Plasma Hyaluronan-binding Protein Research Articles

Lac color inhibits development of rat thyroid carcinomas through targeting activation of plasma hyaluronan-binding protein

Coccid-derived natural food colorants contain active ingredients that potentiate inhibition of tissue proteolysis mediated by activation of plasma hyaluronan-binding protein (PHBP). In the present study, we examined the effect of lac color (LC) and cochineal extract (CE), representative coccid-derived colorants containing laccaic acid and carminic acid as active ingredients, in an intracapsular invasion model of experimental thyroid cancers using rats. One week after initiation with N-bis(hydroxypropyl)nitrosamine, male F344/NSIc rats were fed a powdered diet containing 5.0% LC or 3.0% CE during promotion with 0.15% sulfadimethoxine (SDM) in the drinking water for 13 weeks. Capsular invasive carcinomas (CICs) and lung metastases were decreased by LC treatment and accompanied by transcript downregulation on angiogenesis and PHBP-related tissue proteolysis in CICs. In contrast, CE upregulated angiogenesis-related genes in CICs. PHBP was expressed in capsular macrophages and thyroid proliferative lesions with increased intensity in CICs, and LC decreased PHBP-expressing CICs. The size of CICs and their proliferation activity, however, were unchanged compared with those treated with SDM alone. Suppression of cancer by invasion by LC was more evident after an eight-week treatment, exhibiting a profound decrease in tenascin-C-positive early invasive foci and marked reductions in capsular inflammation and fibrosis. These results suggest that LC and CE exerted dissimilar effects on CIC development, the former suppressing the initial step of neoplastic cell invasion into the capsule by targeting PHBP activity of macrophages and neoplastic cells on tissue proteolysis involving inflammatory responses and angiogenesis, and the latter promoting angiogenesis of developed CICs at later stages.

Factor seven activating protease (FSAP): does it activate factor VII?

Factor seven activating protease (FSAP) was initially reported as an activator of single-chain urokinase-type plasminogen activator (scuPA) and factor VII (FVII). Subsequently, numerous additional substrates have been identified, and multiple other biological effects have been reported. Due to the apparent lack of specificity, the physiological role of FSAP has become increasingly unclear. Rigorous studies have been limited by the difficulty of obtaining intact FSAP from blood or recombinant sources. Our aim was to produce intact recombinant human FSAP, and to assess its role as a trigger of coagulation and fibrinolysis. Expression of wild-type FSAP in various mammalian cells invariably resulted in the accumulation of degraded FSAP due to autoactivation and degradation. To overcome this problem, we constructed a variant in which Arg(313) at the natural activation site was replaced by Gln, creating a cleavage site for the bacterial protease thermolysin. HEK293 cells produced FSAP(R313Q) in its intact form. Thermolysin-activated FSAP displayed the same reactivity toward the substrate S-2288 as plasma-derived FSAP, and retained its ability to activate scuPA. Polyphosphate and heparin increased V(max) by 2-3-fold, without affecting K(m) (62 nm) of scuPA activation. Surprisingly, FVII activation by activated FSAP proved negligible, even in the presence of calcium ions, phospholipid vesicles and recombinant soluble tissue factor. On membranes of 100% cardiolipin FVII cleavage did occur, but this resulted in transient activation and rapid degradation. While FSAP indeed activates scuPA, FVII appears remarkably resistant to activation. Therefore, reappraisal of the putative role of FSAP in hemostasis seems appropriate.

Promoting effects of carminic acid-enriched cochineal extracts on capsular invasive thyroid carcinomas through targeting activation of angiogenesis in rats

Cochineal extracts (CE) is a coccid-derived natural food colorant containing carminic acid (CA) as an active ingredient that potentiates inhibition of tissue proteolysis mediated by activation of plasma hyaluronan-binding protein (PHBP). In our previous study, dietary administered CE (CA: 28.5% in CE) has shown to promote the macroscopic development of capsular invasive carcinomas (CICs) associated with up-regulation of angiogenesis-related genes in an intracapsular invasion model of experimental thyroid cancers using rats. However, the promoting effect of CE could not be confirmed histopathologically. The purpose of the present study was to confirm the promoting effect of CE through direct injections to animals on the development of CICs using this cancer invasion model. One week after initiation with N-bis(hydroxypropyl)nitrosamine, male F344/NSlc rats were administered CA-enriched CE (CA: 52.6% in CE) by intraperitoneal injections every other day (10 mg/kg body weight) during the promotion with 0.15% sulfadimethoxine in the drinking water for 8 weeks. The multiplicities of macroscopical CICs and the mean area of early capsular invasive foci estimated by Tenascin (TN)-C-immunoreactivity in the thyroid significantly increased with CE-treatment, while the number of TN-C-positive foci did not change with CE. Transcript level of Phbp and downstream genes unchanged; however, transcript level of angiogenesis-related genes, i.e, Vegfb and its transcription factor gene, Hif1a, those being downstream of phosphatase and tensin homolog (PTEN)/Akt signaling, up-regulated in the thyroid tissue with CE-administration. These results suggest that CE potentiates promotion activity by facilitating angiogenesis through activation of PTEN/Akt signaling without accompanying modification of PHBP-related proteolysis.

Elucidation of Crucial Structures for a Catechol-Based Inhibitor of Plasma Hyaluronan-Binding Protein (Factor VII Activating Protease) Autoactivation

Plasma hyaluronan-binding protein (PHBP) is a serine protease the activation of which is implicated in inflammation. Previous investigations have suggested the presence of catechol-binding sites in its proenzyme form, pro-PHBP. Here we found that compounds with plural catechol groups conjugated with strong electron-withdrawing groups, such as tyrphostin AG 537 (IC(50)=18 nM), were potent inhibitors of pro-PHBP activation.

Extracellular histone induces plasma hyaluronan-binding protein (factor VII activating protease) activation in vivo

Plasma hyaluronan-binding protein (PHBP), an activator of factor VII and prourokinase, is a serine protease circulating as a single-chain proenzyme (pro-PHBP). Pro-PHBP converts to the active two-chain form through autoproteolysis, and effectors that modulate autoactivation can regulate PHBP-mediated processes. Here, we show that histone promotes pro-PHBP autoactivation in vivo. Histone bound to pro-PHBP and promoted intermolecular pro-PHBP binding. Histone-mediated pro-PHBP activation in plasma leads to the formations of bradykinin and PHBP−α2-antiplasmin complex as well as histone degradation. Pro-PHBP activation was observed in the circulation of mice after injection of histone or lipopolysaccharide, which induced septic response accompanying extracellular histone release. Our results suggest pathophysiological relevance of histone-dependent pro-PHBP activation in hyperinflammatory process.

Inhibitors of Autoactivation of Plasma Hyaluronan-Binding Protein (Factor VII Activating Protease)

Plasma hyaluronan-binding protein (PHBP), a serine protease that can activate coagulation factor VII and prourokinase, circulates in a single-chain form (pro-PHBP) and autoproteolytically converts to an active two-chain form with the aid of an effector such as spermidine and heparin. It has been postulated that PHBP plays roles in regulating inflammation, vascular function, fibrosis and atherosclerosis. From the comprehensive screening of natural sources for inhibitors of spermidine-induced pro-PHBP autoactivation, we identified several compounds with a polyphenol feature. Of these inhibitors, tannic acid (IC(50)=0.020 µM), delphinidin (IC(50)=0.079 µM), hamamelitannin (IC(50)=0.19 µM), (-)-epicatechin gallate (IC(50)=0.24 µM), and 3,5-di-O-caffeoylquinic acid (IC(50)=1.0 µM) were potent and selective, and did not inhibit heparin-induced pro-PHBP autoactivation and the active form of PHBP at concentrations 100 times higher than the respective IC(50) values. From evaluation of the activities of related compounds, it has been suggested that a compound with multiple aromatic rings with plural phenolic hydroxyl substituents exhibits potent activity. The inhibitory actions of delphinidin, hamamelitannin, (-)-epicatechin gallate and 3,5-di-O-caffeoylquinic acid were attenuated by catechol, a minimum polyphenol unit. Thus, it is likely that pro-PHBP binds these potent inhibitors through its site(s) that recognize a catechol-like structure. Our results would facilitate understanding of the molecular mechanism of pro-PHBP autoactivation and rational design of a compound for suppressing unregulated pro-PHBP activation.

Inhibition of Plasma Hyaluronan-Binding Protein Autoactivation by Laccaic Acid

Plasma hyaluronan-binding protein (PHBP) is a serine protease implicated in proteolysis under inflammatory conditions. We identified laccaic acid, a widely used food coloring from scale insects, as a potent inhibitor of the protease in terms of both autoactivation of the PHBP proenzyme (IC(50) = 0.35-0.55 µg/ml) and the catalytic activity of the active enzyme (IC(50) = 1.1 µg/ml).

The cyclopentapeptide plactin enhances cellular binding and autoactivation of the serine protease plasma hyaluronan-binding protein

Plactin, a family of cyclopentapeptides of fungal origin, enhances fibrinolytic activity by promoting of single-chain urokinase-type plasminogen activator (scu-PA) activation on the cell surface. For this activity, factor(s) in the blood plasma is absolutely required. In the previous studies, we identified prothrombin as a plasma cofactor involved in this mechanism, while the presence of another independent cofactor was suggested. The objective of this study was to identify the second cofactor and investigate the mechanism involved. Using plactin-affinity and ion-exchange chromatographies, we purified plasma hyaluronan-binding protein (PHBP) ~ 4,000-fold from human plasma as an independent plactin cofactor. PHBP, at ~ 10 nM, was effective in plactin-dependent promotion of scu-PA activation by U937 cells. PHBP is a serine protease that is produced as a single-chain proenzyme (pro-PHBP) and autoproteolytically converted to an active two-chain form. Pro-PHBP was comparable to PHBP in activity to promote plactin-dependent scu-PA activation by U937 cells. Plactin enhanced both cellular binding and autoproteolytic activation of pro-PHBP. The two activities were obtained with a plactin concentration at ~ 30 μM, which resulted in a significant increase in intrinsic fluorescence and self association of pro-PHBP. Thus, it is suggested that such changes account for both enhanced cellular binding and autoactivation of pro-PHBP, resulting in an enhancement of scu-PA activation.

Purpurin as a Specific Inhibitor of Spermidine-Induced Autoactivation of the Protease Plasma Hyaluronan-Binding Protein

Plasma hyaluronan-binding protein (PHBP), a serine protease that can activate coagulation factor VII and prourokinase, circulates as a single-chain form (pro-PHBP), and is autoproteolytically converted to an active two-chain form with the aid of an effector such as spermidine and heparin. In this study, we screened natural sources for inhibitors of spermidine-induced pro-PHBP autoactivation. As an active agent, we purified bikaverin from a culture of a fungus. Bikaverin inhibited spermidine-induced autoactivation with an IC(50) of 0.45 microM, while it also inhibited the active form of PHBP (IC(50)=0.8 microM). Additional screening of related compounds led to the identification of purpurin, a plant anthraquinone, as a specific inhibitor: IC(50)=6.6 microM for spermidine-induced autoactivation; no inhibition of heparin-induced autoactivation and active PHBP. Alizarin and emodin, which structurally differed from purpurin in the position or the number of the hydroxyl groups, were less active and nonspecific. Thus, the position and/or the number of the hydroxyl group affect both the potency and selectivity of the anthraquinone inhibitors.

Polyamine-promoted autoactivation of plasma hyaluronan-binding protein.

Plasma hyaluronan-binding protein (PHBP), a protease implicated in extracellular proteolysis, consists of multiple domains: an N-terminal region (NTR), three epidermal growth factor (EGF)-like domains, a kringle domain, and a protease domain. PHBP circulates as a single-chain proenzyme (pro-PHBP), which is converted to an active, two-chain form through autoproteolysis. To understand the mechanism of autoactivation. Here, we report that polyamine induces the formation of pro-PHBP autoactivation complex, in which an intermolecular interaction between NTR and the third EGF-like domain (E3) plays a role. Using a series of pro-PHBP mutants that partially lack functional domains, polyamine-induced pro-PHBP autoactivation was investigated in terms of enzyme activity, protein interaction, and inhibition by carminic acid, an anthraquinone compound identified in this study. Polyamine enhanced intermolecular binding of pro-PHBP, but not of mutant pro-PHBP that partially lacked NTR (DeltaN). Carminic acid inhibited intermolecular pro-PHBP binding and specifically abolished polyamine-induced autoactivation. NTR bound to pro-PHBP and DeltaN, but its binding was minimal to a mutant that lacked E3. The NTR-DeltaN binding was inhibited by a combination of polyamine and carminic acid, but each compound alone was ineffective. We infer from the data that (i) polyamine modulates intramolecular NTR-E3 interaction to allow intermolecular binding between NTR and E3 in another pro-PHBP molecule to form an autoactivation complex, and (ii) carminic acid inhibits polyamine-modulated intermolecular NTR-E3 binding. Polyamine concentrations are higher in cells and tissues with inflammation and malignancy. Polyamine leakage from legions through cell death or tissue injury may account for physiologically relevant pro-PHBP activation.

Anti-angiogenic action of plasma hyaluronan binding protein in human umbilical vein endothelial cells

The kringle domain is a triple loop structure present in angiostatin and endostatin. The disulfide bond-linked kringle architectures have been known to be essential for anti-angiogenic activity. Plasma hyaluronan binding protein (PHBP) is a novel serine protease which consists of three epidermal growth factor (EGF) domains, a kringle domain, and a serine protease domain. PHBP can be cleaved autocatalytically to generate activity and is highly expressed in the human blood and liver. To determine the anti-angiogenic activities of PHBP, we purified recombinant mouse PHBP from stable cell line overexpressing PHBP and used protein in vivo and in vitro angiogenesis assays. We found that recombinant PHBP inhibits not only angiogenesis in vivo in chorioallantoic membrane (CAM) assay but also the basic fibroblast growth factor (bFGF)-induced proliferation, invasion and tube formation of human umbilical vein endothelial cells (HUVECs) in a dose-dependant manner. Moreover, we found that the kringle domain of PHBP was essential for the anti-angiogenic action of PHBP by the deletion mutants. These findings unravel a new function of PHBP as an inhibitor of the proangiogenic phenotype of vascular endothelial cells and demonstrate that the kringle domain of PHBP might be a potent novel inhibitor of activated endothelial cells in vitro and in vivo.

Novel human plasma proteins, IHRP (acute phase protein) and PHBP (serine protease), which bind to glycosaminoglycans.

A novel human plasma protein was found in the eluate from the dextran sulfate column, which was used for the treatment of the patients with hypercholesteremia to reduce plasma low density lipoprotein. The results of sequence analysis revealed that this protein was a homologue of heavy chains of inter-alpha-trypsin inhibitor (ITI) family, and it was termed IHRP (ITI family heavy chain related protein). IHRP was identified as an acute-phase protein in animals, and slightly increased concentrations in human plasmas were observed in the patients with inflammatory disorders. IHRP bound to actin and inhibited its polymerization, and IHRP suppressed the phagocytosis and chemotaxis of polymorphonuclear cells. These results suggest that IHRP may function as an anti-inflammatory protein. Plasma hyaluronan binding protein (PHBP) is a novel serine protease which was also found in human plasma. It is consisted of three epidermal growth factor domains, one kringle domain and one serine protease domain from its amino terminus. The amino acid sequence of PHBP is homologous to that of hepatocyte growth factor activator. Purified 75-kDa single chain pro-form of PHBP was auto-activated (auto-cleaved) to 50-kDa heavy chain and 25-kDa light chain, both of them are bridged by a disulfied bond. PHBP digested alpha-chain and beta-chain of fibrinogen to prevent coagulation and cleaved single chain urokinase type plasminogen activator (scuPA) to the active hetero dimer form (tcuPA). The auto-activation of PHBP was accelerated in the presence of dextran sulfate or phosphatidylethanolamine as well as factor XII of the coagulation system. C1 inhibitor of the complement system was identified as the main inhibitor of PHBP in human plasma. Partial hepatectomy and administration of carbon tetrachloride or galactosamine caused the conversion of pro-PHBP to the active form in mouse but administrations of turpentine and mercury chloride did not, suggesting the hepatic injury specific activation of PHBP. These results indicate that PHBP participates not only in the fibrinolytic system but also in the degradation cascade of extracellular matrix (ECM), i.e., PHBP activates scuPA to tcuPA, tcuPA activates matrix metalloproteases (MMPs) and activated MMPs degrade ECM for the tissue remodeling after hepatic injury.

Hepatic injury-specific conversion of mouse plasma hyaluronan binding protein to the active hetero-dimer form.

Plasma hyaluronan binding protein (PHBP) is produced only in liver and kidney in mouse. The induction of PHBP mRNA and the conversion of pro PHBP to the active hetero-dimer form were studied after CCl4, D-galactosamine, HgCl2 or turpentine administration and after partial hepatectomy. The results indicated that the administrations of CCl4 and D-galactosamine, which caused hepatic failure, and the partial hepatectomy enhanced the conversion of pro PHBP to the active two-chain form in the plasma. On the other hand, HgCl2 which injured kidney and turpentine which led to inflammation were not involved in the activation of PHBP. The weak induction and suppression of PHBP mRNA were observed in the liver at 3 h and 12 h, respectively, after the CCl4 administration. However, HgCl2 and turpentine did not influence the amount of PHBP mRNA. These results suggested the hepatic injury-specific activation of PHBP in plasma. PHBP may act as an early factor in the cascade for the tissue remodeling in liver following hepatic injury, i.e., PHBP activates urokinase, urokinase activates matrix metalloproteinases (MMPs) and MMPs degrade extracellular matrix for liver regeneration.

Regulation mechanism of the serine protease activity of plasma hyaluronan binding protein.

The inhibitor for the serine protease activity of plasma hyaluronan binding protein (PHBP) was purified from human plasma by polyethylene glycol (PEG) fractionation, diethylaminoethyl (DEAE)-Sephacel ion-exchange chromatography, Phenyl Toyopearl 650M hydrophobic chromatography, Bio Gel A-0.5 m gel-filtration and hydroxyapatite chromatography. The serine protease activity of PHBP was measured with Boc-Phe-Ser-Arg-methylcoumarine amide (MCA) as the synthetic substrate of PHBP. The results of the amino acid sequence analyses of the purified PHBP inhibitor indicated that it was C1 inhibitor of the serpin family. C1 inhibitor formed a complex with PHBP, suggesting that it is the actual inhibitor of PHBP in human plasma. On the other hand, dextran sulfate and phosphatidylethanolamine enhanced the auto-fragmentation and the serine protease activity of pro-PHBP, but kaolin did not. These results suggested that the serine protease activity of PHBP was regulated in a similar manner to that of factor XII of the coagulation system.

Identification of the substrates for plasma hyaluronan binding protein.

Plasma hyaluronan biding protein (PHBP) is a novel serine protease, which has an amino acid sequence homology to that of hepatocyte growth factor activator (HGFA), and has a similar domain structure to that of urinary plasminogen activator (u-PA), found in human plasma. We searched the PHBP substrate in human plasma by measuring the digested protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that fibrinogen and fibronectin were the major substrates of PHBP. PHBP cleaved the alpha-chain at multiple sites and the beta-chain between lysine53 and lysine54 but not the gamma-chain of fibrinogen. Therefore, PHBP did not initiate the formation of the fibrin clot and did not cause the fibrinolysis directly. PHBP did not cleave (activate) prothrombin and plasminogen, but it converted the inactive single chain urinary plasminogen activator to the active two chain form.

Proteolytic activation and inactivation of the serine protease activity of plasma hyaluronan binding protein.

We prepared anti-plasma hyaluronan binding protein (PHBP) mouse monoclonal antibodies and studied the fragmentation profile of PHBP with them. PHBP is present in human plasma as a single polypeptide chain (70 kDa). During the purification, PHBP partially fragmentated into the 50-kDa N-terminal fragment and the 27-kDa C-terminal fragment. After the incubation of the purified PHBP, the 70-kDa precursor form was completely cleaved to the 50- and 27-kDa fragments, followed by the 50-kDa to the 26-kDa, and the 27-kDa to the 17-kDa plus the 8-kDa fragments, respectively. Because the purified PHBP contained no other detectable proteins and PHBP has a typical serine protease domain, we concluded that the fragmentation of PHBP was caused by own serine protease activity. PHBP cleaved the C-terminal side of Arg in the peptide effectively and that of Lys weakly. The results of the pre-incubation experiments of PHBP suggested that the single-chain form of PHBP is a precursor, the two-subunit structure is an active form and the three- or four-chain structure is an inactive form of a serine protease.

Plasma Hyaluronan-binding Protein Is a Serine Protease

CTCF is an essential factor for optimal transcription from the amyloid beta-protein precursor promoter. A proteolytic activity detected in bovine, rabbit, horse, and human serum cleaves CTCF at three major sites, resulting in a modified mobility shift pattern of the fragments that retain DNA binding ability. The protease was purified to electrophoretic homogeneity, partially sequenced, and identified as the plasma hyaluronan-binding protein. The proteolytic activity was selectively abolished by various serine protease inhibitors, including the Kunitz-type protease inhibitor domain of amyloid beta-protein precursor. Reduction with beta-mercaptoethanol showed that the 70-kDa protein consists of two polypeptides with apparent molecular masses of 44 and 30 kDa. The serine protease domain was localized to the 30-kDa polypeptide as determined by [(3)H]diisopropylfluorophosphate binding.

Isolation and characterization of the plasma hyaluronan-binding protein (PHBP) gene (HABP2).

PHBP is a novel human plasma hyaluronan-binding protein that shows significant homology in amino acid sequence to hepatocyte growth factor activator. Two overlapping clones that encode the human plasma hyaluronan-binding protein (PHBP) gene (HABP2) were isolated and characterized. The PHBP gene spans 35 kb and is composed of 13 exons from 37 to 1,394 bp in size with consensus splice sites. The gene's regulatory sequences contain putative promoter elements, but no typical TATA box. Some exons of this gene showed significant similarities to those of coagulation factor XII, tissue-type plasminogen activator, and urokinase genes in nucleotide length and in intron phasing. We also report the chromosome mapping of this gene by fluorescence in situ hybridization (FISH) using a genomic DNA fragment as a probe. The PHBP gene (HABP2) was located on chromosome 10q25-q26.

Cloning of the cDNA for a mouse homologue of human PHBP: a novel hyaluronan-binding protein.

The cDNA which encodes the mouse counterpart of human plasma hyaluronan-binding protein (PHBP) was isolated and characterized. The clone contained an insert of 2153 bp, which contained the 1674-bp open reading frame coding for a polypeptide of 558 amino acid residues. The amino acid sequence of mouse PHBP predicted from the nucleotide sequence of cDNA shows reasonable homology to that of human PHBP. Like human PHBP, the amino acid sequence predicted from the nucleotide sequence of mouse PHBP cDNA exhibited significant homology to that of human hepatocyte growth factor activator (HGFA).