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,Neta Ilan

doi:10.1074/jbc.m414546200

Abstract

The endo-beta-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 (Lys(158)-Asp(171)) 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 Lys(158)-Asn(162) as amino acids that firmly bound heparin. Deletion of a second heparin binding domain sequence (Gln(270)-Lys(280)) 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.

Highlights

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
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
Addition of the C-terminal cysteine residue shown to promote peptide dimerization and to improve the interaction with heparin [44] only slightly enhanced heparin binding (Fig. 2B, QPKAC). These results suggest that only the KKDC peptide exhibits high affinity to and physically interacts with heparin

Summary

Introduction

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. For inhibition of heparanase binding to heparin, 20 ng of heparanase protein were incubated with heparinSepharose 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 added, and the mixture was boiled for 5 min and centrifuged, and the supernatant was subjected to immunoblotting with anti-heparanase antibodies.

Results

Conclusion