The Development of Assays for Heparanase Enzymatic Activity: Towards a Gold Standard

Abstract

The enzyme heparanase, an endo-β-glucuronidase, degrades heparan sulfate (HS) chains on the cell surface and in the extracellular matrix. Heparanase regulates numerous biological processes that drive tumour growth, metastasis and angiogenesis. In addition to its key role in cancer progression, it has also been implicated in an ever-growing number of other diseases, particularly those associated with inflammation. The importance of heparanase in biology has led to numerous efforts over the years to develop assays to monitor its activity and to screen for new inhibitors as potential drug candidates. Despite these efforts and the commercialization of a few kits, most heparanase assays are still complex, labour intensive, costly or have limited application. Herein we review the various methods for assaying heparanase enzymatic activity, focusing on recent developments towards new assays that hold the promise of accelerating research into this important enzyme.

Forty Years of Basic and Translational Heparanase Research

“…These assays are limited by their heterogeneity, semiquantitative nature, multiple enzyme cleavage sites and inappropriateness for use in biological samples. Advances in the synthesis of simple synthetic oligosaccharide substrates with a single point of cleavage will ultimately lead to a "gold standard" assay for detailed kinetic analyses [23]. Also, undesirable effects of anti-heparanase therapy should be considered.…”

“…But getting the pure enzyme proved to be difficult. Not only is heparanase unstable, but the only assay then available was slow and cumbersome [22,23]. Nevertheless, the Israeli group finally managed to purify heparanase from a human liver cancer cell line and also from human placenta [4], while the Australian group purified it from human platelets [24].…”

Three-Dimensional Models as a New Frontier for Studying the Role of Proteoglycans in the Normal and Malignant Breast Microenvironment

“…Perlecan, a HSPG found largely in the basement membrane, captures anionic growth factors such as stromal fibroblast-growth factor 10 (FGF10), delivering it to the epithelium for growth ( Patel et al, 2007 ). The complex interplay between such HSPG-bound growth factors is modulated by the enzyme heparanase, which cleaves HS side chains and liberates the free growth factors ( Chhabra and Ferro, 2018 ).…”

Ultrasensitive small molecule fluorogenic probe for human heparanase

“…However, the progress on the development of heparanase probes is disproportionate to the biological importance of the enzyme. Several heparanase assays have emerged over the past two decades, but these assays have encountered disadvantages that limit their broad application 1,13 .…”

“…A more recent homogeneous time-resolved fluorescence assay, currently considered the most convenient approach available to detect heparanase activity 16,17 , requires tedious multi-step reagent addition and the output signal and substrate degradation lacks linear correlation 18 . Another common drawback of these assays developed so far is the use of heterogeneous heparan sulfate polysaccharides, suffering from difficulties in the standardization of the assays due to the structural complexity and diversity of the polymer substrates 13,19,20 . To avoid the disadvantages resulting from heterogeneity, a colorimetric assay 21 using a pentasaccharide fondaparinux, a commonly used anticoagulant, instead of heparan sulfate as the substrate 22 , but this assay has not gained broad application due to its low sensitivity and requirement of high substrate concentration, high temperature, and long incubation time.…”