Plasma Protein Histidine-rich Glycoprotein Research Articles

Quartz Crystal Microbalance Measurement of Histidine-Rich Glycoprotein and Stanniocalcin-2 Binding to Each Other and to Inflammatory Cells.

The plasma protein histidine-rich glycoprotein (HRG) is implicated in the polarization of macrophages to an M1 antitumoral phenotype. The broadly expressed secreted protein stanniocalcin 2 (STC2), also implicated in tumor inflammation, is an HRG interaction partner. With the aim to biochemically characterize the HRG and STC2 complex, binding of recombinant HRG and STC2 preparations to each other and to cells was explored using the quartz crystal microbalance (QCM) methodology. The functionality of recombinant proteins was tested in a phagocytosis assay, where HRG increased phagocytosis by monocytic U937 cells while STC2 suppressed HRG-induced phagocytosis. The binding of HRG to STC2, measured using QCM, showed an affinity between the proteins in the nanomolar range, and both HRG and STC2 bound individually and in combination to vitamin D3-treated, differentiated U937 monocytes. HRG, but not STC2, also bound to formaldehyde-fixed U937 cells irrespective of their differentiation stage in part through the interaction with heparan sulfate. These data show that HRG and STC2 bind to each other as well as to U937 monocytes with high affinity, supporting the relevance of these interactions in monocyte/macrophage polarity.

Marginal zone lymphoma expression of histidine-rich glycoprotein correlates with improved survival.

PurposeThe abundant hepatocyte‐expressed plasma protein histidine‐rich glycoprotein (HRG) enhances antitumor immunity by polarizing inflammatory and immune cells in several mouse models, however, the clinical relevance of HRG in human cancer is poorly explored. The expression and role of HRG in human B‐cell lymphomas was investigated in order to find new tools for prognosis and treatment.FindingsImmunohistochemical (IHC) analysis and RNA hybridization of tissue microarrays showed that (i) HRG was expressed by tumor cells in marginal zone lymphoma (MZL), in 36% of 59 cases. Expression was also detected in follicular lymphoma (22%), mantle cell lymphoma (19%), and indiffuse large B‐cell lymphoma (DLBCL;5%) while primary CNS lymphoma (PCNSL) lacked expression of HRG. (ii) MZL patients positive for HRG showed a superior overall survival outcome (HR = 0.086, 95% CI = 0.014‐0.518, P‐value = .007), indicating a protective role for HRG independent of stage, age and sex. (iii) HRG‐expressing MZL displayed significantly increased transcript and protein levels of the host defense peptide alpha defensin 1. In addition, global transcript analyses showed significant changes in gene ontology terms relating to immunity and inflammation, however, infiltration of immune and inflammatory cells detected by IHC was unaffected by HRG expression.ConclusionHRG expression by MZL tumor cells correlates with an altered transcription profile and improved overall survival.

Histidine-Rich Glycoprotein Inhibits HIV-1 Infection in a pH-Dependent Manner.

Histidine-rich glycoprotein (HRG) is an abundant plasma protein with a multidomain structure, allowing its interaction with many ligands, including phospholipids, plasminogen, fibrinogen, IgG antibodies, and heparan sulfate. HRG has been shown to regulate different biological responses, such as angiogenesis, coagulation, and fibrinolysis. Here, we found that HRG almost completely abrogated the infection of Ghost cells, Jurkat cells, CD4+ T cells, and macrophages by HIV-1 at a low pH (range, 6.5 to 5.5) but not at a neutral pH. HRG was shown to interact with the heparan sulfate expressed by target cells, inhibiting an early postbinding step associated with HIV-1 infection. More importantly, by acting on the viral particle itself, HRG induced a deleterious effect, which reduces viral infectivity. Because cervicovaginal secretions in healthy women show low pH values, even after semen deposition, our observations suggest that HRG might represent a constitutive defense mechanism in the vaginal mucosa. Of note, low pH also enabled HRG to inhibit the infection of HEp-2 cells and Vero cells by respiratory syncytial virus (RSV) and herpes simplex virus 2 (HSV-2), respectively, suggesting that HRG might display broad antiviral activity under acidic conditions.IMPORTANCE Vaginal intercourse represents a high-risk route for HIV-1 transmission. The efficiency of male-to-female HIV-1 transmission has been estimated to be 1 in every 1,000 episodes of sexual intercourse, reflecting the high degree of protection conferred by the genital mucosa. However, the contribution of different host factors to the protection against HIV-1 at mucosal surfaces remains poorly defined. Here, we report for the first time that acidic values of pH enable the plasma protein histidine-rich glycoprotein (HRG) to strongly inhibit HIV-1 infection. Because cervicovaginal secretions usually show low pH values, our observations suggest that HRG might represent a constitutive antiviral mechanism in the vaginal mucosa. Interestingly, infection by other viruses, such as respiratory syncytial virus and herpes simplex virus 2, was also markedly inhibited by HRG at low pH values, suggesting that extracellular acidosis enables HRG to display broad antiviral activity.

Leukocyte Differentiation by Histidine-Rich Glycoprotein/Stanniocalcin-2 Complex Regulates Murine Glioma Growth through Modulation of Antitumor Immunity

The plasma-protein histidine-rich glycoprotein (HRG) is implicated in phenotypic switching of tumor-associated macrophages, regulating cytokine production and phagocytotic activity, thereby promoting vessel normalization and antitumor immune responses. To assess the therapeutic effect of HRG gene delivery on CNS tumors, we used adenovirus-encoded HRG to treat mouse intracranial GL261 glioma. Delivery of Ad5-HRG to the tumor site resulted in a significant reduction in glioma growth, associated with increased vessel perfusion and increased CD45+ leukocyte and CD8+ T-cell accumulation in the tumor. Antibody-mediated neutralization of colony-stimulating factor-1 suppressed the effects of HRG on CD45+ and CD8+ infiltration. Using a novel protein interaction-decoding technology, TRICEPS-based ligand receptor capture (LRC), we identified Stanniocalcin-2 (STC2) as an interacting partner of HRG on the surface of inflammatory cells in vitro and colocalization of HRG and STC2 in gliomas. HRG reduced the suppressive effects of STC2 on monocyte CD14+ differentiation and STC2-regulated immune response pathways. In consequence, Ad5-HRG-treated gliomas displayed decreased numbers of IL35+ Treg cells, providing a mechanistic rationale for the reduction in GL261 growth in response to Ad5-HRG delivery. We conclude that HRG suppresses glioma growth by modulating tumor inflammation through monocyte infiltration and differentiation. Moreover, HRG acts to balance the regulatory effects of its partner, STC2, on inflammation and innate and/or acquired immunity. HRG gene delivery therefore offers a potential therapeutic strategy to control antitumor immunity. Mol Cancer Ther; 17(9); 1961-72. ©2018 AACR.

Functional Regulation of the Plasma Protein Histidine-Rich Glycoprotein by Zn2+ in Settings of Tissue Injury

Divalent metal ions are essential nutrients for all living organisms and are commonly protein-bound where they perform important roles in protein structure and function. This regulatory control from metals is observed in the relatively abundant plasma protein histidine-rich glycoprotein (HRG), which displays preferential binding to the second most abundant transition element in human systems, Zinc (Zn2+). HRG has been proposed to interact with a large number of protein ligands and has been implicated in the regulation of various physiological and pathological processes including the formation of immune complexes, apoptotic/necrotic and pathogen clearance, cell adhesion, antimicrobial activity, angiogenesis, coagulation and fibrinolysis. Interestingly, these processes are often associated with sites of tissue injury or tumour growth, where the concentration and distribution of Zn2+ is known to vary. Changes in Zn2+ levels have been shown to modify HRG function by altering its affinity for certain ligands and/or providing protection against proteolytic disassembly by serine proteases. This review focuses on the molecular interplay between HRG and Zn2+, and how Zn2+ binding modifies HRG-ligand interactions to regulate function in different settings of tissue injury.

Histidine-Rich Glycoprotein Prevents Septic Lethality through Regulation of Immunothrombosis and Inflammation

Sepsis is a major cause of death worldwide. We show that a plasma protein histidine-rich glycoprotein (HRG) was decreased significantly in septic mice with cecal ligation and puncture (CLP) and supplementary treatment of septic mice with exogenous HRG improved survival, with strong inhibition of tight attachment of neutrophils to pulmonary vasculatures, subsequent immunothrombosis, DIC state, lung inflammation, hypercytokinemia, and activation of vascular endothelial cells (VECs). In contrast, knockdown of HRG by siRNA exacerbated lethality. Purified human HRG reversibly induced morphological changes in human neutrophils in vitro; induction of spherical shape with reduced microvilli and adhesiveness to VECs. HRG maintained the passage of neutrophils through microcapillaries and abolished production of reactive oxygen species. These results suggested that the supplementary therapy with HRG may provide a novel strategy for the treatment of sepsis through suppression of excessive systemic inflammation and immunothrombosis by keeping circulating neutrophils quiescent and preventing uncontrolled activation of VECs.

Histidine-rich glycoprotein blocks collagen-binding integrins and adhesion of endothelial cells through low-affinity interaction with α2 integrin

The plasma protein histidine-rich glycoprotein (HRG) affects the morphology and function of both endothelial cells (ECs) and monocytes/macrophages in cancer. Here, we examined the mechanism of action of HRG's effect on ECs. HRG suppressed adhesion, spreading and migration of ECs specifically on collagen I (COL I) whereas ECs seeded on other extracellular matrix proteins were insensitive to HRG. HRG did not bind specifically to COL I or to the α-integrin binding site on collagen, GFOGER. Furthermore, HRG's inhibition of EC adhesion was not dependent upon heparan sulfate (HS) moieties as heparitinase-treated ECs remained sensitive to HRG. C2C12 cells expressing α2 integrin, the major collagen-binding α-integrin subunit in ECs, showed increased binding of HRG compared with wild type C2C12 cells lacking the α2 subunit. Recombinant α2 I-domain protein bound HRG and to a higher extent when in active conformation. However, the α2 I-domain bound weakly to HRG compared with COL I and the purified α2β1 ectodomain complex failed to retain HRG. We conclude that HRG binds to α2 integrin through low-affinity interactions in a HS-independent manner, thereby blocking EC-adhesion to COL I.

Histidine-rich glycoprotein prevents septic lethality through neutrophil regulation

Although there have been many clinical trials for treatment of sepsis, no drugs are available at present. Sepsis is thought to be complex disorders; activation/lesion of vascular endothelial cells, accelerated coagulation and platelet aggregation, leukocyte activation or paralysis, and hypercytokinemia. To treat the pathological status of sepsis, it must be necessary to control these plural disorders simultaneously or sequentially. In the present study, we identified and characterized a plasma protein histidine-rich glycoprotein (HRG) as a factor that decreases dramatically in septic condition and maintains neutrophil's shape and functional quiescence. We clarified the involvement of HRG in septic pathogenesis and propose a novel therapy for sepsis based on that.

Enhanced Platelet Activation Mediates the Accelerated Angiogenic Switch in Mice Lacking Histidine-Rich Glycoprotein

BackgroundThe heparin-binding plasma protein histidine-rich glycoprotein (HRG; alternatively, HRGP/HPRG) can suppress tumor angiogenesis and growth in vitro and in vivo. Mice lacking the HRG gene are viable and fertile, but have an enhanced coagulation resulting in decreased bleeding times. In addition, the angiogenic switch is significantly enhanced in HRG-deficient mice.Methodology/Principal FindingsTo address whether HRG deficiency affects tumor development, we have crossed HRG knockout mice with the RIP1-Tag2 mouse, a well established orthotopic model of multistage carcinogenesis. RIP1-Tag2 HRG−/− mice display significantly larger tumor volume compared to their RIP1-Tag2 HRG+/+ littermates, supporting a role for HRG as an endogenous regulator of tumor growth. In the present study we also demonstrate that platelet activation is increased in mice lacking HRG. To address whether this elevated platelet activation contributes to the increased pathological angiogenesis in HRG-deficient mice, they were rendered thrombocytopenic before the onset of the angiogenic switch by injection of the anti-platelet antibody GP1bα. Interestingly, this treatment suppressed the increase in angiogenic neoplasias seen in HRG knockout mice. However, if GP1bα treatment was initiated at a later stage, after the onset of the angiogenic switch, no suppression of tumor growth was detected in HRG-deficient mice.ConclusionsOur data show that increased platelet activation mediates the accelerated angiogenic switch in HRG-deficient mice. Moreover, we conclude that platelets play a crucial role in the early stages of tumor development but are of less significance for tumor growth once angiogenesis has been initiated.

The Anti-angiogenic His/Pro-rich Fragment of Histidine-rich Glycoprotein Binds to Endothelial Cell Heparan Sulfate in a Zn2+-dependent Manner

The plasma protein histidine-rich glycoprotein (HRGP), which has been identified as an angiogenesis inhibitor, binds to heparan sulfate (HS) in a Zn(2+)-dependent manner. We wished to test whether this interaction is mechanistically important in mediation of the anti-angiogenic effect of HRGP. Inhibition of angiogenesis by HRGP is exerted through its central His/Pro-rich domain, which is proteolytically released. A 35-amino-acid residue synthetic peptide, HRGP330, derived from the His/Pro-rich domain retains the inhibitory effect on blood vessel formation in vitro and in vivo, an effect dependent on the presence of Zn(2+). We now show that HRGP330 binds heparin/HS with the same capacity as full-length HRGP, and the binding is Zn(2+)-dependent. Peptides derived from the His/Pro-rich domain of HRGP downstream of HRGP330 fail to inhibit endothelial cell migration and display a significantly reduced heparin-binding capacity. An even shorter peptide, HRGP335, covering a 26-amino-acid sequence within HRGP330 retains full heparin/HS-binding capacity. Characterization of the HS interaction shows that there is a tissue-specific HS pattern recognized by HRGP335 and that the minimal length of heparin/HS required for binding to HRGP335 is an 8-mer oligosaccharide. Saturation of the HS binding sites in HRGP330 by pre-incubation with heparin abrogates the HRGP330-induced rearrangement of endothelial cell focal adhesions, suggesting that interaction with cell surface HS is needed for HRGP330 to exert its anti-angiogenic effect.

Differential Binding of Histidine-rich Glycoprotein (HRG) to Human IgG Subclasses and IgG Molecules Containing κ and λ Light Chains

In previous studies we showed that the plasma protein histidine-rich glycoprotein (HRG) binds strongly to pooled human IgG. In the present work myeloma proteins consisting of different human IgG subclasses were examined for their ability to interact with human HRG. Using an IAsys optical biosensor we found initially that IgG subclasses differ substantially in their affinity of interaction with HRG. However, the most striking finding was the observation that the kinetics of the HRG interaction was dramatically affected by whether the IgG subclasses contained the kappa or lambda light (L)-chains. Thus, the on-rate for the binding of HRG to the kappa L-chain containing IgG1 and IgG2 (IgG1kappa and IgG2kappa) was approximately 4- and approximately 10-fold faster than that for the binding of HRG to lambda L-chain containing IgG1 and IgG2 (IgG1lambda and IgG2lambda), respectively, with the dissociation constants (K(d)) in the range 3-5 nM and 112-189 nM for the kappa and lambda isoforms, respectively. In contrast, the on-rate for the binding of HRG to IgG3kappa and IgG4kappa was found to be 9- and 20-fold slower than that for the binding of HRG to IgG3lambda and IgG4lambda, respectively, with the K(d) in the range 147-268 nM and 96-109 nM for the kappa and lambda isoforms, respectively. The binding of HRG to immunoglobulins containing the kappa L-chain (particularly IgG1kappa) was generally potentiated in the presence of a physiological concentration (20 microM) of Zn(2+) (K(d) decreased to 0.60 +/- 0.01 for IgG1kappa), but Zn(2+) had no effect or slightly inhibited the binding of HRG to immobilized IgG subclasses possessing the lambda L-chain. Interestingly, HRG also bound differentially to Bence Jones (BJ) proteins containing kappa and lambda L-chains, with HRG having a 14-fold lower K(d) for BJkappa than for BJlambda when 20 microM Zn(2+) was present. HRG also bound to IgM (IgMkappa), but the affinity of this interaction (K(d) approximately 1.99 +/- 0.05 microM) was markedly lower than the interaction with IgG, and the affinity was actually decreased 4-fold in the presence of Zn(2+). The results demonstrate that both the heavy (H)- and L-chain type have a profound effect on the binding of HRG to different IgG subclasses and provide the first evidence of a functional difference between the kappa and lambda L-chains of immunoglobulins.

Modified Crossed Immunoelectrophoresis to Study with Whole Plasma the Reversible Complex Formation of Histidine-Rich Glycoprotein with Plasminogen

To study the reversible complex formation between the plasma protein histidine-rich glycoprotein (HRG) and plasminogen, crossed immunoelectrophoresis of HRG was modified. In the modification, purified plasminogen was introduced into the gel of the first dimension electrophoresis. Two molecular forms of plasminogen, Glu- and Lys-plasminogen, induced a dose-dependent reduction of the electrophoretic mobility of HRG, with a half maximal retardation for both plasminogens at 0.50-0.55 microM of added plasminogen to the agarose gel. HRG in plasma behaved as a uniform fraction with respect to plasminogen binding. In contrast, with the same modified technique another plasma protein, alpha 2-antiplasmin, separated into a retarded plasminogen-binding form and a non-retarded non-plasminogen-binding form. The method can be used to assess several aspects of reversible complex formation between plasma proteins, as demonstrated for plasminogen binding of HRG and alpha 2-antiplasmin in whole plasma.

Neutralization of heparin-related saccharides by histidine-rich glycoprotein and platelet factor 4.

Heparin and heparin oligosaccharides prepared by nitrous acid depolymerization were fractionated by affinity chromatography on immobilized antithrombin and by gel chromatography. The anticoagulant activities of high affinity heparin of Mr greater than or equal to 7,800 could be readily neutralized by the plasma protein histidine-rich glycoprotein (see also Lijnen, H.R., Hoylaerts, M., and Collen, D. (1983) J. Biol. Chem. 258, 3803-3808), whereas oligosaccharides falling below 18 saccharide units (Mr 5,400) became increasingly resistant to neutralization. An octasaccharide with characteristic marked ability to accelerate the inactivation of Factor Xa by antithrombin retained greater than 50% of its activity even at a histidine-rich glycoprotein/oligosaccharide molar ratio of 500:1. Histidine-rich glycoprotein, like the platelet-derived heparin neutralizing protein platelet factor 4 (Lane, D.A., Denton, J., Flynn, A.M., Thunberg, L. and Lindahl, U. (1984) Biochem J. 218, 725-732), therefore requires interaction with saccharide sequences in addition to the antithrombin-binding pentasaccharide of heparin in order to efficiently express its antiheparin activity. Heparan sulfate isolated from pig intestinal mucosa (HS I, Mr approximately 20,000) and from human aorta (HS II, Mr approximately 40,000) exhibited anti-Factor Xa activities of 180 and 20 units/micromol [corrected], respectively. A fraction corresponding to about 5% of HS I bound with high affinity to immobilized antithrombin and contained all of the anticoagulant activity of the starting material. While these heparan sulfates were readily neutralized by platelet factor 4, they were relatively resistant to neutralization by histidine-rich glycoprotein, although complete neutralization could be attained in the presence of molar excess of this protein. These findings may be of importance in relation (a) to the functional role of endogenous anticoagulant polysaccharides at the vascular wall and (b) to clinical situations in which heparin or heparin-related compounds are administered as exogenous anticoagulants.