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J. Biol. Chem., Vol. 280, Issue 21, 20457-20466, May 27, 2005

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Identification and Characterization of Heparin/Heparan Sulfate Binding Domains of the Endoglycosidase Heparanase^*

Flonia Levy-Adam, Ghada Abboud-Jarrous, Marco Guerrini¶, Daniela Beccati¶, Israel Vlodavsky||, and Neta Ilan

From the Cancer and Vascular Biology Research Center, The Bruce Rappaport Faculty of Medicine, Technion, Haifa 31096, Israel, the Department of Oncology, Hadassah-Hebrew University Medical Center, Jerusalem 91120, Israel, and ^¶G. Ronzoni Institute for Chemical and Biochemical Research, via G. Colombo, 81, 20133 Milan, Italy

The endo--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.

Received for publication, December 27, 2004 , and in revised form, March 7, 2005.

^* This work was supported by United States Public Health Service Grant RO1 CA106456-01 from NCI, National Institutes of Health, Grant 532/02 from the Israel Science Foundation, The Susan G. Komen Breast Cancer Foundation, The European Commission 5th Framework Program, Contract QLKCT-2002-02049, and by a charitable fund established in memory of Rachel Litvin. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed: Cancer and Vascular Biology Research Center Faculty of Medicine, Technion, P. O. Box 9649, Haifa 31096, Israel. Tel.: 972-4-8295410; Fax: 972-4-8523947; E-mail: vlodavsk{at}cc.huji.ac.il.

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