smith C.W. Bannow C.A. Heinrikson R.L. J. Biol. Chem. 1999; 274: 29587-29590Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 31Levy-Adam F. Miao H.Q. Heinrikson R.L. Vlodavsky I. Ilan N. Biochem. Biophys. Res. Commun. 2003; 308: 885-891Crossref PubMed Scopus (102) Google Scholar, 32McKenzie E. Young K. Hircock M. Bennett J. Bhaman M. Felix R. Turner P. Stamps A. McMillan D. Saville G. Ng S. Mason S. Snell D. Schofield D. Gong H. Townsend R. Gallagher J. Page M. Parekh R. Stubberfield C. Biochem. J. 2003; 373: 423-435Crossref PubMed Scopus (104) Google Scholar, 33Parish C.R. Freeman C. Hulett M.D. Biochim. Biophys. Acta. 2001; 1471: M99-M108PubMed Google Scholar). The present study was undertaken to identify functional domains that would serve as targets for drug development. Based on published consensus sequences that mediate the interaction between polypeptides and heparin, we have identified three potential heparin binding domains mapped at Lys158–Asp171, Gln270–Lys280, and Lys411–Arg432 of the 50-kDa heparanase subunit. Here we provide evidence that the Lys158–Asp171 N-terminal peptide physically associates with heparin and HS more strongly than the other two peptides. Moreover, the peptide inhibits heparanase enzymatic activity in a dose-responsive manner, presumably through competition with the HS substrate. Furthermore, antibodies directed to this region inhibit heparanase activity (34Zetser A. Levy-Adam F. Kaplan V. Gingis-Velitski S. Bashenko Y. Schubert S. Flugelman M.Y. Vlodavsky I. Ilan N. J. Cell Sci. 2004; 117: 2249-2258Crossref PubMed Scopus (190) Google Scholar), and a deletion construct lacking this domain exhibits no enzymatic activity. NMR titration experiments performed with a synthetic pentasaccharide confirmed residues Lys158–Asn162 as the amino acids important for heparin binding, indicating this recognition domain as a potentially attractive target for the development of heparanase inhibitors. Antibodies and Reagents—The rabbit anti-heparanase antibody (1453) recognizing the 65- and 50-kDa heparanase proteins and the rabbit anti 8-kDa (810) antibody have been characterized previously (31Levy-Adam F. Miao H.Q. Heinrikson R.L. Vlodavsky I. Ilan N. Biochem. Biophys. Res. Commun. 2003; 308: 885-891Crossref PubMed Scopus (102) Google Scholar, 34Zetser A. Levy-Adam F. Kaplan V. Gingis-Velitski S. Bashenko Y. Schubert S. Flugelman M.Y. Vlodavsky I. Ilan N. J. Cell Sci. 2004; 117: 2249-2258Crossref PubMed Scopus (190) Google Scholar). The anti-Myc epitope tag (sc-40) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). 10–20% Tris-Tricine gels were purchased from Bio-Rad. Heparin, heparin-Sepharose beads, and hyaluronic acid were purchased from Sigma. HS-Sepharose was kindly provided by Dr. Hua Quan Miao (ImClone Systems Inc., New York), and bovine lung HS was kindly provided by Dr. Jin-Ping Li (Biomedical Chemistry, University of Uppsala, Sweden). Glycol-split modified heparin (compound ST1514) was kindly provided by Claudio Pisano (Sigma-Tau, Research Department, Pomezia, Rome, Italy) (35Casu B. Guerrini M. Guglieri S. Naggi A. Perez M. Torri G. Cassinelli G. Ribatti D. Carminati P. Giannini G. Penco S. Pisano C. Belleri M. Rusnati M. Presta M. J. Med. Chem. 2004; 47: 838-848Crossref PubMed Scopus (82) Google Scholar, 36Casu B. Guerrini M. Naggi A. Perez M. Torri G. Ribatti D. Carminati P. Giannini G. Penco S. Pisano C. Belleri M. Rusnati M. Presta M. Biochemistry. 2002; 41: 10519-10528Crossref PubMed Scopus (80) Google Scholar, 37Pisano C. Aulicino C. Vesci L. Casu B. Naggi A. Torri G. Ribatti D. Belleri M. Rusnati M. Presta M. Glycobiology. 2005; 15: C1-C6Crossref PubMed Scopus (61) Google Scholar). Recombinant active (8 + 50) heparanase was produced in insect cells and purified as described (32McKenzie E. Young K. Hircock M. Bennett J. Bhaman M. Felix R. Turner P. Stamps A. McMillan D. Saville G. Ng S. Mason S. Snell D. Schofield D. Gong H. Townsend R. Gallagher J. Page M. Parekh R. Stubberfield C. Biochem. J. 2003; 373: 423-435Crossref PubMed Scopus (104) Google Scholar, 38Goshen R. Hochberg A. Korner G. Levy E. Ishai-Michaeli R. Elkin M. de Groot N. Vlodavsk Y.I. Mol. Hum. Reprod. 1996; 2: 679-684Crossref PubMed Scopus (56) Google Scholar). Recombinant latent (65 kDa) heparanase was produced in 293 cells and purified as described (39Zetser A. Bashenko Y. Miao H.-Q. Vlodavsky I. Ilan N. Cancer Res. 2003; 63: 7733-7741PubMed Google Scholar). Heparanase Deletion Mutants—Human heparanase cDNA constructs were generated using splice overlap extension PCR (40Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 61-68Crossref PubMed Scopus (2641) Google Scholar), applying the pcDNA3-Hpa plasmid (31Levy-Adam F. Miao H.Q. Heinrikson R.L. Vlodavsky I. Ilan N. Biochem. Biophys. Res. Commun. 2003; 308: 885-891Crossref PubMed Scopus (102) Google Scholar) as a template. Briefly, two PCRs were performed to generate overlapping 5′ and 3′ fragments encoding each mutation, followed by a third PCR for the fusion of the two fragments. The following primers were used in each PCR: for the Lys158–Val172 deletion (65Δ15 variant): PCR1, 8F (31Levy-Adam F. Miao H.Q. Heinrikson R.L. Vlodavsky I. Ilan N. Biochem. Biophys. Res. Commun. 2003; 308: 885-891Crossref PubMed Scopus (102) Google Scholar), and 65Δ15R, 5′-TGCAAAAGTGTATAGCTGGTAGTGTTCTCGGAGTA-3′; PCR2, 65Δ15F, 5′-CGAGAACACTACCAGCTATACACTTTTGCAAACTGCT-3′ and 50R (31Levy-Adam F. Miao H.Q. Heinrikson R.L. Vlodavsky I. Ilan N. Biochem. Biophys. Res. Commun. 2003; 308: 885-891Crossref PubMed Scopus (102) Google Scholar); for the Gln270–Lys280 deletion: PCR1, 8F (31Levy-Adam F. Miao H.Q. Heinrikson R.L. Vlodavsky I. Ilan N. Biochem. Biophys. Res. Commun. 2003; 308: 885-891Crossref PubMed Scopus (102) Google Scholar), and 65Δ10R, 5′-CACCAGCCTTCAGGAAGCTACCAACATCAGGACC-3′; PCR2, 65Δ10F, 5′-CTCTATGGTCCTGATGTTGGTAGCTTCCTGAAGGCT-3′ and 50R (31Levy-Adam F. Miao H.Q. Heinrikson R.L. Vlodavsky I. Ilan N. Biochem. Biophys. Res. Commun. 2003; 308: 885-891Crossref PubMed Scopus (102) Google Scholar); for the Lys411–Arg432 deletion: PCR1, 8F (31Levy-Adam F. Miao H.Q. Heinrikson R.L. Vlodavsky I. Ilan N. Biochem. Biophys. Res. Commun. 2003; 308: 885-891Crossref PubMed Scopus (102) Google Scholar), and 65Δ20R, 5′-GCAATGAAGGTATACGAACAGAAGAGATAGCCAATA-3′; PCR2, 65Δ20F, CTATCTCTTCTGTTCGTATACCTTCATTGCACAAAC-3′ and 50R (31Levy-Adam F. Miao H.Q. Heinrikson R.L. Vlodavsky I. Ilan N. Biochem. Biophys. Res. Commun. 2003; 308: 885-891Crossref PubMed Scopus (102) Google Scholar). The final PCR (PCR3) for the fusion of the above fragments was performed with 8F and 50R primers. For introducing the deletions in the 50-kDa heparanase the following primers were used: 50Δ15F, 5′-GGAATTCTATACACTTTTGCAAACTGCT-3′, and 50R primers, for generation of the 50Δ15-kDa variant, PCR1, 50F (31Levy-Adam F. Miao H.Q. Heinrikson R.L. Vlodavsky I. Ilan N. Biochem. Biophys. Res. Commun. 2003; 308: 885-891Crossref PubMed Scopus (102) Google Scholar), and 65Δ10R; PCR2, 65Δ10F and 50R, PCR3, 50F and 50R for the generation of the 50Δ10-kDa construct; PCR1, 50F and 65Δ20R; PCR2, 65Δ20F and 50R; PCR3, 50F and 50R were applied to generate the 50Δ20-kDa construct. Expression Vectors—The forward primers (8F and 50F) contained an inserted EcoRI restriction site, and the reverse primer (50R) contained an XhoI restriction site that enabled cloning in-frame into the pSecTag2A vector (Invitrogen). Proteins cloned in this vector contained c-Myc and His6 tags in their C terminus. Following PCR with a proofreading enzyme (Pfu, Promega, Madison, WI), the vector and the constructs were digested with EcoRI and XhoI and ligated with T4 ligase. The clones were propagated in DH5α Escherichia coli strain. DNA sequencing of the constructs was performed using vector- and construct-specific primers. Cell Lines and Transfection—HEK 293 and B16 melanoma cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, glutamine, pyruvate, and antibiotics. Transient or stable transfection was performed using FuGENE reagent, according to the manufacturer instructions (Roche Applied Science). 48 h following transfection, cells were harvested, lysed, and analyzed by immunoblotting and heparanase enzyme activity assays or were subjected to selection with 500 μg/ml Zeocin (Invitrogen). Stable transfectant pools were obtained after 2–3 weeks and used for further experiments. Immunoblotting, Metabolic Labeling, and Immunoprecipitation— Cell extracts were prepared using a lysis buffer containing 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 0.5% Triton X-100, and a mixture of protease inhibitors (Roche Applied Science). Protein concentration was determined (Bradford reagent, Bio-Rad), and 30 μg of protein were resolved by SDS-PAGE under reducing conditions. After electrophoresis, proteins were transferred to polyvinylidene difluoride membrane (Bio-Rad) and probed with the appropriate antibody followed by horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA) and an enhanced chemiluminescent substrate (Pierce). Metabolic labeling was performed essentially as described (34Zetser A. Levy-Adam F. Kaplan V. Gingis-Velitski S. Bashenko Y. Schubert S. Flugelman M.Y. Vlodavsky I. Ilan N. J. Cell Sci. 2004; 117: 2249-2258Crossref PubMed Scopus (190) Google Scholar). Briefly, confluent cell cultures were methionine-starved for 30 min prior to the addition of 150 μCi/ml [35S]methionine (Amersham Biosciences) and pulsed for 20 min. For immunoprecipitation, equal volumes (0.1 ml) or equal trichloroacetic acid-precipitable counts/min of lysate samples were brought to a volume of 1 ml with 50 mm Tris-HCl, pH 7.4, 5 mm EDTA, 150 mm NaCl, and 0.5% Nonidet P-40 (buffer A) and incubated with anti-Myc tag antibody for 2 h at 4 °C. Protein A/G-Sepharose beads (Santa Cruz Biotechnology) were then added for an additional 30 min. Beads were collected by centrifugation and washed three times with buffer A supplemented with 300 mm NaCl and 5% sucrose and once again with buffer A. Sample buffer was then added, and after boiling at 100 °C for 5 min, samples were subjected to electrophoresis as described above. Gels were fixed (30 min, 25% isopropyl alcohol + 10% acetic acid) and fluorographed (30 min, Amplify, Amersham Biosciences) following drying and autoradiography. Heparanase Activity Assay—Preparation of ECM-coated 35-mm dishes and determination of heparanase activity were performed as described in detail elsewhere (14Vlodavsky I. Friedmann Y. Elkin M. Aingorn H. Atzmon R. Ishai-Michaeli R. Bitan M. Pappo O. Peretz T. Michal I. Spector L. Pecker I. Nat. Med. 1999; 5: 793-802Crossref PubMed Scopus (725) Google Scholar, 31Levy-Adam F. Miao H.Q. Heinrikson R.L. Vlodavsky I. Ilan N. Biochem. Biophys. Res. Commun. 2003; 308: 885-891Crossref PubMed Scopus (102) Google Scholar). For inhibition studies, heparanase (20 ng) was incubated (2 h, 37 °C) with 35S-labeled ECM in the presence of the indicated concentration of peptides. For heparanase inhibition studies with intact cells, B16 melanoma cells (2 × 106) were resuspended in RPMI medium and incubated (18 h, 37 °C) with 35S-labeled ECM in the presence of the indicated peptide concentration. To evaluate heparanase activity in cell extracts, heparanase-transfected 293 cells (1 × 106) expressing the WT or deletion constructs were lysed by three freeze/thaw cycles, and the resulting cell extracts were incubated (18 h, 37 °C) with 35S-labeled ECM. The incubation medium (1 ml) containing sulfate-labeled degradation fragments was subjected to gel filtration on a Sepharose CL-6B column. Fractions (0.2 ml) were eluted with PBS, and their radioactivity was counted in a β-scintillation counter. Degradation fragments of HS side chains are eluted at 0.5 < Kav < 0.8 (peak II, fractions 15–30) and represent heparanase degradation products. Nearly intact HSPGs are eluted just after the V0 (Kav < 0.2, peak I, fractions 3–15) (14Vlodavsky I. Friedmann Y. Elkin M. Aingorn H. Atzmon R. Ishai-Michaeli R. Bitan M. Pappo O. Peretz T. Michal I. Spector L. Pecker I. Nat. Med. 1999; 5: 793-802Crossref PubMed Scopus (725) Google Scholar). These high molecular weight products are released by proteases that cleave the HSPG core protein. Heparin/HS Binding—Peptides (50 μm) were incubated (2 h, 4 °C) with heparin/HS-Sepharose beads in PBS, washed with PBS supplemented with NaCl to a final concentration of 0.35 m, followed by one wash with PBS. Dye-free sample buffer was added, and the beads were boiled for 5 min and centrifuged, and the supernatants were loaded on Tris-Tricine gel. Subsequently, gels were stained with Coomassie Blue to visualize bound peptides. For inhibition of heparanase binding to heparin, 20 ng of heparanase protein were incubated with heparin-Sepharose beads in the presence of increasing concentrations of the peptide of interest or its scrambled control peptide, followed by two washes with PBS supplemented with 0.6 m NaCl. Sample buffer was then added, and the mixture was boiled for 5 min and centrifuged, and the supernatant was subjected to immunoblotting with anti-heparanase antibodies. Extracellular accumulation of heparanase in the presence of heparin was examined as described (41Gingis-Velitski S. Zetser A. Kaplan V. Ben-Zaken O. Cohen E. Levy-Adam F. Bashenko Y. Flugelman M.Y. Vlodavsky I. Ilan N. J. Biol. Chem. 2004; 279: 44084-44092Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Briefly, stable transfected 293 cells expressing the 65-kDa WT, point-mutated, or the deletion (Δ10, Δ15, and Δ20) variants were incubated (18 h, 37 °C) with heparin, HS, and hyaluronic acid (HA) or glycol-split modified heparin under serum-free conditions. Medium was then collected and subjected to immunoblotting without or with prior precipitation with 10% trichloroacetic acid. Cell extracts were prepared, and 30 μg of protein were similarly analyzed. Heparanase Uptake—Uptake experiments were carried out essentially as described (34Zetser A. Levy-Adam F. Kaplan V. Gingis-Velitski S. Bashenko Y. Schubert S. Flugelman M.Y. Vlodavsky I. Ilan N. J. Cell Sci. 2004; 117: 2249-2258Crossref PubMed Scopus (190) Google Scholar, 41Gingis-Velitski S. Zetser A. Kaplan V. Ben-Zaken O. Cohen E. Levy-Adam F. Bashenko Y. Flugelman M.Y. Vlodavsky I. Ilan N. J. Biol. Chem. 2004; 279: 44084-44092Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Briefly, CHO K1 cells were incubated with 1 μg of 65-kDa WT or the 65Δ10 heparanase for the indicated time under serum-free conditions. At each time point the medium was aspirated, cells were washed twice with ice-cold PBS, and cell extracts were analyzed by immunoblotting as described above. For uptake inhibition studies, heparanase was iodinated to a high specific activity by the chloramine T (Sigma) method. Iodinated heparanase was added to CHO cells together with the Lys158–Asp171 (KKDC) peptide or its scrambled control peptide. Following incubation (1 h, 4 °C), cells were washed, lysed, and counted with γ-counter. NMR Spectra—NMR samples of the unlabeled Lys158–Asp171 peptide (KKDC) and the corresponding scrambled sequence were dissolved in 20 mm sodium phosphate buffer, pH 5.8, supplemented with 0.15 m NaCl and 10% D2O. All NMR spectra were recorded on a Bruker Avance 600 spectrometer equipped with a quadruple resonance proton-carbon-nitrogen-deuterium 5-mm probe (Bruker TCI cryo-probe®), with cooled coil and preamplifier and using a self-shielded z gradient coil. Spectra were acquired nonspinning at temperatures of 295 K. For two-dimensional homonuclear 1H experiments, DQF-COSY, TOCSY, and ROESY were performed according to the Bruker library pulse sequences. For ROESY, mixing times of 200 ms and a spin-lock of 150 ms obtained by continuous irradiation were used. COSY/TOCSY and ROESY experiments were acquired using 8 and 16 scans per series of 1Kx512W data points, respectively. Data were zero filled in F1, and a shifted squared cosine function was applied prior to Fourier transformation. Water suppression was carried out using excitation sculpting sequence with gradients (42Hwang T.L. Shaka A.J. J. Magn. Reson. 1998; 135: 280-287Crossref PubMed Scopus (55) Google Scholar). Two-dimensional 1H-15N HSQC experiments were acquired with 96 scans for each of 320 increments on F1 dimension. The matrix size 1Kx512 was zero filled to 4Kx2K by application of a squared cosine function prior to Fourier transformation. Titration Experiments—For titration of both the KKDC and scrambled control peptide with the synthetic pentasaccharide: GlcNSO3,6SO3-GlcA-GlcNSO3,3,6SO3-IdoA2SO3-GlcNSO3,6SO3-OMe (AGA*IA) (Sanofi-Synthelabo®), peptides concentration were 1.3 mm in the buffer described above. To minimize dilution, titration steps were carried out by adding small quantities (8 μl) of a concentrated (58 mm) solution of AGA*IA pentasaccharide to reach a peptide/pentasaccharide molar ratio of 1:5 (asterisk indicates a trisulfated saccharide). Proton and nitrogen chemical shifts of the amide cross-peaks of HSQC spectra were measured. Values were referred to trimethylsilyl propionate sodium salt and urea for proton and nitrogen, respectively. For each NH cross-peak, the composite chemical shift variation vector ΔCS (ΔCS = [(ΔδHN2 + ΔδN2/25)/2]1/2) for both peptides was calculated. Identification of Heparin Binding domains—Sequence alignment of heparin binding domains from several proteins had led to the characterization of two consensus sequences, XBBXBX and XBBBXXBX, where B is basic and X is hydropathic (neutral and hydrophobic) amino acid (43Cardin A.D. Weintraub H.J. Arteriosclerosis. 1989; 9: 21-32Crossref PubMed Google Scholar). Analysis of the primary protein sequence of heparanase revealed the existence of two domains that match the consensus sequence for heparin binding (Fig. 1). These are mapped at Lys158–Asp162 (KKFKN), which is the N terminus region of the 50-kDa heparanase subunit, and at Pro271–Met278 (PRRKTAKM) (the boldface letters represent basic amino acids that comprise a consensus sequence for heparin binding) (Fig. 1). A third domain contains two clusters of basic amino acids in tandem (Lys411–Lys417 and Lys427–Arg432) (Fig. 1) and is considered as a potential heparin binding domain as well. The KKDC (Lys158–Asp171) Peptide Physically Interacts with HS—In order to study the relevance of the above sequences for heparanase/HS interaction, we synthesized three peptides that contained the putative heparin binding domain (Fig. 2A). Peptides (50 μm), or their control scra" @default.
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• W1971939635 date "2005-05-01" @default.
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• W1971939635 title "Identification and Characterization of Heparin/Heparan Sulfate Binding Domains of the Endoglycosidase Heparanase" @default.
• W1971939635 cites W1491748226 @default.
• W1971939635 cites W1513452909 @default.
• W1971939635 cites W1535606309 @default.
• W1971939635 cites W1580514690 @default.
• W1971939635 cites W1869562643 @default.
• W1971939635 cites W1973037169 @default.
• W1971939635 cites W1976831807 @default.
• W1971939635 cites W1977997233 @default.
• W1971939635 cites W1982423863 @default.
• W1971939635 cites W1988176449 @default.
• W1971939635 cites W1989676517 @default.
• W1971939635 cites W1996937282 @default.
• W1971939635 cites W1998591058 @default.
• W1971939635 cites W2008581828 @default.
• W1971939635 cites W2009345525 @default.
• W1971939635 cites W2011208401 @default.
• W1971939635 cites W2012339228 @default.
• W1971939635 cites W2021014181 @default.
• W1971939635 cites W2027261163 @default.
• W1971939635 cites W2037813339 @default.
• W1971939635 cites W2041402726 @default.
• W1971939635 cites W2043451019 @default.
• W1971939635 cites W2049811381 @default.
• W1971939635 cites W2053175158 @default.
• W1971939635 cites W2053811100 @default.
• W1971939635 cites W2054649531 @default.
• W1971939635 cites W2058143304 @default.
• W1971939635 cites W2062799405 @default.
• W1971939635 cites W2068818299 @default.
• W1971939635 cites W2070389683 @default.
• W1971939635 cites W2072160034 @default.
• W1971939635 cites W2089944271 @default.
• W1971939635 cites W2091857312 @default.
• W1971939635 cites W2092751827 @default.
• W1971939635 cites W2093270567 @default.
• W1971939635 cites W2094496000 @default.
• W1971939635 cites W2100959766 @default.
• W1971939635 cites W2102291969 @default.
• W1971939635 cites W2105301755 @default.
• W1971939635 cites W2114297048 @default.
• W1971939635 cites W2123204118 @default.
• W1971939635 cites W2125279675 @default.
• W1971939635 cites W2132643319 @default.
• W1971939635 cites W2137907593 @default.
• W1971939635 cites W2146240095 @default.
• W1971939635 cites W2148405949 @default.
• W1971939635 cites W2150802762 @default.
• W1971939635 cites W2158862842 @default.
• W1971939635 cites W2159959764 @default.
• W1971939635 cites W4232261933 @default.
• W1971939635 doi "https://doi.org/10.1074/jbc.m414546200" @default.
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