Cellular mechanisms of mammalian heparanase uptake and activation

Heparanase-1 is the only known endoglycosidase that cleaves the heparan sulfate (HS) side chains of heparan sulfate proteoglycans (HSPGs). HSPGs play a key role in the self-assembly and integrity of extracellular matrices and sequester a variety of biological mediators such as growth factors, chemokines and cytokines in these support structures. Consistently, heparanase-1 has been strongly implicated in inflammatory processes, wound healing, tumor metastasis and angiogenesis. Understanding how heparanase-1 activity is generated and controlled is thus of major medical interest. Cells can generate the active form of heparanase-1 from the larger inactive precursor protein by a process of secretion, (re-)capture, internalization and proteolytic processing in late endosomes / lysosomes. By heparanase-1 transfection, binding and uptake experiments and by using a combination of specific inhibitors and receptor-defective cells, we found that the receptors involved in the binding and internalization of secreted heparanase-1 precursor include HSPGs, low density lipoprotein receptor-related protein (LRP), possibly other receptor-associated protein-sensitive receptor(s), and mannose-6-phosphate (Man-6-P) receptors. We noted that each of these receptors can work separately, but also provided evidence that LRP cooperates with HSPGs to form a dual receptor-complex that binds pro-heparanase-1 with an affinity (Kd = ~0.10nM) that is ~20 times higher than that of LRP alone (Kd = ~2.09nM). We found the Man-6-P receptors to work as separate receptors, which have a lower affinity (Kd = ~23.3nM), but are ~10 fold more abundant that the LRP-based receptors. Additionally, we demonstrated that the mature, active form of heparanase-1 (which can be secreted from intracellular compartments in response to a proper and effective stimulus) can only be (re-)captured by the LRP∙HSPG dual receptor complex or by LRP alone. Moreover, our data led us to conclude that the recognition of (activated) heparanase-1 by LRP is based on a conformation-dependent determinant. We also explored the spectrum of heparanase activities that might occur in mammalian cells, by evaluating whether the findings for heparanase-1 are also valid for heparanase-2, a close but as yet uncharacterized homologue of heparanase-1. For this purpose, we investigated the HS-degrading activity, post-translational processing/activation and trafficking route of the four different splice-variants of human heparanase-2 (hep-2O, hep-2A, hep-2B and hep-2AB). We found none of these variants to mimic heparanase-1 in these respects. Yet, we noted that, (secreted) hep-2B and hep-2AB bind avidly to cell surfaces, and that (as for heparanase-1) HSPGs play a role in this binding. Since heparanase-1 also contributes to tumor metastasis and angiogenesis by binding to cell surface receptors, activating signalling pathways independently of its enzymatic activity, we surmise, by extension, this might reveal a HS/heparin requirement for heparanase-2 to bind to a signalling receptor and to exert maximal activity. Finally, encouraged by the above findings, we attempted to develop a highly effective and specific inhibitor(s) of heparanase-1 activation and/or activity. Therefore, human heparanase-1 specific nanobodies were raised, using recombinant purified heparanase-1 precursor as antigen in llamas and as substrate for the selection (panning) of the nanobodies. We showed that several of these nanobodies are suitable reagents for detecting heparanase-1 in different in vitro molecular biological assays (i.e. Western blotting, immunoprecipitation and/or immunocytochemistry). Furthermore, the panel of nanobodies recognizes several different epitopes of pro-heparanase-1, providing means to develop a high-throughput, cheap and sensitive ELISA, suitable for the detection and quantification of human heparanase-1, e.g. in tissue extracts and body fluids of patients. None of the current nanobodies significantly inhibits heparanase-1 enzymatic activity. However, we found that one of these nanobodies, recognizing a conformational epitope in (pro-)heparanase-1, effectively blocks LRP-mediated binding and uptake of (pro-)heparanase-1, and thus blocks the major route of entry and consequent activation of heparanase-1 by cells. In conclusion, our studies add several, new insights into the understanding of how heparanase-1 activity is generated and controlled. Moreover, the identification of the uptake-blocking nanobody might have major implications for the development of novel strategies to inhibit heparanase-1 activation, cancer progression and inflammatory processes.status: publishe

Mammalian heparanase: what is the message?

Heparan sulphate proteoglycans are ubiquitous macromolecules of cell surfaces and extracellular matrices. Numerous extracellular matrix proteins, growth factors, morphogens, cytokines, chemokines and coagulation factors are bound and regulated by heparan sulphate. Degradation of heparan sulphate thus potentially profoundly affects cell and tissue function. Although there is evidence that several heparan sulphate-degrading endoglucuronidases (heparanases) might exist, so far only one transcript encoding a functional heparanase has been identified: heparanase-1. In the first part of this review, we discuss the current knowledge about heparan sulphate proteoglycans and the functional importance of their versatile interactions. In the second part, we summarize recent findings that have contributed to the characterization of heparanase-1, focusing on the molecular properties, working mechanism, substrate specificity, expression pattern, cellular activation and localization of this enzyme. Additionally, we review data implicating heparanase-1 in several normal and pathological processes, focusing on tumour metastasis and angiogenesis, and on evidence for a potentially direct signalling function of the molecule. In that context, we also briefly discuss heparanase-2, an intriguing close homologue of heparanase-1, for which, so far, no heparan sulphate-degrading activity could be demonstrated.status: publishe