The Development of a Diagnostic Test for Heparanase Activity

Abstract

Heparan sulfate proteoglycans (HSPGs) are ubiquitous macromolecules located in the extracellular matrices and basement membranes; they present a physical barrier to the movement of cells (e.g., tumour cells and leukocytes) into tissues and play an important role in a variety of biological processes including inflammation, the metastatic potential of tumour cells, and angiogenesis. The basic HSPG structure consists of a protein core, to which are attached several linear glycosaminoglycan (GAG) chains that confer most of the biological properties of HSPGs. Cleavage of heparan sulfate (HS) chains is critical for the modulation of the biological function of HS-binding proteins, and profoundly affects cell and tissue function involving migration and response to changes in the extracellular matrix. It is also essential in the degradation of the extracellular matrix by invading cells, particularly metastatic tumour cells and leukocytes entering inflammatory sites. Heparanase is an endo-β-D-glucuronidase that degrades HS-GAG chains; elevated levels of heparanase expression correlates with metastatic potential, tumour vascularity, and reduced post-operative survival of cancer patients. Heparanase inhibitors reduce the incidence of tumour metastases; hence, heparanase is a promising target for anticancer drug development. The most clinically advanced heparanase inhibitor, PI-88, is currently undergoing clinical trials for melanoma, myeloma, and lung carcinoma. The lack of a convenient, functional assay has hampered progress into the investigation of heparanase. Although several heparanase assays have been developed, they rely on either radiolabelled substrates or separation of enzymatically degraded substrates on the basis of molecular size. The primary objective of this investigation was to develop a simple fluorometric or colourimetric assay for heparanase activity. The development of such an assay would be extremely beneficial in studies on heparanase, including the kinetic evaluation of potential inhibitors and the correlation of heparanase with various disease states. Chapter One provides a general introduction to heparan sulfate, the synthesis of heparin and heparan sulfate oligosaccharides, the structure and function of heparanase, heparanase substrate specificity, inhibition of heparanase, current heparanase assays, and the scope of the thesis. A library of monosaccharide glucuronides and a library of disaccharide glycosides were synthesised as putative heparanase substrates. These glycosides had various aryl aglycons that could be measured spectrophotometrically upon hydrolysis of the glycosidic linkage by an enzyme such as heparanase. The synthesis of these monosaccharide and disaccharide libraries of aryl glycosides is detailed in Chapters Two and Three, respectively. In order to obtain sufficient quantities of heparanase for enzyme assays, human heparanase cDNA was cloned into a baculovirus expression vector according to a published procedure. The expression of heparanase in insect cells was contracted by the industry partner, Progen Pharmaceuticals Ltd. Purification of recombinant human heparanase was performed by Progen Pharmaceuticals Ltd. The cloning of human heparanase is detailed in Chapter Four. The evaluation of the monosaccharide and disaccharide libraries as heparanase substrates is presented in Chapter Five. It was found that the N-sulfated 4-nitrophenyl glycosyl glucuronide 154 and the N-sulfated methylumbelliferyl glycosyl glucuronide 158 were hydrolysed by recombinant human heparanase with specific activities of 17 and 48 nmol/h/mg, respectively. These compounds are the simplest substrates of heparanase that have been reported, and may be beneficial in studies on heparanase, including the kinetic evaluation of potential inhibitors and the correlation of heparanase activity with various disease states. Experimental data supporting the synthesis of the libraries of aryl glycosides presented in Chapters Two and Three is detailed in Chapter Six. Selected 1H NMR spectra are presented in the Appendix.