Histidine-rich glycoprotein binds heparanase and regulates its enzymatic activity and cell surface interactions.

Heparanase, an endo-beta-D-glucuronidase, is involved in numerous normal physiological and pathological processes, such as inflammation, wound healing and tumour metastasis/angiogenesis, through its ability to mediate the degradation of heparan sulfate, a key structural component of the extracellular matrix and on the surface of cells. Identifying endogenous molecules that can regulate heparanase activity will aid the understanding of its molecular function in health and disease and provide the potential for development of novel anti-cancer and anti-inflammatory therapeutics. The ability of the extracellular heparanase to tether onto cell surface heparan sulfate proteoglycans and other receptor(s), such as the cation-independent mannose-6-phosphate receptor, is key to its activation, function and uptake into intracellular compartments. Here we describe experiments demonstrating that a relatively abundant plasma glycoprotein, histidine-rich glycoprotein, directly interacts with platelet-derived heparanase and enhances its enzymatic activity. The findings in this study also show that histidine-rich glycoprotein interferes with heparanase binding to cell surface receptors, particularly heparan sulfate proteoglycans. Thus, the interaction between histidine-rich glycoprotein and heparanase can potentially regulate the role of heparanase in a variety of physiological and pathological conditions.

Histidine-rich glycoprotein specifically binds to necrotic cells via its amino-terminal domain and facilitates necrotic cell phagocytosis.

Cells that become necrotic or apoptotic through tissue damage or during normal cellular turnover are usually rapidly cleared from the circulation and tissues by phagocytic cells. A number of soluble proteins have been identified that facilitate the phagocytosis of apoptotic cells, but few proteins have been defined that selectively opsonize necrotic cells. Previous studies have shown that histidine-rich glycoprotein (HRG), an abundant (approximately 100 microg/ml) 75-kDa plasma glycoprotein, binds to cell surface heparan sulfate on viable cells and cross-links other ligands, such as plasminogen, to the cell surface. In this study we have demonstrated that HRG also binds very strongly, in a heparan sulfate-independent manner, to cytoplasmic ligand(s) exposed in necrotic cells. This interaction is mediated by the amino-terminal domain of HRG and results in enhanced phagocytosis of the necrotic cells by a monocytic cell line. In contrast, it was found that HRG binds poorly to and does not opsonize early stage apoptotic cells. Thus, HRG has the unique property of selectively recognizing necrotic cells and may play an important physiological role in vivo by facilitating the uptake and clearance of necrotic, but not apoptotic, cells by phagocytes.

Evidence that the cellular ligand for the human NK cell activation receptor NKp30 is not a heparan sulfate glycosaminoglycan.

NKp30 (NCR3, CD337) is a natural cytotoxicity receptor, expressed on subsets of human peripheral blood NK cells, involved in NK cell killing of tumor cells and immature dendritic cells. The cellular ligand for NKp30 has remained elusive, although evidence that membrane-associated heparan sulfate (HS) proteoglycans are involved in the recognition of cellular targets by NKp30 was recently reported. The data presented in this report show conclusively that HS glycosaminoglycans (GAG) are not ligands for NKp30. We show that removing HS completely from the cell surface of human 293-EBNA cells with mammalian heparanase does not affect binding of rNKp30/human IgG1 Fc chimera complexes or binding of multimeric liposome-rNKp30 complexes. Removing HS from 293-EBNA cells, culture-generated DC, MM-170 malignant melanoma cells, or HeLa cells does not affect the NKp30-dependent killing of these cells by NK cells. We show further that the GAG-deficient hamster pgsA-745 cells that lack HS and the GAG-expressing parent CHO-K1 cells are both killed by NK cells, with killing of both cell lines inhibited to the same extent by anti-NKp30 mAb. From these results we conclude that HS GAG are not ligands for NKp30, leaving open the question as to the nature of the cellular ligand for this important NK cell activation receptor.

Histidine-rich glycoprotein: A novel adaptor protein in plasma that modulates the immune, vascular and coagulation systems.

Histidine-rich glycoprotein (HRG) is an abundant plasma glycoprotein that has a multidomain structure, interacts with many ligands, and has been shown to regulate a number of important biological processes. HRG ligands include Zn(2+) and haem, tropomyosin, heparin and heparan sulphate, plasminogen, plasmin, fibrinogen, thrombospondin, IgG, FcgammaR and complement. In many cases, the histidine-rich region of the molecule enhances ligand binding following interaction with Zn(2+) or exposure to low pH, conditions associated with sites of tissue injury or tumour growth. The multidomain nature of HRG indicates that it can act as an extracellular adaptor protein, bringing together disparate ligands, particularly on cell surfaces. HRG binds to most cells primarily via heparan sulphate proteoglycans, binding which is also potentiated by elevated free Zn(2+) levels and low pH. Recent reports have shown that HRG can modulate angiogenesis and additional studies have shown that it may regulate other physiological processes such as cell adhesion and migration, fibrinolysis and coagulation, complement activation, immune complex clearance and phagocytosis of apoptotic cells. This review outlines the molecular, structural, biological and clinical properties of HRG as well as describing the role of HRG in various physiological processes.

Plasminogen is tethered with high affinity to the cell surface by the plasma protein, histidine-rich glycoprotein.

Plasminogen has been implicated in extracellular matrix degradation by invading cells, but few high affinity cell surface receptors for the molecule have been identified. Previous studies have reported that the plasma protein, histidine-rich glycoprotein (HRG), interacts with plasminogen and cell surfaces, raising the possibility that HRG may immobilize plasminogen/plasmin to cell surfaces. Here we show, based on optical biosensor analyses, that immobilized HRG interacts with soluble plasminogen with high affinity and with an extremely slow dissociation rate. Furthermore, the HRG-plasminogen interaction is lysine-dissociable and involves predominately the amino-terminal domain of HRG, and the fifth kringle domain of plasminogen, but not the carboxyl-terminal lysine of HRG. HRG was also shown to tether plasminogen to cell surfaces, with this interaction being potentiated by elevated Zn(2+) levels and low pH, conditions that prevail at sites of tissue injury, tumor growth, and angiogenesis. Based on these data we propose that HRG acts as a soluble adaptor molecule that binds to cells at sites of tissue injury, tumor growth, and angiogenesis, providing a high affinity receptor for tethering plasminogen to the cell surface and thereby enhancing the migratory potential of cells.

Histidine-rich glycoprotein binds to cell-surface heparan sulfate via its N-terminal domain following Zn2+ chelation.

Histidine-rich glycoprotein (HRG) is an alpha2-glycoprotein found in mammalian plasma at high concentrations (approximately 150 microg/ml) and is distinguished by its high content of histidine and proline. Structurally, HRG is a modular protein consisting of an N-terminal cystatin-like domain (N1N2), a central histidine-rich region (HRR) flanked by proline-rich sequences, and a C-terminal domain. HRG binds to cell surfaces and numerous ligands such as plasminogen, fibrinogen, thrombospondin, C1q, heparin, and IgG, suggesting that it may act as an adaptor protein either by targeting ligands to cell surfaces or by cross-linking soluble ligands. Despite the suggested functional importance of HRG, the cell-binding characteristics of the molecule are poorly defined. In this study, HRG was shown to bind to most cell lines in a Zn(2+)-dependent manner, but failed to interact with the Chinese hamster ovary cell line pgsA-745, which lacks cell-surface glycosaminoglycans (GAGs). Subsequent treatment of GAG-positive Chinese hamster ovary cells with mammalian heparanase or bacterial heparinase III, but not chondroitinase ABC, abolished HRG binding. Furthermore, blocking studies with various GAG species indicated that only heparin was a potent inhibitor of HRG binding. These data suggest that heparan sulfate is the predominate cell-surface ligand for HRG and that mammalian heparanase is a potential regulator of HRG binding. Using recombinant forms of full-length HRG and the N-terminal N1N2 domain, it was shown that the N1N2 domain bound specifically to immobilized heparin and cell-surface heparan sulfate. In contrast, synthetic peptides corresponding to the Zn(2+)-binding HRR of HRG did not interact with cells. Furthermore, the binding of full-length HRG, but not the N1N2 domain, was greatly potentiated by physiological concentrations of Zn2+. Based on these data, we propose that the N1N2 domain binds to cell-surface heparan sulfate and that the interaction of Zn2+ with the HRR can indirectly enhance cell-surface binding.