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.

Individual low molecular weight heparins (LMWHs) exhibit distinct pharmacological and biochemical profiles because of manufacturing differences. Correlation of biological properties with particular structural motifs is a major challenge in the design of new LMWHs as well as in the development of generic versions of proprietary LMWHs. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy permits identification and quantification of structural peculiarities of LMWH preparations. In this article, heteronuclear single quantum coherence spectroscopy, previously used to determine variously substituted monosaccharide components of heparan sulfate (HS) and HS-like glycosaminoglycan mimics, has been applied to the structural characterization of three commercially available LMWHs (enoxaparin, dalteparin, and tinzaparin). Relevant residues belonging to the parent heparin, as well as minor residues generated by each depolymerization procedure, have been characterized and quantified. The use of a high-sensitivity NMR spectrometer (600 MHz equipped with cryoprobe) allowed the accurate quantification of residues with sensitivity better than 1 to 2%.