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Modulation of the Heparanase-inhibiting Activity of Heparin through Selective Desulfation, Graded N-Acetylation, and Glycol Splitting

2005; Elsevier BV; Volume: 280; Issue: 13 Linguagem: Inglês

10.1074/jbc.m414217200

ISSN

1083-351X

Autores

Annamaria Naggi, Benito Casu, Marta Pérez, Giangiacomo Torri, Giuseppe Cassinelli, Sergio Penco, Claudio Pisano, Giuseppe Giannini, Rivka Ishai-Michaeli, Israël Vlodavsky,

Tópico(s)

Carbohydrate Chemistry and Synthesis

Resumo

Heparanase is an endo-β-glucuronidase that cleaves heparan sulfate (HS) chains of heparan sulfate proteoglycans on cell surfaces and in the extracellular matrix (ECM). Heparanase, overexpressed by most cancer cells, facilitates extravasation of blood-borne tumor cells and causes release of growth factors sequestered by HS chains, thus accelerating tumor growth and metastasis. Inhibition of heparanase with HS mimics is a promising target for a novel strategy in cancer therapy. In this study, in vitro inhibition of recombinant heparanase was determined for heparin derivatives differing in degrees of 2-O- and 6-O-sulfation, N-acetylation, and glycol splitting of nonsulfated uronic acid residues. The contemporaneous presence of sulfate groups at O-2 of IdoA and at O-6 of GlcN was found to be non-essential for effective inhibition of heparanase activity provided that one of the two positions retains a high degree of sulfation. N-Desulfation/ N-acetylation involved a marked decrease in the inhibitory activity for degrees of N-acetylation higher than 50%, suggesting that at least one NSO3 group per disaccharide unit is involved in interaction with the enzyme. On the other hand, glycol splitting of preexisting or of both preexisting and chemically generated nonsulfated uronic acids dramatically increased the heparanase-inhibiting activity irrespective of the degree of N-acetylation. Indeed N-acetylated heparins in their glycol-split forms inhibited heparanase as effectively as the corresponding N-sulfated derivatives. Whereas heparin and N-acetylheparins containing unmodified d-glucuronic acid residues inhibited heparanase by acting, at least in part, as substrates, their glycol-split derivatives were no more susceptible to cleavage by heparanase. Glycol-split N-acetylheparins did not release basic fibroblast growth factor from ECM and failed to stimulate its mitogenic activity. The combination of high inhibition of heparanase and low release/potentiation of ECM-bound growth factor indicates that N-acetylated, glycol-split heparins are potential antiangiogenic and antimetastatic agents that are more effective than their counterparts with unmodified backbones. Heparanase is an endo-β-glucuronidase that cleaves heparan sulfate (HS) chains of heparan sulfate proteoglycans on cell surfaces and in the extracellular matrix (ECM). Heparanase, overexpressed by most cancer cells, facilitates extravasation of blood-borne tumor cells and causes release of growth factors sequestered by HS chains, thus accelerating tumor growth and metastasis. Inhibition of heparanase with HS mimics is a promising target for a novel strategy in cancer therapy. In this study, in vitro inhibition of recombinant heparanase was determined for heparin derivatives differing in degrees of 2-O- and 6-O-sulfation, N-acetylation, and glycol splitting of nonsulfated uronic acid residues. The contemporaneous presence of sulfate groups at O-2 of IdoA and at O-6 of GlcN was found to be non-essential for effective inhibition of heparanase activity provided that one of the two positions retains a high degree of sulfation. N-Desulfation/ N-acetylation involved a marked decrease in the inhibitory activity for degrees of N-acetylation higher than 50%, suggesting that at least one NSO3 group per disaccharide unit is involved in interaction with the enzyme. On the other hand, glycol splitting of preexisting or of both preexisting and chemically generated nonsulfated uronic acids dramatically increased the heparanase-inhibiting activity irrespective of the degree of N-acetylation. Indeed N-acetylated heparins in their glycol-split forms inhibited heparanase as effectively as the corresponding N-sulfated derivatives. Whereas heparin and N-acetylheparins containing unmodified d-glucuronic acid residues inhibited heparanase by acting, at least in part, as substrates, their glycol-split derivatives were no more susceptible to cleavage by heparanase. Glycol-split N-acetylheparins did not release basic fibroblast growth factor from ECM and failed to stimulate its mitogenic activity. The combination of high inhibition of heparanase and low release/potentiation of ECM-bound growth factor indicates that N-acetylated, glycol-split heparins are potential antiangiogenic and antimetastatic agents that are more effective than their counterparts with unmodified backbones. Heparanase is a mammalian endo-β-d-glucuronidase that cleaves heparan sulfate (HS) 1The abbreviations used are: HS, heparan sulfate; HSPG, heparan sulfate proteoglycan; ECM, extracellular matrix; FGF-2, basic fibroblast growth factor; FGF-1, acidic fibroblast growth factor; IdoA, l-iduronic acid; GlcA, d-glucuronic acid; GlcN, d-glucosamine; GlcNAc, N-acetyl-d-glucosamine; GlcNSO3, d-glucosamine N-sulfate; LMWH, low molecular weight heparin; GPC-HPLC, gel permeation chromatography-high performance liquid chromatography; NA, N-acetylated; NAH, N-acetylheparin; NS, N-sulfated; gs, glycol-split; RO-H, reduced oxyheparin; PBS, phosphate-buffered saline; OdeS, O-desulfated; GalA, l-galacturonic acid; M̄w, weight average molecular weight. chains at a limited number of sites (1.Vlodavsky I. Friedmann Y. J. Clin. Investig. 2001; 108: 341-347Crossref PubMed Scopus (550) Google Scholar, 2.Parish C.R. Freeman C. Hulett M.D. Biochim. Biophys. Acta. 2001; 1471: M99-M108PubMed Google Scholar, 3.Dempsey L.A. Brunn G.T. Platt J.L. Trends Biol. Sci. 2000; 25: 349-351Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Cloning of the heparanase cDNA by several groups (1.Vlodavsky I. Friedmann Y. J. Clin. Investig. 2001; 108: 341-347Crossref PubMed Scopus (550) Google Scholar, 2.Parish C.R. Freeman C. Hulett M.D. Biochim. Biophys. Acta. 2001; 1471: M99-M108PubMed Google Scholar, 3.Dempsey L.A. Brunn G.T. Platt J.L. Trends Biol. Sci. 2000; 25: 349-351Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 4.Vlodavsky I. Friedmann Y. Elkin M. Aingorn H. Atzmon R. Ishai-Michaeli R. Spector L. Pecker I. Nat. Med. 1999; 5: 793-802Crossref PubMed Scopus (731) Google Scholar, 5.Hulett M.D. Freeman C. Hamdorf B.J. Baker R.T. Harris M.J. Parish C.R. Nat. Med. 1999; 5: 803-809Crossref PubMed Scopus (491) Google Scholar, 6.Toyoshima M. Nakajima M. J. Biol. Chem. 1999; 274: 24153-24160Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar) suggests that a single functional HS-degrading endoglycosidase is expressed in mammalian cells. The enzyme is synthesized as a latent 65-kDa precursor that undergoes proteolytic cleavage, yielding 8- and 50-kDa subunits that heterodimerize to form a highly active enzyme (7.McKenzie 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 (105) Google Scholar, 8.Levy-Adam F. Miao H.Q. Heinrikson R.L. Vlodavsky I. Ilan N. Biochim. Biophys. Res. Commun. 2003; 308: 885-891Crossref PubMed Scopus (102) Google Scholar). Heparanase enzymatic activity participates in degradation and remodeling of the extracellular matrix (ECM), facilitating, among other activities, cell invasion associated with cancer metastasis, angiogenesis, and inflammation (1.Vlodavsky I. Friedmann Y. J. Clin. Investig. 2001; 108: 341-347Crossref PubMed Scopus (550) Google Scholar, 2.Parish C.R. Freeman C. Hulett M.D. Biochim. Biophys. Acta. 2001; 1471: M99-M108PubMed Google Scholar, 3.Dempsey L.A. Brunn G.T. Platt J.L. Trends Biol. Sci. 2000; 25: 349-351Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 9.Vlodavsky I. Eldor A. Haimovitz-Friedman A. Matzner Y. Ishai-Michaeli R. Lider O. Naparstek Y. Cohen I.R. Fucks Z. Invasion Metastasis. 1992; 12: 112-127PubMed Google Scholar). Heparanase upregulation has been documented in a variety of human tumors correlating, in some cases, with increased vascular density and poor postoperative survival (10.Koliopanos 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, 11.El-Assal O.N. Yamanoi A. Ono T. Kohno H. Nagasue N. Clin. Cancer Res. 2001; 7: 1299-1305PubMed Google Scholar, 12.Gohji K. Hirano H. Okamoto M. Kitazawa S. Toyoshima M. Dong J. Katsuoka Y. Nakajima M. Int. J. Cancer. 2001; 95: 295-301Crossref PubMed Scopus (111) Google Scholar, 13.Sato 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 other pathologies such as cirrhosis (14.Xiao Y. Kleeff J. Shi X. Buchler M.W. Friess H. Hepatol. Res. 2003; 26: 192-198Crossref PubMed Scopus (34) Google Scholar), nephrosis (15.Levidiotis V. Kanellis J. Ierino F.L. Power D.A. Kidney Int. 2001; 60: 1287-1296Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), and diabetes (16.Katz A. Van-Dijk D.J. Aingorn H. Erman A. Davies M. Darmon D. Hurvitz H. Vlodavsky I. Isr. Med. Assoc. 2002; 4: 996-1002PubMed Google Scholar). In addition to its intimate involvement in the egress of cells from the blood stream, heparanase activity releases from the ECM and tumor microenvironment a multitude of HS-bound growth factors, cytokines, chemokines, and enzymes that affect cell and tissue function, most notably angiogenesis (17.Bernfield M. Gotte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2341) Google Scholar, 18.Elkin M. Ilan N. Ishai-Michaeli R. Friedmann Y. Papo O. Pecker I. Vlodavsky I. FASEB J. 2001; 15: 1661-1663Crossref PubMed Scopus (288) Google Scholar). These observations, the anticancerous effect of heparanase gene silencing (ribozyme and small interfering RNA) (19.Edovitsky E. Elkin M. Zcharia E. Peretz T. Vlodavsky I. J. Natl. Cancer Inst. 2004; 96: 1219-1230Crossref PubMed Scopus (227) Google Scholar) and of heparanase-inhibiting molecules (non-anticoagulant species of heparin and other sulfated polysaccharides) (20.Vlodavsky I. Mohsen M. Lider O. Svahn C.M. Ekre H.P. Vigoda M. Peretz T. Invasion Metastasis. 1994; 14: 290-302PubMed Google Scholar, 21.Nakajima M. Irimura T. Nicolson G.L. J. Cell. Biochem. 1988; 36: 157-167Crossref PubMed Scopus (282) Google Scholar), and the unexpected identification of a predominant functional heparanase (1.Vlodavsky I. Friedmann Y. J. Clin. Investig. 2001; 108: 341-347Crossref PubMed Scopus (550) Google Scholar, 2.Parish C.R. Freeman C. Hulett M.D. Biochim. Biophys. Acta. 2001; 1471: M99-M108PubMed Google Scholar, 3.Dempsey L.A. Brunn G.T. Platt J.L. Trends Biol. Sci. 2000; 25: 349-351Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) suggest that the enzyme is a promising target for development of new anticancer drugs. HS and the structurally related heparin are present in most animal species. They are glycosaminoglycans constituted by repeating disaccharide units of a uronic acid (either d-glucuronic acid (GlcA) or l-iduronic acid (IdoA)) and d-glucosamine (either GlcNAc or d-glucosamine N-sulfate (GlcNSO3)) and bear sulfate substituents in various positions (22.Parish C.R. Freeman C. Brown K.J. Francis D.J. Cowden W.B. Cancer Res. 1999; 59: 3433-3441PubMed Google Scholar, 23.Kjellén L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1686) Google Scholar, 24.Casu B. Lindahl U. Adv. Carbohydr. Chem. Biochem. 2001; 57: 159-208Crossref PubMed Scopus (353) Google Scholar, 25.Lindahl U. Glycoconj. J. 2000; 17: 597-605Crossref PubMed Scopus (62) Google Scholar). Although derived from the common biosynthetic precursor N-acetylheparosan (-GlcA-GlcNAc)n, HS and heparin have different structures: HS is less sulfated and more heterogeneous than heparin. The two glycosaminoglycans also have different locations in tissues: whereas HS is a component of the ECM and of the surface of most cells, heparin is stored in granules of mast cells and co-released with histamine into the circulation upon cellular degranulation mainly in cases of allergic and inflammatory reactions and anaphylactic stress. On the other hand, exogenous heparin is widely used as an anticoagulant and antithrombotic drug and is of increasing interest for novel therapeutical applications (24.Casu B. Lindahl U. Adv. Carbohydr. Chem. Biochem. 2001; 57: 159-208Crossref PubMed Scopus (353) Google Scholar, 25.Lindahl U. Glycoconj. J. 2000; 17: 597-605Crossref PubMed Scopus (62) Google Scholar, 26.Lever R. Page C.P. Nat. Rev. Drug Discov. 2002; 1: 140-148Crossref PubMed Scopus (308) Google Scholar, 27.Linhardt R.J. J. Med. Chem. 2003; 46: 2551-2564Crossref PubMed Scopus (446) Google Scholar). As an analog of the natural substrate of heparanase, heparin is commonly considered to be a potent inhibitor of heparanase (20.Vlodavsky I. Mohsen M. Lider O. Svahn C.M. Ekre H.P. Vigoda M. Peretz T. Invasion Metastasis. 1994; 14: 290-302PubMed Google Scholar, 21.Nakajima M. Irimura T. Nicolson G.L. J. Cell. Biochem. 1988; 36: 157-167Crossref PubMed Scopus (282) Google Scholar, 28.Bar-Ner M. Eldor A. Wasserman L. Matzner Y. Vlodavsky I. Blood. 1987; 70: 551-557Crossref PubMed Google Scholar, 29.Vlodavsky I. Fuks Z. Bar-Ner M. Ariav Y. Schirrmacher V. Cancer Res. 1983; 43: 2704-2711PubMed Google Scholar, 30.Irimura T. Nakajima M. Nicolson G.L. Biochemistry. 1986; 25: 5322-5328Crossref PubMed Scopus (136) Google Scholar, 31.Parish C.R. Coombe D.R. Jakobsen K.B. Bennett F.A. Underwood P.A. Int. J. Cancer. 1987; 40: 511-518Crossref PubMed Scopus (163) Google Scholar). This activity is attributed, in part, to its high affinity interaction with the enzyme and limited degradation, serving as an alternative substrate. Early reports (20.Vlodavsky I. Mohsen M. Lider O. Svahn C.M. Ekre H.P. Vigoda M. Peretz T. Invasion Metastasis. 1994; 14: 290-302PubMed Google Scholar, 21.Nakajima M. Irimura T. Nicolson G.L. J. Cell. Biochem. 1988; 36: 157-167Crossref PubMed Scopus (282) Google Scholar, 30.Irimura T. Nakajima M. Nicolson G.L. Biochemistry. 1986; 25: 5322-5328Crossref PubMed Scopus (136) Google Scholar, 31.Parish C.R. Coombe D.R. Jakobsen K.B. Bennett F.A. Underwood P.A. Int. J. Cancer. 1987; 40: 511-518Crossref PubMed Scopus (163) Google Scholar) showed that heparin and some chemically modified species of heparin as well as other sulfated polysaccharides (22.Parish C.R. Freeman C. Brown K.J. Francis D.J. Cowden W.B. Cancer Res. 1999; 59: 3433-3441PubMed Google Scholar, 32.Miao H.Q. Elkin M. Aingorn E. Ishai-Michaeli R. Stein C.A. Vlodavsky I. Int. J. Cancer. 1999; 83: 424-431Crossref PubMed Scopus (179) Google Scholar) that inhibit tumor cell heparanase also inhibit experimental metastasis in animal models, while other related compounds that lack heparanase-inhibiting activity fail to exert an antimetastatic effect (20.Vlodavsky I. Mohsen M. Lider O. Svahn C.M. Ekre H.P. Vigoda M. Peretz T. Invasion Metastasis. 1994; 14: 290-302PubMed Google Scholar, 21.Nakajima M. Irimura T. Nicolson G.L. J. Cell. Biochem. 1988; 36: 157-167Crossref PubMed Scopus (282) Google Scholar, 22.Parish C.R. Freeman C. Brown K.J. Francis D.J. Cowden W.B. Cancer Res. 1999; 59: 3433-3441PubMed Google Scholar, 30.Irimura T. Nakajima M. Nicolson G.L. Biochemistry. 1986; 25: 5322-5328Crossref PubMed Scopus (136) Google Scholar, 31.Parish C.R. Coombe D.R. Jakobsen K.B. Bennett F.A. Underwood P.A. Int. J. Cancer. 1987; 40: 511-518Crossref PubMed Scopus (163) Google Scholar, 32.Miao H.Q. Elkin M. Aingorn E. Ishai-Michaeli R. Stein C.A. Vlodavsky I. Int. J. Cancer. 1999; 83: 424-431Crossref PubMed Scopus (179) Google Scholar). Regardless of the mode of action, heparin and low molecular weight heparin (LMWH) were reported to exert a beneficial effect in cancer patients (33.Altinbas M. Coskun H.S. Er O. Ozkan M. Eser B. Unal A. Cetin M. Soyuer S.A. J. Thromb. Haemostasis. 2004; 2: 1266-1271Crossref PubMed Scopus (421) Google Scholar), stimulating research on the potential use of modified, non-anticoagulant species of heparin and HS in cancer therapy. Screening of heparin derivatives has permitted the identification of some of the structural features of heparin associated with inhibition of the enzyme. As a general trend, the heparanase-inhibiting activity increases with increasing degrees of O-sulfation. However, N-sulfates seems to exert little effect since they can be replaced by N-acyl (N-acetyl, N-succinyl, or N-hexanoyl) groups without substantial loss of inhibitory activity (20.Vlodavsky I. Mohsen M. Lider O. Svahn C.M. Ekre H.P. Vigoda M. Peretz T. Invasion Metastasis. 1994; 14: 290-302PubMed Google Scholar, 34.Ishai-Michaeli R. Svahn C.M. Weber M. Chajek-Shaul T. Korner G. Ekre H.-P. Vlodavsky I. Biochemistry. 1992; 31: 2080-2088Crossref PubMed Scopus (75) Google Scholar). No significant differences were found between the currently used unfractionated heparins and low molecular weight heparins and a tetradecasaccharidic fragment (34.Ishai-Michaeli R. Svahn C.M. Weber M. Chajek-Shaul T. Korner G. Ekre H.-P. Vlodavsky I. Biochemistry. 1992; 31: 2080-2088Crossref PubMed Scopus (75) Google Scholar). 2-O-Desulfated derivatives were shown to retain the inhibitory activity, whereas N-desulfated, N-acetylated derivatives displayed a reduced activity (35.Lapierre F. Holme K. Lam L. Tressler R.J. Storm N. Wee J. Stack R.J. Castellot J. Tyrrell D.J. Glycobiology. 1996; 6: 355-366Crossref PubMed Scopus (140) Google Scholar). In the present study, relationships between structure and heparanase-inhibiting activity of heparin were studied using a larger number of heparins and heparin derivatives, including some with various degrees of 6-O-sulfation of GlcN and 2-O-sulfation of IdoA residues as well as "glycol-split" derivatives obtained by controlled periodate oxidation/borohydride reduction of natural (36.Casu B. Diamantini G. Fedeli G. Mantovani M. Prino G. Oreste P. Pescador R. Torri G. Zoppetti G. Arzneim.-Forsch. (Drug Res.). 1985; 36: 637-642Google Scholar) or partially 2-O-desulfated heparins (37.Casu 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, 38.Casu B. Guerrini M. Naggi A. Perez M. Torri G. Ribatti D. Carminati C. Giannini G. Penco S. Pisano C. Belleri M. Rusnati M. Presta M. Biochemistry. 2002; 41: 10519-10528Crossref PubMed Scopus (80) Google Scholar). Glycol splitting of C-2–C-3 bonds of nonsulfated uronic acid residues was suggested to interfere with the biological interactions of heparin by providing flexible joints between protein binding sequences (37.Casu 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, 38.Casu B. Guerrini M. Naggi A. Perez M. Torri G. Ribatti D. Carminati C. Giannini G. Penco S. Pisano C. Belleri M. Rusnati M. Presta M. Biochemistry. 2002; 41: 10519-10528Crossref PubMed Scopus (80) Google Scholar, 39.Casu B. Harenberg J. Heene D.L. Stehle G. Schettler G. New Trends in Hemostasis. Springer Verlag, Heidelberg, Germany1990: 2-11Google Scholar). When framing heparin sequences that bind FGF-2, glycol-split residues were shown not to impair the binding to FGF-2. However, they prevented activation of FGF-2 and FGF-2-induced angiogenic activity (37.Casu 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, 38.Casu B. Guerrini M. Naggi A. Perez M. Torri G. Ribatti D. Carminati C. Giannini G. Penco S. Pisano C. Belleri M. Rusnati M. Presta M. Biochemistry. 2002; 41: 10519-10528Crossref PubMed Scopus (80) Google Scholar). The present study shows that glycol splitting enhances the heparanase-inhibiting activity of heparin. Based on the observation that N-acetyl groups do not prevent and may even assist recognition by heparanase (40.Sandbäck-Pikas 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, 41.Okada Y. Yamada S. Toyoshima M. Dong J. Nakajima M. Sugahara K. J. Biol. Chem. 2002; 277: 42488-42495Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) and taking into account that N-acetylheparin, as opposed to heparin, does not release angiogenic factors from ECM (34.Ishai-Michaeli R. Svahn C.M. Weber M. Chajek-Shaul T. Korner G. Ekre H.-P. Vlodavsky I. Biochemistry. 1992; 31: 2080-2088Crossref PubMed Scopus (75) Google Scholar), we prepared and tested heparins with various degrees of N-acetylation/N-sulfation together with some of their glycol-split derivatives. N-Acetylated, glycol-split heparins were shown to inhibit heparanase more efficiently than the corresponding non-glycol-split N-acetylated heparins. All chemicals were of reagent grade from Sigma and were used as supplied. Heparins were commercial preparations from pig mucosa (H-1 to H-3 from Laboratori Derivati Organici, Trino Vercellese, Italy, and H-6 from Hepar), from beef mucosa (H-4 and H-5, Laboratori Derivati Organci), and from beef lung (H-7, The Upjohn Co.). The corresponding contents of major sulfate groups, as evaluated by 13C NMR spectroscopy (42.Casu B. Guerrini M. Naggi A. Torri G. De Ambrosi L. Boveri G. Gonella S. Cedro A. Ferro L. Lanzarotti E. Paternò M. Attolini M. Valle M.G. Arzneim.-Forsch. (Drug Res.). 1996; 46: 472-477PubMed Google Scholar) and expressed as mole percent of IdoA2SO3, GlcNSO3, and GlcN(SO3 or Ac)6SO3 per disaccharide unit based on quantification of underlined sulfate groups, were: H-1: 69, 89, 79; H-2: 68, 85, 82; H-3: 64, 85, 82; H-4: 62, 89, 60; H-5: 66, 92, 60; H-6: 65, 86, 82; and H-7: 86, 98, 95. The weight average molecular weights (M̄w, by GPC-HPLC (43.Bertini S. Bisio A. Torri G. Bensi D. Terbojevich M. Biomacromolecules. 2005; 6: 168-173Crossref PubMed Scopus (82) Google Scholar)) were: H-1, 14,200; H-2, 18,100; H-3, 19,600; H-4, 18,800; H-5, 18,200; H-6, 23,200; and H-7, 21,600. Sample desalting was carried out by dialysis against water with 1000-Da cut-off tubes or by fractionation on a 2.5 × 100-cm Sephadex G-25 column (Amersham Biosciences) using 10% ethanol in water as eluent and UV detection at 210 nm. Molecular weight determinations were performed by GPC-HPLC on a Viscotex instrument equipped with a VE1121 pump, Rheodyne valve (100 μl), and triple detector array 302 equipped with IR, viscosimeter, and 90° light-scattering systems. Two 300 × 7.8-mm TSK GMPWXL Viscotek columns were used with 0.1 m NaNO3 as eluent (flow, 0.6 ml/min). Samples were dissolved in the eluent solution at the concentration of 15 mg/ml (43.Bertini S. Bisio A. Torri G. Bensi D. Terbojevich M. Biomacromolecules. 2005; 6: 168-173Crossref PubMed Scopus (82) Google Scholar). NMR spectra were recorded at 500 MHz for 1H and 125 MHz for 13C with a Bruker AMX spectrometer equipped with a 5-mm 1H/X inverse probe. The spectra were obtained at 45 °C from D2O solutions (15 mg/0.5 ml D2O, 99.99% D). Chemical shifts, given in parts per million down field from sodium-3-(trimethylsilyl)propionate, were measured indirectly with reference to acetone in D2O (δ 2.235 for 1H and δ 30.20 for 13C). The 13C NMR spectra were recorded at 300 or 400 MHz with a Bruker AC-300 or AMX-400 spectrometer. Recombinant enzymatically active heparanase was purified from heparanase-transfected Chinese hamster ovary cells (4.Vlodavsky I. Friedmann Y. Elkin M. Aingorn H. Atzmon R. Ishai-Michaeli R. Spector L. Pecker I. Nat. Med. 1999; 5: 793-802Crossref PubMed Scopus (731) Google Scholar). Briefly Chinese hamster ovary cells were harvested with trypsin and centrifuged, and the cell pellet was suspended in 20 mm citrate-phosphate buffer pH 5.4. The suspension was subjected to four cycles of freeze/thaw (–70/37 °C, 5 min each), the cell extract was centrifuged (18,000 rpm, 15 min, 2–8 °C), and the supernatant was collected and filtered through a 0.45-μm filter. The filtrate was applied onto a Source 15 S column (Amersham Biosciences) equilibrated with 20 mm phosphate buffer, pH 6.8. The column was washed (20 mm phosphate buffer, pH 6.8, followed by 20 mm phosphate buffer, pH 8.0), and heparanase was eluted with a linear gradient (0–35%) of 8 column volumes of 1.5 m NaCl in 20 mm phosphate buffer, pH 8.0. Active fractions were pooled and applied onto a Fractogel EMD SO–3 (Merck) column equilibrated with 20 mm citratephosphate buffer, pH 5.4. Heparanase was eluted with a linear gradient (0–22%) of 1 column volume followed by 10 column volumes (22–25%) of 1.5 m NaCl in 20 mm phosphate buffer, pH 8.0. Finally heparanase eluted from the Fractogel column was applied onto a HiTrap heparin column (Amersham Biosciences) equilibrated with 20 mm phosphate buffer, pH 8.0, and eluted with a linear gradient of 1 column volume (0–20%) and 15 column volumes (20–28%) of 1.5 m NaCl in 20 mm phosphate buffer, pH 8.0. Eluted fractions were analyzed by gradient SDS-PAGE, stained with Gelcode® (Pierce), and pooled according to their purity. An at least 90% pure, highly active heparanase preparation was obtained, containing the active 50- and 8-kDa heparanase subunits and, to a lower extent, the 65-kDa proheparanase (8.Levy-Adam F. Miao H.Q. Heinrikson R.L. Vlodavsky I. Ilan N. Biochim. Biophys. Res. Commun. 2003; 308: 885-891Crossref PubMed Scopus (102) Google Scholar). Active recombinant human heparanase was also produced in insect cells as described previously (7.McKenzie 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 (105) Google Scholar). The construct encoding the 8- and 50-kDa heparanase subunits was kindly provided by Dr. E. McKenzie (Oxford GlycoSciences Ltd., Abingdon, Oxon, UK) (7.McKenzie 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 (105) Google Scholar). Similar results were obtained with both preparations. Procedure A—An extensively 6-O-desulfated heparin also partially (∼15%) 2-O-desulfated (716OdeS-H(A) where the superscript denotes the degree of 6-O-desulfation), M̄w 16,000, was prepared according to Nagasawa et al. (44.Nagasawa K. Inoue Y. Kamata T. Carbohydr. Res. 1977; 58: 47-55Crossref PubMed Scopus (264) Google Scholar), starting from the pyridinium salt of heparin H-1, under solvolytic conditions (10 mg/ml in Me2SO:water 9:1) at 100 °C for 2.5 h followed by resulfation of free amino groups with sulfur trioxidetrimethylamine complex in alkaline aqueous medium (45.Lloyd A.G. Emberg G. Fowler L.J. Biochem. Pharmacol. 1971; 20: 637Crossref PubMed Scopus (72) Google Scholar). Procedure B—6-O-Desulfated-heparins (776OdeS-H(B), M̄w 19,000; 736OdeS-H(B), M̄w 17,700; and 466OdeS-H(B), M̄w 20,400) were prepared according to Matsuo et al. (46.Matsuo M. Takano R. Kamei-Hayashi K. Hara S. Carbohydr. Res. 1993; 241: 209-215Crossref PubMed Scopus (45) Google Scholar) by O-desulfation through activation with N-methyl-N-(trimethylsilyl)trifluoroacetamide or N,O-bis (trimethylsilyl)acetamide without N-desulfation. Heparin H-1 (200 mg) was converted into its pyridinium salt and soaked in pyridine (20 ml). After addition of 4 ml of N-methyl-N-(trimethylsilyl)trifluoroacetamide, the solution was heated for 4 h at 80 °Cto yield 736OdeS-H or for 8 h at 60 °C to yield 776OdeS-H. Heparin (H-1) was converted into its pyridinium salt and soaked in pyridine (30 ml). After addition of 6 ml of N,O-bis(trimethylsilyl)acetamide, the solution was heated for 2 h at 60 °C to yield 466OdeS-H. Procedure A—2-O-Desulfated heparin in the IdoA form (H, IdoA(A), M̄w 17,700) was prepared according to Jaseja et al. (47.Jaseja M. Rej R.N. Sauriol F. Perlin A.S. Can. J. Chem. 1989; 67: 1449-1456Crossref Scopus (139) Google Scholar). Heparin (500 mg) was simply dissolved in 500 ml of 0.1 m NaOH, and the solution was frozen and lyophilized. The residue dissolved in 500 ml of distilled water was dialyzed, and the product was isolated by evaporation under reduced pressure. Its 13C NMR spectrum closely corresponded to the one reported in the literature (48.Piani S. Casu B. Marchi E.G. Torri G. Ungarelli F. J. Carbohydr. Chem. 1993; 12: 507-521Crossref Scopus (29) Google Scholar), indicating an essentially complete conversion of the original IdoA2SO3 residues into IdoA residues. Procedure B—2-O-Desulfated heparin in the GalA form (H, GalA(B), M̄w 12,600) was prepared by a modification of methods used by Jaseja et al. (47.Jaseja M. Rej R.N. Sauriol F. Perlin A.S. Can. J. Chem. 1989; 67: 1449-1456Crossref Scopus (139) Google Scholar) and Rej and Perlin (49.Rej R.N. Perlin A.S. Carbohydr. Res. 1990; 200: 437-447Crossref Scopus (41) Google Scholar) essentially as described previously (48.Piani S. Casu B. Marchi E.G. Torri G. Ungarelli F. J. Carbohydr. Chem. 1993; 12: 507-521Crossref Scopus (29) Google Scholar). Heparin (500 mg) was dissolved in 10 ml of 1 m NaOH and then heated at 85 °C for 1 h. After cooling below 30 °C, the solution was brought to pH 7 with 0.1 m HCl and heated at 70 °C for 48 h to give (after cooling, dialysis, and freeze-drying) the GalA derivative with a typical 13C NMR spectrum (48.Piani S. Casu B. Marchi E.G. Torri G. Ungarelli F. J. Carbohydr. Chem. 1993; 12: 507-521Crossref Scopus (29) Google Scholar). N-Acetylated heparins (xNAH, where the su

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