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.     

Simplified procedures for the isolation of HF3, bothropasin, disintegrin-like/cysteine-rich protein and a novel P-I metalloproteinase from Bothrops jararaca venom

HF3 and bothropasin are P-III hemorrhagic snake venom metalloproteinases (SVMPs) of Bothrops jararaca. The DC protein is composed of the disintegrin-like/cysteine-rich domains derived from the autolysis of P-III SVMPs. Here we describe simplified procedures for the isolation of HF3, bothropasin, the DC protein, and BJ-PI, a novel P-I SVMP. The isolated proteins were identified by mass spectrometry. BJ-PI is a potent caseinolytic enzyme devoid of hemorrhagic activity. HF3, bothropasin and BJ-PI show distinct fibrinogenolytic activities.     

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.     

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.     

Phylogenetic conservation of a snake venom metalloproteinase epitope recognized by a monoclonal antibody that neutralizes hemorrhagic activity.

Snake venom metalloproteinases (SVMPs) are present in large quantities in venoms of viper snakes and also in some elapids. Jararhagin is a representative of a P-III multidomain hemorrhagic SVMP present in Bothrops jararaca venom. It is comprised of a catalytic, a disintegrin-like and a cysteine-rich domain. Seven anti-jararhagin monoclonal antibodies (MAJar 1-7) were produced, of which six reacted with the disintegrin domain. MAJar 3 recognized an epitope present at the C-terminal part of the disintegrin-like domain, and neutralized jararhagin-induced hemorrhage. In this study, we evaluated the reactivity of these monoclonal antibodies with venoms from 27 species of snakes belonging to different families. MAJar 3 recognized most of the hemorrhagic venoms. By ELISA, MAJar 3 reacted strongly with venoms from Viperidae family and weakly with Colubridae and Elapidae venoms. This recognition pattern was due to bands between 50 and 80 kDa, corresponding to P-III SVMPs. This antibody preferentially neutralized the hemorrhage induced by venoms of Bothrops snakes. This fact suggests that the epitope recognized by MAJar 3 is present in other metalloproteinases throughout snake phylogeny. However, slight structural differences in the epitope may result in insufficient affinity for neutralization of biological activities.     

Modulation of Adhesion Molecules Expression by Different Metalloproteases Isolated from Bothrops Snakes

Snake venom metalloproteinases (SVMP) are involved in local inflammatory reactions observed after snakebites. Based on domain composition, they are classified as PI (pro-domain + proteolytic domain), PII (PI + disintegrin-like domains), or PIII (PII + cysteine-rich domains). Here, we studied the role of different SVMPs domains in inducing the expression of adhesion molecules at the microcirculation of the cremaster muscle of mice. We used Jararhagin (Jar)—a PIII SVMP with intense hemorrhagic activity, and Jar-C—a Jar devoid of the catalytic domain, with no hemorrhagic activity, both isolated from B. jararaca venom and BnP-1—a weakly hemorrhagic P1 SVMP from B. neuwiedi venom. Toxins (0.5 µg) or PBS (100 µL) were injected into the scrotum of mice, and 2, 4, or 24 h later, the protein and gene expression of CD54 and CD31 in the endothelium, and integrins (CD11a and CD11b), expressed in leukocytes were evaluated. Toxins induced significant increases in CD54, CD11a, and CD11b at the initial time and a time-related increase in CD31 expression. In conclusion, our results suggest that, despite differences in hemorrhagic activities and domain composition of the SVMPs used in this study, they behave similarly to the induction of expression of adhesion molecules that promote leukocyte recruitment.     

Hemorrhage induced by snake venom metalloproteinases: biochemical and biophysical mechanisms involved in microvessel damage

Zinc-dependent metalloproteinases are responsible for the hemorrhagic activity characteristic of viperid snake venoms. Snake venom metalloproteinases (SVMPs) are classified in various groups (P-I–IV), according to their domain composition. P-III SVMPs, comprising metalloproteinase, disintegrin-like and cysteine-rich domains, exert more potent hemorrhagic activity than P-I SVMPs, which present only the metalloproteinase domain. SVMPs degrade various components of the basement membrane and are also able to hydrolyze endothelial cell membrane proteins, such as integrins and cadherins, involved in cell–matrix and cell–cell adhesion. In addition, disintegrin-like and cysteine-rich domains interact with endothelial cell integrins, interfering with their adhesion to extracellular matrix. Hemorrhage induced by SVMPs is an extremely rapid event in vivo, with capillary endothelial cells showing drastic structural alterations within few minutes. In contrast, observations in cell culture conditions do not evidence such rapid endothelial cell damage. Instead, the main effect is detachment and rounding of these cells; it is only after several hours of incubation that cells show evidence of apoptotic damage. This apparent discrepancy between in vivo and in vitro observations can be explained if biophysical forces operating on microvessels in vivo are taken into consideration. It is proposed that SVMP-induced hemorrhage occurs in vivo by a ‘two-step’ mechanism. Initially, SVMPs degrade basement membrane and adhesion proteins, thus weakening the capillary wall and perturbing the interactions between endothelial cells and the basement membrane. Then, transmural pressure acting on the weakened capillary wall causes distention. As a consequence, endothelial cells become very thin, until the integrity of the capillary wall is lost at some points, where extravasation occurs. In addition, endothelial cells become more susceptible to blood flow-dependent shear stress, which further contributes to capillary wall disruption.     

蛇毒解离素的结构特征及其与生物学活性的关系

目前已从蛇毒中分离出 80余种解离素多肽,根据其结构及来源可分为五大类,即短链、中链、长链解离素、金属蛋白酶P Ⅲ的类解离素区释放的解离素以及二聚体解离素。不同解离素的分子结构共同点包括RGD活性中心、半胱氨酸残基配对形成的二硫键、RGD三肽的C 端和N 端的氨基酸序列以及解离素多肽分子的C 末端。这些结构决定了解离素的抑制血小板聚集、抑制细胞粘附以及血管形成等生物学活性。     

Albocollagenase, a novel recombinant P-III snake venom metalloproteinase from green pit viper (Cryptelytrops albolabris), digests collagen and inhibits platelet aggregation

Molecular cloning and functional characterization of P-III snake venom metalloproteinases (SVMPs) will give us deeper insights in the pathogenesis of viper bites. This may lead to novel therapy for venom-induced local tissue damages, the complication refractory to current antivenom. The aim of this study was to elucidate the in vitro activities of a new SVMP from the green pit viper (GPV) using recombinant DNA technology. We report, here, a new cDNA clone from GPV (Cryptelytrops albolabris) venom glands encoding 614 amino acid residues P-III SVMP, termed albocollagenase. The conceptually translated protein comprised a signal peptide and prodomain, followed by a metalloproteinase domain containing a zinc-binding motifs, HEXGHXXGXXH-CIM and 9 cysteine residues. The disintegrin-like and cysteine-rich domains possessed 24 cysteines and a DCD (Asp-Cys-Asp) motif. The albocollagenase deduced amino acid sequence alignments showed approximately 70% identity with other P-III SVMPs. Notably, the prodomain was highly conserved, while the metalloproteinase, disintegrin-like and cysteine-rich domains contained several differences. Albocollagenase without the signal peptide and prodomain was expressed in Pichia pastoris with an N-terminal six-histidine tag. After affinity purification from the supernatant of methanol-induced media, SDS-PAGE and Western blot analysis in both reducing and non-reducing conditions showed a protein band of approximately 62 kDa. The recombinant albocollagenase could digest human type IV collagen from human placenta basement membrane within 1 min. After 10-min incubation, it also inhibited collagen-induced platelet aggregation with 50% inhibitory concentration (IC₅₀) of 70 nM. This is the first report of the active recombinant SVMP enzymes expressed in P. pastoris. The results suggest the significant roles of P-III SVMP in local and systemic pathology of envenomated patients. Inhibitors of this SVMP will be investigated in further studies to find a better treatment for viper bites.     

Molecular cloning and characterization of cDNAs encoding metalloproteinases from snake venom glands

Snake venom metalloproteinases (SVMPs) are a superfamily of zinc-dependent proteases and participate in a number of important biological, physiological and pathophysiological processes. In this work, we simultaneously amplified nine cDNAs encoding different classes of metalloproteinases from glands of four different snake species (Agkistrodon contortrix laticinctus, Crotalus atrox, Crotalus viridis viridis and Agkistrodon piscivorus leucostoma) by RT-PCR with a pair of primers. Among the encoded metalloproteinases, two enzymes (AclVMP-I and AplVMP-I), three enzymes (CaVMP-II, CvvVMP-II and AplVMP-II) and four enzymes (AclVMP-III, CaVMP-III, CvvVMP-III and AplVMP-III) with the characteristic motif (HEXXHXXGXXH) of metalloproteinase belong to type P-I, P-II and P-III enzymes, respectively. Disintegrin domains of CaVMP-II and CvvVMP-II from two Crotatus snakes contain RGD-motif whereas AplVMP-II from Agkistrodon snake has KGD-motif. Instead of R/KGD-motif within disintegrin domain of SVMP-II enzyme, CaVMP-III, CvvVMP-III and AplVMP-III enzymes contain SECD-motif, while AclVMP-III has DDCD-motif in their corresponding position of disintegrin-like domains. There are 12 Cys amino acids in cysterin-rich domains of each P-III enzyme. Moreover, a disintegrin precursor (AplDis) with RGD-motif also simultaneously amplified from the glands of A.p. leucostoma while amplifying AplVMP-II and AplVMP-III, which indicated that different types of SVMPs and related genes are present in a single species of snake and share a consensus sequence at the 3′ and 5′ untranslated regions. RT-PCR result also showed that P-III is highly expressed in Crotalus snakes than in Agkistrodon snakes. Aligning the deduced amino acid sequence of these enzymes with other SVMPs from GenBank database indicated that this is the first report on the isolation of cDNAs encoding P-II and P-III enzymes from C.v. viridis and A.p. leucostoma snakes. The availability of these SVMP sequences directly facilitated further studies of structure characterization and diversified function analysis.     

Proteomic profiling of snake venom metalloproteinases (SVMPs): Insights into venom induced pathology

Bothrops sp. snakebites account for the majority of envenomations in South and Central America. Bothrops jararaca accidents are characterized by edema, hemorrhage and necrosis, mainly attributed to the action of hemorrhagic snake venom metalloproteinases (SVMPs). Interestingly, accidents involving Bothrops lanceolatus (Fer-de-Lance) have a prothrombotic profile with necrosis and hemorrhage rarely reported. Here we describe biochemical and proteomic approaches to compare the venom composition of these snakes, focusing on the presence and activity of SVMPs. The total relative amount of SVMPs was found to be approximately the same in the venom of both species, the difference being in the distribution of SVMPs subgroups. Fer-de-Lance venom has relatively more PI SVMPs peptides identified (23–16%) while Jararaca venom has a higher amount of PIII SVMPs (54–43%). Gelatinolytic activity in the PIII mass range is also higher in Jararaca venom. Interestingly, the homologous band region in the Fer-de-Lance zymogram was only very weakly gelatinolytic. According to these findings it is feasible that the different distribution of SVMPs subgroups and their particular biochemical and pharmacological characteristics are two of the main factors contributing to these two radically different venom induced pathologies.     

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.     

Importance of jararhagin disintegrin-like and cysteine-rich domains in the early events of local inflammatory response

Jararhagin is a multi-domain SVMP from Bothrops jararaca venom comprising catalytic, disintegrin-like and cysteine-rich domains, which cause a local reaction manifested by hemorrhage, edema, cytokine release and inflammatory cell recruitment. In this study, the importance of disintegrin-like/cysteine-rich domains of jararhagin was addressed by analyzing the effects of jararhagin-C, which lacks the catalytic domain, in induction of leukocyte rolling and release of pro-inflammatory cytokines. Jararhagin-C was isolated from B. jararaca venom conserving the same ability of complete jararhagin molecule in inhibiting collagen-induced platelet-aggregation. Treatment of trans-illuminated cremaster muscle in vivo with jararhagin-C increased number of rolling leukocytes (approximately 250%) in post-capillary venules in all periods analyzed, without interfering with microvasculature haemodinamic, like vessel diameter, the erythrocyte speed or the blood flow rate. The release of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 was significantly enhanced in the local of jararhagin-C injection, showing the maximum levels in periods between 2 and 4 h after treatment. Besides the action of jararhagin-C, the presence of the inactivated catalytic domain in o-phenanthrolin-treated jararhagin was related to a higher increase in the number of rolling leukocytes. Moreover, the levels of IL-6 and IL-1β induced by catalytically active jararhagin were higher than those induced by jararhagin-C. In conclusion, our findings suggest that the disintegrin-like/cysteine-rich domains of jararhagin are sufficient to locally activate the early events of an acute inflammatory response as leukocyte rolling and pro-inflammatory cytokine release and this action may add to the effect of catalysis, which enhances the primary cell activation.     

A novel high molecular weight metalloproteinase cleaves fragment F1 of activated human prothrombin.

A hemorrhagic proteinase, jerdohagin, was purified from Trimeresurus jerdonii venom by gel filtration and ion-exchange chromatographies. It was a single chain polypeptide with an apparent molecular weight of 96 kDa as estimated by SDS-PAGE under the non-reducing and reducing conditions. Internal peptide sequencing indicated that it consisted of metalloproteinase, disintegrin-like and cysteine-rich domains and belonged to the class III snake venom metalloproteinases (class P-III SVMPs). Like other typical metalloproteinases, hemorrhagic activities of jerdohagin were completely inhibited by EDTA, but not by PMSF. Jerdohagin preferentially degraded alpha-chain of human fibrinogen. Interestingly, jerdohagin did not activate human prothrombin, whereas it cleaved human prothrombin and fragment F1 of activated human prothrombin.     

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.     

Isolation and characterization of a novel P-II class snake venom metalloproteinase from Trimeresurus stejnegeri.

Stejnitin, a novel class P-II snake venom metalloproteinase (SVMP) with a molecular weight of about 35kDa, was purified from Trimeresurus stejnegeri venom. The cDNA of stejnitin encoded a polypeptide of 295 amino acid residues which comprises a signal peptide, proprotein, metalloproteinase domain, spacer and disintegrin domain. The protein sequence deduced from cDNA was confirmed by peptide mass fingerprinting analysis. It is highly homologous to the members of subclass P-IIa SVMPs which comprises metalloproteinase and disintegrin together. Results from DNA fragmentation and flow cytometry analysis also indicated that stejnitin is able to induce apoptosis of ECV304 cells (R=0.908, P=0.012).     

Releasing or expression modulating mediator involved in hemostasis by Berythractivase and Jararhagin (SVMPs).

PIII snake venom metalloproteases (SVMPs) are structurally related to ADAMs (a disintegrin and metalloprotease human family of proteins). Berythractivase and Jararhagin are PIII SVMPs with 69% homology with different hemostatic properties. In order to clarify these differences and further characterize the biological effects of these proteins, we compared the effect of both proteases on human umbilical vein endothelial cell (HUVEC) for evaluating the release and modulation of coagulation and fibrinolysis mechanisms as well as the expression of their correlated genes. We found that both proteins increase the von Willebrand factor liberation, but did not modulate gene expression. Berythractivase, differently from Jararhagin increased the expression of tissue factor. Our results showed that both SVMPs (Berythractivase and Jararhagin) activate HUVEC releasing or modulating mediators involved in hemostasis. Meanwhile, we can suggest through the up-regulation of TF gene that the studied SVMP acts in a specific manner, suggesting that Jararhagin has preferentially a local action, while Berythractivase can be assumed as a systemic pro-coagulant protein with activity on the surface of HUVECs.     

Comparative analysis of the catalytic domain of hemorrhagic and non-hemorrhagic snake venom metallopeptidases using bioinformatic tools.

Snake venom metalloproteases (SVMPs) are a set of interesting enzymes that are one of the major components of venom affecting hemostasis. A great challenge since their discovery has been to find molecular features responsible for their hemorrhagic potency and many attempts have been made without any consistent result. Here we describe a series of comparisons between the catalytic domains of hemorrhagic and non-hemorrhagic SVMPs made with the help of bioinformatics. These involved sequence and structure-based multiple alignments, phylogenetic reconstruction, predicted physical and chemical properties, motif scanning and structural analyses. Although hemorrhagic activity seems to be complex, involving multiple factors, we found some molecular characteristics that may influence the toxic effects. Among these findings, it was possible to use a molecular surface feature to subdivide the P-I class in hemorrhagic and non-hemorrhagic SVMPs. It was also possible to suggest a role for the conserved Asp148 and Ser176 residues in the stabilization of the active site.     

Inhibition of lung tumor colonization and cell migration with the disintegrin crotatroxin 2 isolated from the venom of Crotalus atrox.

Disintegrins are low molecular weight proteins (4-15 kDa) with an RGD binding region at their binding loop. Disintegrin and disintegrin-like proteins are found in the venom of four families of snakes: Atractaspididae, Elapidae, Viperidae, and Colubridae. This report describes the biological activity of a disintegrin, crotatroxin 2, isolated by a three-step chromatography procedure from the venom of the Western diamondback rattlesnake (Crotalus atrox). The intact molecular mass for crotatroxin 2 was 7.384 kDa and 71 amino acids. Crotatroxin 2 inhibited human whole blood platelet aggregation with an IC(50) of 17.5 nM, inhibited cell (66.3p) migration by 63%, and inhibited experimental lung tumor colonization in BALB/c mice at 1000 microg/kg. Our data suggest that while crotatroxin 2 inhibits platelet aggregation, cancer cell migration, and lung tumor colonization, it is done via different integrins.