Purification, cloning, and molecular characterization of a high molecular weight hemorrhagic metalloprotease, jararhagin, from Bothrops jararaca venom. Insights into the disintegrin gene family.

A large hemorrhagin, jararhagin, has been cloned from a Bothrops jararaca venom gland cDNA expression library. The cDNA sequence predicts a 421-amino acid residue molecule with strong amino acid sequence homology and similar domain structure to HR1B, a high molecular weight hemorrhagic metalloprotease isolated from Trimeresurus flavoviridis (Habu) venom. Like HR1B, jararhagin contains enzyme, disintegrin, and cysteine-rich carboxyl-terminal regions. In the disintegrin region, the Arg-Gly-Asp sequence is replaced by Glu-Cys-Asp, as found in non-Arg-Gly-Asp disintegrin regions of HR1B and a guinea pig sperm fusion protein PH-30 beta. The cDNA sequence of jararhagin predicts a precursor protein (proprotein) with striking similarity to cryptic regions in precursors of the disintegrin peptides trigramin and rhodostomin. Comparison of jararhagin with disintegrin precursors highlights the modular arrangement of proprotein, metalloprotease, and disintegrin domains in the metalloprotease/disintegrin family and provides an insight into their biosynthesis and evolution.     

新城疫病毒F蛋白中两段七肽重复序列的克隆和表达

从新城疫病毒 (NDV)中国强毒株F4 8E9和弱毒株长春株F蛋白的cDNA中亚克隆出两段七肽重复序列(HeptadRepeatRegion ,HR1,HR2 ) ,将HR1和HR2分别插入表达载体pGEX 6p 1,在大肠杆菌BL2 1(DE3 )中表达 ,将与载体中的GST(GlutathioneS Trasferase)融合表达的可溶性融合蛋白用GST亲和层析柱纯化。纯化的融合蛋白用蛋白酶酶切后 ,先用GST亲和层析柱除去GST ,再加热进一步纯化。纯化的HR1和HR2质谱分析其分子量 ,结果表明 ,强株的HR1和HR2的分子量分别为 7 10 3kD和 6 3 0 1kD ,弱株的HR1和HR2的分子量分别为 7 10 7kD和6 3 0 9kD ,强弱株HR1和HR2的分子量都基本一致。本工作为研究HR1、HR2的结构以及它们在NDV与宿主细胞融合中的作用奠定了基础。     

Isolation and characterization of pepsinogen from Trimeresurus flavoviridis (Habu snake)

Pepsinogen was isolated from the gastric mucosa of Trimeresurus flavoviridis (Habu snake) by DEAE-cellulose and DEAE-Sepharose ion-exchange chromatographies, and Sephacryl S-200 gel-chromatography. The yield calculated from the crude extract was 29% with 6.2-fold purification. The purified pepsinogen gave a single band on both native- and SDS-PAGE. As no other active enzyme was detected on the chromatographies, it was concluded that the Habu snake has one major pepsinogen. The molecular mass of the pepsinogen was estimated to be 38 kDa by SDS-PAGE. The sequence of the N-terminal 26 amino acid residues was determined and compared with those of other pepsinogens. The N-terminal structure of Habu snake pepsinogen was more homologous with those of mammalian pepsinogens C than those of mammalian pepsinogens A. The pepsinogen was rapidly converted to pepsin by way of an intermediate form induced by acidification. The optimum pH of Habu snake pepsin for bovine hemoglobin was 1.5-2.0, and it retained full activity at pH 6.2 and 30 degrees C on incubation for 30 min. The optimum temperature for the snake pepsin was 50 degrees C and it was stable at 40 degrees C on incubation for 10 min. The proteolytic activity of the pepsin toward bovine hemoglobin was about two times higher than that of porcine pepsin A, however, the activity toward oxidized bovine insulin B-chain was lower than that of porcine pepsin A, and it did not hydrolyze oligopeptides. The specificity for oxidized bovine insulin B-chain of the pepsin was different from that of porcine pepsin A. Habu snake pepsin was inhibited by pepstatin A but not by serine, cysteine, or metallo protease inhibitors.     

Sequence of a cDNA clone encoding the zinc metalloproteinase hemorrhagic toxin e from Crotalus atrox: evidence for signal, zymogen, and disintegrin-like structures.

The sequence of two overlapping cDNA clones for the zinc metalloproteinase hemorrhagic toxin e (also known as atrolysin e, EC 3.4.24.44) from the venom gland of Crotalus atrox, the Western diamondback rattlesnake, is presented. The assembled cDNA sequence is 1975 nucleotides in length and encodes an open reading frame of 478 amino acids. The mature hemorrhagic toxin e protein as isolated from the crude venom has a molecular weight of approximately 24,000 and thus represents the processed product of this open reading frame. From the deduced amino acid sequence, it can be hypothesized that the enzyme is translated with a signal sequence of 18 amino acids, an amino-terminal propeptide of 169 amino acids, a central hemorrhagic proteinase domain of 202 amino acids, and a carboxy-terminal sequence of 89 amino acids. The propeptide has a short region similar to the region involved in the activation of matrix metalloproteinase zymogens. The proteinase domain is similar to other snake venom metalloproteinases, with over 57% identity to the low molecular weight proteinases HR2a and H2-proteinase from the Habu snake Trimeresurus flavoviridis. The carboxy-terminal region, which is not observed in the mature protein, strongly resembles the protein sequence immediately following the proteinase domain of HR1B (a high molecular weight hemorrhagic proteinase from the venom of T. flavoviridis) and the members of a different family of snake venom polypeptides known for their platelet aggregation inhibitory activity, the disintegrins. The cDNA sequence bears striking similarity to a previously reported sequence for a disintegrin cDNA. This report is evidence that this subfamily of venom metalloproteinases is synthesized in a proenzyme form which must be proteolytically activated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid sequence of Trimeresurus flavoviridis phospholipase A2

The amino acid sequence of phospholipase A2 from the venom of Trimeresurus flavoviridis (the Habu snake) was determined. The enzyme subunit has a molecular weight of 13,764 and consists of a single polypeptide chain of 122 amino acids and seven disulfide bonds. The fragmentation was conducted by digesting the reduced and S-carboxymethylated derivative of the protein with Achromobacter protease I, chymotrypsin, and trypsin, respectively. Achromobacter protease I peptides were used for alignment and to establish overlaps over chymotryptic and tryptic peptides. The automated Edman degradation of the S-carboxymethylated protein, which was extended to the N-terminal 30 amino acid residues, supplemented the deletions found with the enzymatic peptides alone. T. flavoviridis phospholipase A2 was found to be highly (65-67%) homologous in sequence to the enzymes from T. okinavensis, Crotalus adamanteus, and Crotalus atrox (viperid family) and less (35-44%) homologous to those from elapid snakes and mammalian pancreas. The T. flavoviridis enzyme appears to be similar in secondary structure composition to the C. atrox enzyme.     

Purification and characterization of a coagulant enzyme from Trimeresurus flavoviridis venom

An enzyme bearing thrombin-like specificity has been purified to homogeneity from the venom of Trimeresurus flavoviridis (the Habu snake). The enzyme is a monomer with a molecular weight of 23,500 as determined by analytical gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein contains approximately 210 amino acid residues and has a relatively high content of aspartic acid and glutamic acid. The isoelectric point was 4.8 and the extinction coefficient at 280 nm for a 1% solution was 11.5. The enzyme acted directly on fibrinogen to form a fibrin clot with 2.0 NIH units. Analysis by high performance liquid chromatography of enzyme-treated fibrinogen revealed the release of a peptide identical in composition to thrombin-induced fibrinopeptide A, but no peptide corresponding to fibrinopeptide B was detected. The enzyme showed esterase and amidase activities on synthetic substrates containing arginine. The enzyme exhibited higher activity toward tosyl-L-arginine methyl ester (TAME) but 6-times lower activity toward benzoyl-L-arginine p-nitroanilide when compared with bovin thrombin. The esterase activity was inhibited by diisopropylfluorophosphate and at a slower rate by phenylmethanesulfonyl fluoride, but was least affected by tosyl-L-lysine chloromethyl ketone, showing that the enzyme is a serine protease like thrombin. The enzyme showed a bell-shaped pH dependence of kcat/Km for hydrolysis of TAME, with a maximum around pH 8.5.     

Serotriflin, a CRISP family protein with binding affinity for small serum protein-2 in snake serum

Habu (Trimeresurus flavoviridis) serum contains 3 small serum proteins (SSP-1, SSP-2, and SSP-3) with molecular masses of 6.5 to 10 kDa. Gel filtration analysis showed that all the SSPs exist in high molecular mass forms of approximately 60 kDa in the serum. Ultrafiltration of Habu serum showed that SSPs dissociated from the complex below a pH of 4. An SSP-binding protein was purified from Habu serum by gel filtration, ion exchange, and reverse-phase HPLC. N-terminal sequencing yielded a 39-amino acid sequence, similar to the N-terminal region of triflin, which is a snake venom-derived Ca2+ channel blocker that suppresses smooth muscle contraction. The amino acid sequence of this protein, termed serotriflin, was established by peptide analysis and cDNA cloning. Serotriflin is a glycosylated protein and consists of 221 amino acids. Among the 3 SSPs, only SSP-2 formed a noncovalent complex with serotriflin. It was bound to triflin and serotriflin with high affinity, as evidenced by surface plasmon resonance. SSP-2 is considered to be a protein that prevents self injury by accidental leaking of venom into the blood.     

The complete amino acid sequence of the high molecular mass hemorrhagic protein HR1B isolated from the venom of Trimeresurus flavoviridis

Hemorrhage is a common occurrence in a victim bitten by crotalid and viperid snakes, and hemorrhagic components in these various venoms have been isolated and characterized. Previously, we have shown that a low molecular weight hemorrhagic protein (HR2a, 202 amino acid residues) isolated from the venom of Trimeresurus flavoviridis is a member of a new subfamily of metalloproteinases. We now report the complete amino acid sequence of a high molecular mass hemorrhagic protein isolated from the same venom. This protein, HR1B, is a mosaic protein composed of 416 residues containing four asparagine-linked oligosaccharide chains. The amino-terminal half (residues 1-203) of HR1B contains a metalloproteinase domain, the sequence of which is 62% identical to that of HR2a and 52% identical to that of hemorrhagic toxin d isolated from Crotalus atrox venom. The most interesting finding is that the middle region (residues 204-300) of HR1B shows a striking similarity to disintegrins, Arg-Gly-Asp-containing platelet aggregation inhibitors, recently found in several viper venoms. Interestingly, however, this region of HR1B does not contain the Arg-Gly-Asp sequence which is known to be a putative binding site in the disintegrins for the platelet fibrinogen receptor, the glycoprotein IIb-IIIa complex. We also found that the carboxyl-terminal region (residues 213-336) of the middle part of HR1B shows 30% identity to residues 1543-1656 of von Willebrand factor and that the remaining region at the carboxyl-terminal end is unique and has a cysteine-rich sequence. These results suggest that the middle portion of HR1B, which shows structural similarities to the disintegrins and von Willebrand factor, may be important in synergistically stimulating hemorrhagic activity in the NH2-terminal metalloproteinase domain.     

Amino acid sequences of the two subunits of a phospholipase A2 inhibitor from the blood plasma of Trimeresurus flavoviridis. Sequence homologies with pulmonary surfactant apoprotein and animal lectins

Phospholipase A2 inhibitor (PLI), purified from the blood plasma of the Habu snake (Trimeresurus flavoviridis), was separated into two distinct subunits, PLI-A and PLI-B. These subunits were shown to be glycoproteins with molecular weights of around 21,000-22,000. When they were deglycosylated chemically with trifluoromethanesulfonic acid, the molecular weights were found to be 17,000. Their amino acid sequences were determined by alignment of peptides obtained by lysyl endopeptidase digestion and Staphylococcus aureus V8 protease digestion. PLI-A and PLI-B were each composed of 147 amino acid residues with one residue, Asn103, being for N-linked glycosylation, and the molecular weights of their protein portions were calculated to be 16,368 and 16,408, respectively. Each subunit contained four cysteine residues, all of which exist in disulfide linkages (Cys64-Cys141 and Cys119-Cys133). The sequences of PLI-A and PLI-B showed 89.9% homology to each other. When the sequences were compared with those of lipocortins, no significant homologies were detected. But the sequences were significantly homologous to those of COOH-terminal carbohydrate recognition portions of pulmonary surfactant apoprotein and animal lectins.     

An uniquely purified HCV NS3 protease and NS4A(21-34) peptide form a highly active serine protease complex in peptide hydrolysis.

The N-terminal domain of the hepatitis C virus (HCV) polyprotein containing the NS3 protease (residues 1027 to 1206) was expressed in Escherichia coli as a soluble protein under the control of the T7 promoter. The enzyme has been purified to homogeneity with cation exchange (SP-Sepharose HR) and heparin affinity chromatography in the absence of any detergent. The purified enzyme preparation was soluble and remained stable in solution for several weeks at 4 degrees C. The proteolytic activity of the purified enzyme was examined, also in the absence of detergents, using a peptide mimicking the NS4A/4B cleavage site of the HCV polyprotein. Hydrolysis of this substrate at the expected Cys-Ala scissile bond was catalyzed by the recombinant protease with a pseudo second-order rate constant (k(cat)/K(M)) of 205 and 196,000 M(-1) s(-1), respectively, in the absence and presence of a central hydrophobic region (sequence represented by residues 21 to 34) of the NS4A protein. The rate constant in the presence of NS4A peptide cofactor was two orders of magnitude greater than reported previously for the NS3 protease domain. A significantly higher activity of the NS3 protease-NS4A cofactor complex was also observed with a substrate mimicking the NS4B/5A site (k(cat)/K(M) of 5180 +/- 670 M(-1) s(-1)). Finally, the optimal formation of a complex between the NS3 protease domain and the cofactor NS4A was critical for the high proteolytic activity observed.     

Abrogation of hybrid resistance to bone marrow engraftment by graft-vs-host-induced immune deficiency

Lethally irradiated F1 mice, heterozygous at the hematopoietic histocompatibility locus Hh-1, which is linked with H-2Db, reject bone marrow grafts from H-2b parents. This hybrid resistance (HR) is reduced by prior injection of H-2b parental spleen cells. Because injection of parental spleen cells produces a profound suppression of F1 immune functions, we investigated whether parental-induced abrogation of HR was due to graft-vs-host-induced immune deficiency (GVHID). HR was assessed by quantifying engraftment of H-2b bone marrow in F1 mice with the use of splenic [125I]IUdR uptake; GVHID, by the ability of F1 spleen cells to generate cytotoxic T lymphocytes (CTL) in vitro. We observed a correlation in the time course and spleen cell dose dependence between loss of HR and GVHID. Both GVHID and loss of HR were dependent on injection of parental T cells; nude or T-depleted spleen cells were ineffective. The injection of B10 recombinant congenic spleens into (B10 X B10.A)F1 mice, before grafting with B10 marrow, demonstrated that only those disparities in major histocompatibility antigens that generated GVH would result in loss of HR. Thus, spleens from (B10 X B10.A(2R]F1 mice (Class I disparity only) did not induce GVHID or affect HR, whereas (B10 X B10.A(5R))F1 spleens (Class I and II disparity) abrogated CTL generation and HR completely. GVHID produced by a class II only disparity, as in (B10 X B10.A(5R))F1 spleens injected into (B6bm12 X B10.A(5R))F1 mice, was also sufficient to markedly reduce HR to B10 bone marrow. This evidence that GVHID can modulate hematopoietic graft rejection may be relevant to the mechanisms of natural resistance to marrow grafts in man.     

Interaction of Peptides with Sequences from the Newcastle Disease Virus Fusion Protein Heptad Repeat Regions

Typical of many viral fusion proteins, the sequence of the Newcastle disease virus (NDV) fusion protein has several heptad repeat regions. One, HR1, is located just carboxyl terminal to the fusion peptide, while the other, HR2, is located adjacent to the transmembrane domain. The structure and function of a synthetic peptide with a sequence from the region of the NDV HR1 region (amino acids 150 to 173) were characterized. The peptide inhibited fusion with a half-maximal concentration of approximately 2 microM; however, inhibition was observed only if the peptide was added prior to protease activation of the fusion protein. This inhibition was virus specific since the peptide had minimal effect on fusion directed by the Sendai virus glycoproteins. To explore the mechanism of action, the potential HR1 peptide interaction with a previously characterized fusion inhibitory peptide with a sequence from the HR2 domain (J. K. Young, R. P. Hicks, G. E. Wright, and T. G. Morrison, Virology 238:291-304, 1997) was characterized. The results demonstrated an interaction between the two peptides both functionally and directly. First, while the individual peptides each inhibit fusion, equimolar mixtures of the two peptides had minimal effect on fusion, suggesting that the two peptides form a complex preventing their interaction with a target protein. Second, an HR2 peptide covalently linked with biotin was found to bind specifically to HR1 peptide in a Western blot. The structure of the HR1 peptide was analyzed by nuclear magnetic resonance spectroscopy and found to be an alpha helix.     

Protease B of the lysosomelike vacuole of the yeast Saccharomyces cerevisiae is homologous to the subtilisin family of serine proteases.

The PRB1 gene of Saccharomyces cerevisiae encodes the vacuolar endoprotease protease B. We have determined the DNA sequence of the PRB1 gene and the amino acid sequence of the amino terminus of mature protease B. The deduced amino acid sequence of this serine protease shares extensive homology with those of subtilisin, proteinase K, and related proteases. The open reading frame of PRB1 consists of 635 codons and, therefore, encodes a very large protein (molecular weight, greater than 69,000) relative to the observed size of mature protease B (molecular weight, 33,000). Examination of the gene sequence, the determined amino-terminal sequence, and empirical molecular weight determinations suggests that the preproenzyme must be processed at both amino and carboxy termini and that asparagine-linked glycosylation occurs at an unusual tripeptide acceptor sequence.     

Primary structure of the antihemorrhagic factor in serum of the Japanese Habu: a snake venom metalloproteinase inhibitor with a double-headed cystatin domain.

The complete amino acid sequence of an antihemorrhagic factor, HSF, in the serum of the Japanese Habu snake, Trimeresurus flavoviridis, has been determined. The protein is composed of 323 amino acid residues and contains three asparagine-linked oligosaccharide chains at positions 123, 185, and 263. The molecule contains two copies of the cystatin domain in the N-terminal portion up to position 240, and these domains show a remarkable sequence homology (about 50%) to those of plasma glycoproteins such as alpha 2-HS (human) and fetuin (bovine) and to a lesser extent to that of HRG (human). The amino acid sequence of the noncystatin region towards the C-terminus is unique, showing no significant homology with those of the corresponding regions of alpha 2-HS and fetuin. In spite of the presence of cystatin domains, HSF does not inhibit cysteine proteinases such as papain and cathepsin B but does inhibit several metalloproteases in Habu venom. The results suggest that HSF is the first protein found to be functionally related to metalloproteinase inhibitors among the structurally homologous proteins with a double-headed cystatin domain, and is a member of a novel family (family 4) with divergent functions of the cystatin superfamily proteinase inhibitors. Although HSF possesses similar physicochemical properties to those of oprin, a snake venom metalloproteinase inhibitor with antihemorrhagic activity isolated from opossum serum [Catanese & Kress (1992) Biochemistry 31, 410-418], its primary structure is strikingly different from that of oprin.     

Mutational analysis of different regions in the coxsackievirus 2B protein: requirements for homo-multimerization, membrane permeabilization, subcellular localization, and virus replication

The coxsackievirus 2B protein is a small hydrophobic protein (99 amino acids) that increases host cell membrane permeability, possibly by forming homo-multimers that build membrane-integral pores. Previously, we defined the functional role of the two hydrophobic regions HR1 and HR2. Here, we investigated the importance of regions outside HR1 and HR2 for multimerization, increasing membrane permeability, subcellular localization, and virus replication through analysis of linker insertion and substitution mutants. From these studies, the following conclusions could be drawn. (i) The hydrophilic region ((58)RNHDD(62)) between HR1 and HR2 is critical for multimerization and increasing membrane permeability. Substitution analysis of Asn(61) and Asn(62) demonstrated the preference for short polar side chains (Asp, Asn), residues that are often present in turns, over long polar side chains (Glu, Gln). This finding supports the idea that the hydrophilic region is involved in pore formation by facilitating a turn between HR1 and HR2 to reverse chain direction. (ii) Studies undertaken to define the downstream boundary of HR2 demonstrated that the aromatic residues Trp(80) and Trp(82), but not the positively charged residues Arg(81), Lys(84), and Lys(86) are important for increasing membrane permeability. (iii) The N terminus is not required for multimerization but does contribute to the membrane-active character of 2B. (iv) The subcellular localization of 2B does not rely on regions outside HR1 and HR2 and does not require multimerization. (v) Virus replication requires both the membrane-active character and an additional function of 2B that is not connected to this activity.     

Cys-His proteases are among the wired proteins of the cell

Integrated cell protein degradation can be paced by the transfer of reductive energy, as revealed by experimental agents of informative actions. The peptidolytic pair of Cys-His proteases can undergo oxidative reactions to inactive derivatives and inhibitory metal binding. Proton-dependent ionizations can modify ongoing activity. If the reaction rate of a Cys-His protease were found responsive to the ranges of metal/redox/proton factors regulated within the cell, then these factors might serve to link the peptidolytic reaction rate to cell controls. Here, cathepsin B (cat B) was found to be inhibited by Zn2+, Fe3+, and Cu2+ (1-50 microM) under excess GSH or DTT protease activators (6 mM). Under DTT or GSH (6 mM) the initial inhibitory action of Zn2+ is stable indefinitely; however, the inhibitory actions of Fe3+ and Cu2+ are reversed over approximately 1h. The 12-14 min half time of reversal of initial protease inhibition is correlated with the measured reduction of Fe3+ to Fe2+ by DTT or GSH (pH 5.5 or 6.5). Endogenous Fe2+ concentrations (100 microM) inhibit cat B only marginally. However, the inhibitory threshold of several microM Fe3+ is only a few percent oxidation of the endogenous pool. Without metals cat B reaction is reportedly proportional to GSH concentration, and is inhibited by increasing GSSG/GSH redox ratio. Following activation with GSH, cat B can be influenced by Fe3+/Fe2+, Cu2+/Cu+, and GSSG/GSH ratios and concentrations. Results are interpreted in relation to properties of the thiolate-imidazolium pair as illustrated by Dock modeling of their shared Fe3+ binding. It is proposed that the interaction of Cys-His with 1 electron transition between Fe2+ and Fe3+ serves as a sensor, signal integrator and switch wiring cat B reaction rate to the transfer of reductive energy in the presence of excess GSH. Speciated metals might also serve among electron acceptors transferring from reduced protease to oxygen. Results provide a model for pharmacologic redox switching of protease functions with metal-interactive drugs, and other nano-technology engineering.     

Characterization of the novel cardiolipin binding regions identified on the protease and lipid activated PKC‐related kinase 1

Protein kinase C‐related kinase 1 (PRK1) or PKN is a protease and lipid activated protein kinase that acted downstream of the RhoA or Rac1 pathway. PRK1 comprises a unique regulatory domain and a PKC homologous kinase domain. The regulatory domain of PRK1 consists of homologous region ?1 (HR1) and ?2 (HR2). PRK1‐(HR1) features a pseudosubstrate motif that overlapped with the putative cardiolipin and known RhoA binding sites. In fact, cardiolipin is the most potent lipid activator for PRK1 in respect of its either auto‐ or substrate phosphorylation activity. This study was thus aimed to characterize the binding region(s) of cardiolipin that was previously suggested for the regulatory domain of PRK1. The principal findings of this work established (i) PRK1‐(HR1) folded into an active conformation where high affinity binding sites (mainly located in HR1a subdomain) were accessible for cardiolipin binding to protect against limited Lys‐C digestion, (ii) the binding nature between acidic phospholipids and PRK1 (HR1) involved both polar and nonpolar components consistent with the amphipathic nature of the known cardiolipin‐binding motifs, (iii) identification of the molecule masses of the Lys‐C fragments of PRK1‐(HR1) complexed with cardiolipin molecule, and (iv) appreciable reductions in the secondary structural contents at 222 nm measured by circular dichroism analyses demonstrated the binding of cardiolipin elicited the disruptive effect that was most evident among all phospholipids tested, suggestive of a functional correlation between the extents of helical disruption and PRK1 activation.