A snake venom metalloproteinase that inhibited cell proliferation and induced morphological changes of ECV304 cells.

TSV-DM, a basic metalloproteinase with a molecular weight of 110kDa, was purified from Trimeresurus stejnegeri venom. TSV-DM degraded the Aalpha chain of fibrinogen more rapidly than the Bbeta chain in a dose dependent manner. The cDNA of TSV-DM encoded a polypeptide of 622 amino acid residues, which comprises a signal peptide, proprotein, metalloproteinase domain, spacer, disintegrin-like domain and cysteine-rich domain. The protein sequence deduced from cDNA was confirmed by peptide mass fingerprinting analysis. It is highly homologous to the members of subclass P-IIIb snake venom metalloproteinase, which comprises vascular apoptosis-inducing proteins. TSV-DM inhibited cell proliferation and induced cell morphologic changes transiently of ECV304 cells. However, DNA fragmentation and DNA content analysis demonstrated that this metalloproteinase could not induce ECV304 cells apoptosis.     

Molecular cloning and sequence analysis of cDNA encoding flavoridin, a disintegrin from the venom of Trimeresurus flavoviridis.

We isolated a cDNA of 2001bp encoding the full-length precursor of flavoridin, which is one of the four disintegrins in the venom of Trimeresurus flavoviridis, and analyzed the cDNA nucleotide sequence. The deduced amino acid sequence of the open reading frame consisted of a pro-domain (190 residues), a metalloproteinase domain (205 residues), a spacer domain (18 residues) and a disintegrin (flavoridin) domain (70 residues), thus indicating that the flavoridin precursor belongs to the P-II class of snake venom metalloproteinases. The unknown metalloproteinase domain shared strong sequence similarity with HR2a (71.2% identity) and H(2)-proteinase (74.1% identity), a low molecular mass hemorrhagic metalloproteinase and a non-hemorrhagic metalloproteinase in the same snake venom, respectively.     

Molecular diversity of disintegrin-like domains within metalloproteinase precursors of Bothrops jararaca.

Disintegrins are small peptides isolated from the venom of several snake families which act as integrin-antagonists or agonists, interacting with a variety of biological processes mediated by integrins. In this work we describe five new disintegrin-like domains within metalloproteinase precursor sequences, obtained from a Bothrops jararaca venom gland cDNA library. Among the new disintegrin-like domains, four were contained in PIII metalloproteinase precursors, with three of them presenting ECD-motifs and one presenting a new KCD-motif. Moreover, we found three disintegrin-like domains within PII metalloproteinase precursors. Two of them are similar to the already described disintegrins jarastatin and jararacin. The third molecule is unusual, presenting some typical PIII metalloproteinase characteristics but lacking the cysteine-rich domain being, thus, classified as a PII metalloproteinase. Only few reports presented molecules with these characteristics. Sequence analysis suggests that these molecules are intermediate steps between the more ancient PIII and the more recent PII metalloproteinases. We also investigated disintegrin N-terminus diversity in B. jararaca crude venom by purifying jarastatin and jararacin and analyzing them by mass spectrometry.     

“Insularin, a disintegrin from Bothrops insularis venom: Inhibition of platelet aggregation and endothelial cell adhesion by the native and recombinant GST-insularin proteins”

Insularin (INS) was obtained from Bothrops insularis venom by reversed-phase highperformance liquid chromatography using a C₁₈ column and characterized as a disintegrin by peptide mass fingerprint and inhibition of ADP-induced platelet aggregation. A cDNA coding for P-II a metalloproteinase/disintegrin was cloned from a cDNA library from B. insularis venom glands. The deduced protein sequence possesses 73 amino acid residues, including the N-terminal, internal peptides of native insularin, the ARGDNP-sequence and 12 cysteines in a conserved alignment. This cDNA fragment was subcloned in the pGEX-4T-1 vector and expressed in a prokaryotic expression system as a fusion protein with glutathione S-transferase (GST-INS). Both native and recombinant insularin inhibited ADP-induced platelet aggregation and endothelial cells (HUVEC) adhesion with similar activities indicating that GST-INS folded correctly and preserved the integrin-binding loop. Insularin may be a tool in studies that involve platelets and endothelial cell adhesion dependent on alphaIIbeta3 and alphavbeta3 integrins.     

Purification, cloning and biological characterization of a novel disintegrin from Trimeresurus jerdonii venom.

A novel disintegrin, jerdonin, was purified from the Trimeresurus jerdonii venom by means of gel filtration and reverse phase high pressure liquid chromatography. Its coding cDNA was also isolated from the venom gland. The jerdonin coding cDNA is part of a precursor composed of proprotein, metalloproteinase, and disintegrin domains. From the deduced amino acid sequence, jerdonin is composed of 71 amino acid residues including 12 cysteines and the tripeptide sequence Arg-Gly-Asp (RGD), a well-known characteristic of the disintegrin family. Molecular mass of jerdonin was determined to be 7483Da by matrix-assisted laser desorption ionization time of flight mass spectrometry. Jerdonin inhibited ADP- and collagen-induced human platelet aggregation with IC(50) of 220 and 240 nM, respectively. In vivo, jerdonin inhibited the growth of subcutaneously inoculated B16 solid tumor in C57BL/6 mice and improved the survival time of the tumor-bearing mice.     

Primary structure of brevilysin L4, an enzymatically active fragment of a disintegrin precursor from Gloydius halys brevicaudus venom.

Brevilysin L4 (L4) is a non-hemorrhagic P-I class metalloprotease (MP) isolated from Gloydius halys brevicaudus venom. Its complete amino acid sequence has been determined. L4 is a single-chain polypeptide and highly homologous to those of other snake venom MPs. A zinc-binding motif, HExxHxxGxxH, is located at residues 142-152. A characteristic feature of L4 is the presence of a spacer sequence (LRTDTVS) at the C-terminal that links metalloprotease and disintegrin domains and is usually removed by post-translational proteolysis, suggesting that L4 is expressed together with a spacer region and a disintegrin domain at the C-terminal. The nucleotide sequence of a cDNA clone encoding L4 has revealed that L4 is a disintegrin precursor and produced as a P-II class MP. The disintegrin coded after L4 sequence was brevicaudin 1, a disintegrin previously isolated from the same venom. P-II class MPs have been suspected to undergo autoproteolysis to release disintegrins. Although being P-I class MP, L4 itself autocatalytically degrades with a half-life of 30min at pH 8.5 and 37 degrees C in the absence of Ca(2+). Sequence analysis of several fragment peptides produced during the autolysis of L4 indicated that more than 40 peptide bonds were split, and the cleavages of Ser(60)-Asn(61), Thr(99)-Ala(100), and Phe(103)-Asp(104) bonds may trigger the autoproteolysis. Addition of Ca(2+) completely suppressed the cleavage of these particular bonds, resulting in a marked prevention of autoproteolysis. Thus, L4 provides a good model for the investigation of autolysis of some MPs.     

Molecular cloning of albolatin, a novel snake venom metalloprotease from green pit viper (Trimeresurus albolabris), and expression of its disintegrin domain

Disintegrins are snake venom-derived, RGD- or KGD-containing peptides that can inhibit integrin-mediated platelet aggregation and cell–matix interactions. The aim of this study is to analyze the full-length cDNA sequence of a snake venom metalloprotease (SVMP) from green pit viper (Trimeresurus albolabris) venom and characterize functions of its disintegrin domain on human platelets. From the primary cDNA library of venom glands, a partial sequence of a novel SVMP (Albolatin) was obtained. Using the 5′-RACE, the 2040 bp full-length sequence of albolatin mRNA was derived. The deduced amino acid sequence revealed a type P-II SVMP of 484 amino acid residues comprising a signal region, pro-peptide, inactive metalloprotease domain and a disintegrin domain. It showed 85% amino acid identical to Trimeresurus jerdonii jerdonitin and 81% to Gloydius halys agkistin. Sequence alignment revealed that all cysteines were conserved except for an extra cysteine in the protease domain of albolatin. The disintegrin domain of albolatin, which comprised 76 amino acids with a KGDW sequence, was expressed in Pichia pastoris with the yield of 3.3 mg/L of culture medium. The molecular weights were 11 kDa in reduced and 22 kDa in non-reduced states indicating a homodimer. It can inhibit collagen-induced platelet aggregation with IC₅₀ of 976 nM and, therefore, should be investigated for a potential to be a novel therapeutic agent.     

Isolation and cloning of a metalloproteinase from king cobra snake venom.

A 50 kDa fibrinogenolytic protease, ohagin, from the venom of Ophiophagus hannah was isolated by a combination of gel filtration, ion-exchange and heparin affinity chromatography. Ohagin specifically degraded the alpha-chain of human fibrinogen and the proteolytic activity was completely abolished by EDTA, but not by PMSF, suggesting it is a metalloproteinase. It dose-dependently inhibited platelet aggregation induced by ADP, TMVA and stejnulxin. The full sequence of ohagin was deduced by cDNA cloning and confirmed by protein sequencing and peptide mass fingerprinting. The full-length cDNA sequence of ohagin encodes an open reading frame of 611 amino acids that includes signal peptide, proprotein and mature protein comprising metalloproteinase, disintegrin-like and cysteine-rich domains, suggesting it belongs to P-III class metalloproteinase. In addition, P-III class metalloproteinases from the venom glands of Naja atra, Bungarus multicinctus and Bungarus fasciatus were also cloned in this study. Sequence analysis and phylogenetic analysis indicated that metalloproteinases from elapid snake venoms form a new subgroup of P-III SVMPs.     

cDNA cloning, expression and fibrin(ogen)olytic activity of two low-molecular weight snake venom metalloproteinases

Two cDNA clones, AplVMP1 and AplVMP2, were isolated from a snake (Agkistrodon piscivorus leucostoma) venom gland cDNA library. The full-length cDNA sequence of AplVMP1 with a calculated molecular mass of 46.61 kDa is 1233 bp in length. AplVMP1 encodes PI class metalloproteinase with an open reading frame of 411 amino acid residues that includes signal peptide, pro-domain and metalloproteinase domains. The full-length cDNA of the AplVMP2 (1371 bp) has a calculated molecular mass of 51.16 kDa and encodes PII class metalloproteinase. The open reading frame of AplVMP2 with a 457 amino acid residues is composed of signal peptide, pro-domain, metalloproteinase and disintegrin domains. AplVMP1 and AplVMP2 showed 85% and 93% amino acid identical to PI class enzyme Agkistrodon contortrix laticinctus ACLPREF and PII class enzyme Agkistrodon piscivorus piscivorus piscivostatin, respectively. When expressed in Escherichia coli, most of recombinant proteins of AplVMP1 and AplVMP2 were in insoluble inclusion bodies, with soluble yields of 0.7 mg/l and 0.4 mg/l bacterial culture, respectively. Both affinity purified recombinant proteins show proteolytic activity on fibrinogen, although having an activity lower than that of crude A. p. leucostoma venom. Proteolytic activities of AplVMP1 and AplVMP2 were completely abolished after incubation with a final concentration of 100 μM of EDTA or 1,10-phenanthroline. Both AplVMP1 and AplVMP2 were active in a fibrin-agarose plate but devoid of hemorrhagic activity when injected (up to 50 μg) subcutaneously into mice, and had no capacity to inhibit platelet aggregation.     

Fractionation of snake venom metalloproteinases by metal ion affinity: A purified cobra metalloproteinase, Nk, from Naja kaouthia binds Ni-agarose

Snake venom metalloproteinases represent unique probes for analyzing platelet adhesion receptors regulating hemostasis and thrombosis. Snake venom metalloproteinase-disintegrins consist of a propeptide domain, a catalytic domain containing a metal ion-coordination sequence (HEXXHXXGXXH), a disintegrin domain, and a Cys-rich domain. Here, we investigate whether metal ion-affinity chromatography may be used to fractionate venom metalloproteinases based on the metal ion-coordination motif. First, we showed that a purified cobra metalloproteinase, Nk, from Naja kaouthia bound Ni²⁺-agarose, and was eluted by 10 mM imidazole, confirming the validity of the approach. Nk cleaved the platelet von Willebrand factor (VWF) receptor, glycoprotein (GP)Ib, with similar activity to the previously reported cobra metalloproteinase, mocarhagin, as shown by EDTA-inhibitable Nk-dependent proteolysis of a purified GPIb extracellular fragment (glycocalicin), and inhibition of ¹²⁵I-VWF binding to GPIb on washed human or canine platelets. Second, crude venom from the viper, Trimeresurus albolabris, was fractionated on Ni²⁺-agarose. Samples of flow-through, wash, and imidazole-eluted (0–30 mM gradient) fractions were analyzed by (i) SDS-polyacrylamide gel electrophoresis, (ii) immunoblotting with a rabbit anti-mocarhagin antibody, and (iii) assessing metalloproteinase activity using human fibrinogen as substrate. The combined results support the general concept of using Ni²⁺-agarose to fractionate snake venom metalloproteinases.     

Molecular cloning and expression of structural domains of bothropasin, a P-III metalloproteinase from the venom of Bothrops jararaca.

Mature P-III snake metalloproteinases are soluble venom components which belong to the Reprolysin sub family and are structurally related to the mammalian membrane-bound A Disintegrin And Metalloproteinase (ADAMs). Here we present the molecular cloning of bothropasin, a metalloproteinase with hemorrhagic and myonecrotic activities isolated from the venom of Bothrops jararaca. The full-length cDNA encoding the bothropasin precursor was cloned by immunoscreening and its authenticity was confirmed by the amino acid sequence of internal fragments obtained from an autolyzed sample of native bothropasin. The predicted bothropasin precursor is comprised of the elements of a P-III venom metalloproteinase: signal sequence, pro-, metalloproteinase, disintegrin-like and cysteine-rich domains. In the autolysis process of native bothropasin, the disintegrin-like and cysteine-rich domains remained intact while the metalloproteinase domain was cleaved at different sites. The attempts made to obtain the recombinant precursor form of bothropasin using bacterial, yeast and mammalian cell expression systems failed to produce it in an amount sufficient to analyze the activation of the zymogen. Nevertheless, the study of the expression of the individual domains of bothropasin using a bacterial system resulted in the production of recombinant pro-and disintegrin-like+cysteine-rich domains but not the metalloproteinase domain. These results along with the autolysis pattern of the native protein suggest a role for the metalloproteinase domain in the structural stability of bothropasin.     

A novel metalloprotease from Vipera lebetina venom induces human endothelial cell apoptosis.

A novel endothelial cell apoptosis inducing metalloprotease (VLAIP) was found in the snake venom of Vipera lebetina. This metalloprotease is a heterodimeric glycoprotein with molecular mass of about 106 kDa. The protease hydrolyzes azocasein, fibrinogen and oxidized insulin B-chain. The enzyme readily hydrolyzes the Aalpha-chain and more slowly Bbeta-chain of fibrinogen. VLAIP does not cleave fibrin. The complete amino acid sequences of the two different monomers of VLAIP are deduced from the nucleotide sequences of cDNAs encoding these proteins. The full-length cDNA sequences of the VLAIP-A and VLAIP-B encode open reading frames of 616 and 614 amino acids that include signal peptide, propeptide and mature metalloproteinase with disintegrin-like and cysteine-rich domains. VLAIP belongs to the metalloprotease/disintegrin family of reprolysins and has high identity with the proteins that induce apoptosis of endothelial cells. Treatment of HUVEC cells with VLAIP induces changes in the attachment of cells to the substrate and causes cell death. We demonstrated that VLAIP inhibits endothelial cell adhesion to extracellular matrix proteins: fibrinogen, fibronectin, vitronectin, collagen I, and collagen IV. The induction of apoptosis by VLAIP was shown by means of a typical DNA fragmentation pattern of apoptotic cells as well as by monitoring phosphatidylserine externalization using annexin V-FITC staining and flow cytometric analysis.     

Characterization of a novel metalloproteinase in Duvernoy's gland of Rhabdophis tigrinus tigrinus

During the characterization of hemorrhagic factor in venom of Rhabdophis tigrinus tigrinus, so-called Yamakagashi in Japan, one of the Colubridae family, a novel metalloproteinase with molecular weight of 38 kDa in the Duvernoy's gland of Yamakagashi was identified by gelatin zymography and by monitoring its proteolytic activity using a fluorescence peptide substrate, MOCAc-PLGLA2pr(Dnp)AR-NH2, which was developed for measuring the well-known matrix metalloproteinase (MMP) activity. After purification by gel filtration HPLC and/or column switch HPLC system consisting of an affinity column, which was immobilized with a synthetic BS-10 peptide (MQKPRCGVPD) originating from propeptide domain of MMP-7 and a reversed-phase column, the N-terminal amino acid sequence of the 38 kDa metalloproteinase was identified as FNTFPGDLK which shared a high homology to Xenopus MMP-9. The 38 kDa metalloproteinase required Zn2+ and Ca2+ ions for its proteolytic activity. In addition, the proteolytic activity was almost completely inhibited by BS-10, a MMP inhibitor, but not by the serine proteinase inhibitors, cysteine proteinase inhibitors and aspartic proteinase inhibitors.Together these results demonstrated that the 38 kDa proteinase is a novel snake verom metalloproteinase (SVMP) containing HExGHxxGxxH motif which possesses high affinity to the BS-10 peptide, into its molecule, and the enzymatic properties are closed to that of MMPs. Based on the results obtained in the present study, we concluded that the 38 kDa metalloproteinase is a novel metalloproteinase whose activity may be regulated by the cysteine switch mechanism, and could be classified as one of the matrix metalloproteinases rather than snake venom metalloproteinases.     

Snake venom metalloproteinases: structure/function relationships studies using monoclonal antibodies.

Snake Venom Metalloproteinases (SVMPs) are synthesized as zymogens and undergo proteolytic processing resulting in a variety of multifunctional proteins. Jararhagin is a P-III SVMP, isolated from the venom of Bothrops jararaca, comprising metalloproteinase, disintegrin-like and cysteine-rich domains. The catalytic domain is responsible for the hemorrhagic activity. The disintegrin-like/cysteine-rich domains block alpha2beta1 integrin binding to collagen and apparently enhance the hemorrhagic activity of SVMPs. The relevance of disintegrin-like domain is described in this paper using a series of mouse anti-jararhagin monoclonal antibodies (MAJar 1-7). MAJar 3 was the only antibody able to completely neutralize jararhagin hemorrhagic activity. Neutralization of catalytic activity was partial by incubation with MAJar 1. MAJars 1 and 3 efficiently neutralized jararhagin binding to collagen with IC50 of 330 and 8.4 nM, respectively. MAJars 1 and 3 recognized the C-terminal portion of the disintegrin domain, which is apparently in conformational proximity with the catalytic domain according to additivity tests. These data suggest that disintegrin-like domain epitopes are in close contact with catalytic site or functionally modulate the expression of hemorrhagic activity in SVMPs.     

Identification of BmKAPi, a novel type of scorpion venom peptide with peculiar disulfide bridge pattern from Buthus martensii Karsch.

A novel cDNA sequence encoding a new type of scorpion venom peptide (BmKAPi) was first isolated from the venom gland of Buthus martensiiKarsch by cDNA library screening combined with 5′-race. The encoded precursor of BmKAPi consisted of 89 amino acid residues including a signal peptide of 24 residues, a putative mature peptide of 64 residues (BmKAPi) and an extra basic residue at the C-terminus which might be removed in the post-translational processing. BmKAPi is stabilized by five disulfide bridges, whereas all other disulfide-bridged scorpion toxins described are cross-linked by three or four disulfide bridges. It suggested the three-dimensinal scaffold of BmKAPi might be different from other scorpion toxins. The amino acid sequence of BmKAPi showed no homology with other scorpion venom peptides, but shared a little similarity with some anticoagulant peptides and proteinase inhibitors isolated from hookworm, honeybee or European frog, respectively. RT-PCR analysis showed that BmKAPi mRNA could be induced by venom extraction suggesting BmKAPi might be a component of scorpion venom. These results suggest that BmKAPi is a new type of scorpion venom peptide different from other described scorpion toxins in structural and functional aspects.     

Cloning of two novel P-III class metalloproteinases from Trimeresurus stejnegeri venom gland.

Hemorrhagic toxins are widely distributed in viperid and crotalid snake venoms. Envenomation of Trimeresurus stejnegeri, a member of Crotalidae family, caused potent systemic and local hemorrhage. Up to now, there is no report on hemorrhage toxins from this venom. In this work, we cloned two cDNAs of P-III metalloproteinase precursors, designated as stejnihagin-A and stejnihagin-B, respectively, from T. stejnegeri venom gland. Both cDNAs encode an opening reading frame of 600 amino acid residues, containing a signal sequence, a proprotein domain, a metalloproteinase domain, a disintegrin-like domain and a cystetine-rich domain. Sequence analysis suggested that these two sequences shared highest similarity to the hemorrhagic toxin HR1b from T. flavoviridis. Aligning the deduced mature protein sequences of stejnihagin-A and stejnihagin-B with other snake venom metalloproteinases (SVMPs), we observed that stejnihagin-A and stejnihagin-B, together with HR1b shared the common cysteinyl residue at the position 100 in the metalloproteinase domain. In combination with the phylogenetic analysis, we presumed that stejnihagin-A, stejnihagin-B and HR1b might constitute a novel subclass of P-III SVMPs, named P-IIIc.     

A preliminary investigation into the venom proteome of Macrovipera lebetina obtusa (Dwigubsky, 1832) from Southeastern Anatolia by MALDI-TOF mass spectrometry and comparison of venom protein profiles with Macrovipera lebetina lebetina (Linnaeus, 1758) from Cyprus by 2D-PAGE

We compared venoms of two subspecies of blunt-nosed viper Macrovipera lebetina (Linnaeus, 1758) from Southeastern Anatolia and Cyprus by two-dimensional gel electrophoresis (2D-PAGE). Additionally, peptide mass fingerprinting analysis was carried out using matrix-assisted laser desorption ionization/time of flight (MALDI-TOF) mass spectrometry in order to achieve preliminary protein identification from M. lebetina obtusa venom from Turkey. As a result of 2D-PAGE, statistical tests revealed some significant differences that can be considered as subspecies-specific biomarker candidates between two subspecies. Using bioinformatic analyses, proteins belonging to 11 families were identified from the venom of M. l. obtusa: phospholipase A₂, metalloproteinase, serin proteinase, disintegrin, cysteine-rich secretory protein, C-type lectin, vascular endothelial growth factor, nerve growth factor, hyaluronidase, l-amino acid oxidase, and trypsin inhibitor. Venom of M. lebetina was studied by 2D-PAGE for the first time in the literature, and also this is the first work aiming to determine regional variations of snake venoms by this method in Turkey and Cyprus. Our preliminary results show that snake venom research deserves more attention in Turkey as well as in the toxinology field in general.     

cDNA cloning of a novel P-I lebetase isoform Le-4.

In order to better understand the function of fibrinolytic enzyme lebetase isoforms and the synthesis of disintegrins we have isolated a cDNA encoding the most basic isoform (Le-4) from the cDNA library prepared from the poly(A)(+) RNA of the venomous gland of an individual Vipera lebetina snake. The truncated 5'-sequence of 1112 basepairs encodes the mature protein with 203 amino acid residues with calculated isoelectric point and size of 5.6 and 22,930 Da, respectively. Multiple comparison of the deduced amino acid sequence of the metalloprotease part of Le-4 is related to short reprolysins, identities were within the range of 60--87%. The two lebetase isoforms are synthesized in different way: Le-4 is synthesized with metalloprotease domain only; Le-3 is synthesized with metalloprotease and disintegrin-like domain and processed posttranslationally. The sequence of the disintegrin-like part of Le-3 is identical to A-chain of the heterodimeric disintegrin VLO5 from Vipera lebetina obtusa venom (Calvete et al., 2003).