title: Production of heparanase constructs suitable for nuclear magnetic resonance and drug discovery studies.
abstract: Heparanase is an endo-β-D-glucosidase capable of specifically degrading heparan sulphate, one of the main components of the extracellular matrix. This 65 kDa polypeptide is implicated in cancer processes such as tumour formation, angiogenesis and metastasis, making it a very attractive target in antitumour treatments. Structure-based approaches to find inhibitors of heparanase have been historically hampered by the lack of success in crystallizing the protein. With the aim to undertake the NMR structural characterisation of heparanase, we have designed and produced, using recombinant methods, smaller constructs of heparanase containing the catalytically active glutamic acids and the two binding sites for heparan sulphate. An extensive range of expression and purification conditions were evaluated to alleviate the intrinsic low solubility and aggregation propensity of heparanase, allowing the obtention of the enzyme in milligram quantities, both unlabelled and ¹⁵N-labelled for NMR studies. Using the smallest of the designed constructs and applying NMR and SPR methodologies, we have demonstrated that known inhibitors of heparanase bind to this construct specifically and selectively with K(D) values in the range of those reported for human heparanase, validating it for future drug discovery projects focused on the identification of novel inhibitors of this enzyme.
authors.affiliation: Medicinal Chemistry Department, Structural Biology Laboratory, Centro de Investigación Príncipe Felipe, Avda. Autopista del Saler 16, E-46012 Valencia, Spain.
authors.forename: Silvia
authors.initials: S
authors.lastname: Mosulén
authors.forename: Leticia
authors.initials: L
authors.lastname: Ortí
authors.forename: Esperanza
authors.initials: E
authors.lastname: Bas
authors.forename: Rodrigo J
authors.initials: RJ
authors.lastname: Carbajo
authors.forename: Antonio
authors.initials: A
authors.lastname: Pineda-Lucena
mesh_terms: D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D055808:Drug Discovery; D005966:Glucuronidase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009587:Nitrogen Isotopes; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D020349:Surface Plasmon Resonance; D000099175:Heparanase
chemical_list: D017931:DNA Primers; D005966:Glucuronidase; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D000099175:Heparanase