Antibodies against synthetic epitopes inhibit the enzymatic activity of mutalysin II, a metalloproteinase from bushmaster snake venom

Mutalysin II (mut-II), a 22.5 kDa zinc endopeptidase isolated from bushmaster (Lachesis muta muta) snake venom, is a direct acting fibrin(ogen)olytic proteinase. It induces monoclonal and polyclonal antibodies which efficiently neutralize the hemorrhagic effect of L. muta and several Bothrops whole venoms. To characterize epitopes of protective antibodies we have used the Spot method of multiple peptide synthesis to prepare 64 overlapping dodecapeptides frameshifted by three residues, covering the complete amino acid sequence of mut-II. The rabbit anti-mut-II antibodies binding pattern to peptides revealed several continuous antigenic regions: one in the N-terminal part, two in the central region and the other in the C-terminal of mut-II. By using homology modelling, a three-dimensional model of mut-II was built which showed that epitopes are surface exposed. Anti-peptide antibodies were raised against three peptides (one representative of each epitope region) covalently coupled as a mixture to keyhole limpet hemocyanin. Purified IgG from the resulting anti- peptide antibodies cross-reacted with mut-II and induced a dose-dependent inhibition of the mut-II catalyzed proteolysis of fibrinogen.     

Jararhagin, a hemorrhagic snake venom metalloproteinase from Bothrops jararaca

Jararhagin is a metalloproteinase isolated from Bothrops jararaca snake venom, which has been extensively studied. These studies showed its involvement on most of the systemic and local damaging effects of snakebite envenomings. In this review we comment on the major targets of jararhagin as the vascular endothelium, platelets and coagulation factors and also its action on other cell systems as inflammatory cells and their mediators, cancer and cell signaling. The mechanisms of jararhagin action are discussed together with structural features essential for the expression of its biological activities. The studies reviewed here denote jararhagin as a prototype for studies of snake venom metalloproteinases, bringing new insights into cellular-matrix interactions and adding for the improvement of snakebite treatment.     

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.     

BnP1, a novel P-I metalloproteinase from Bothrops neuwiedi venom: biological effects benchmarking relatively to jararhagin, a P-III SVMP.

Snake venom metalloproteinases (SVMPs) have been extensively studied and their effects associated with the local bleeding observed in human accidents by viper snakes. Representatives of P-I and P-III classes of SVMPs similarly hydrolyze extracellular matrix proteins or coagulation factors while only P-III SVMPs induce significant hemorrhage in experimental models. In this work, the effects of P-I and P-III SVMPs on plasma proteins and cultures of muscle and endothelial cells were compared in order to enlighten the mechanisms involved in venom-induced hemorrhage. To reach this comparison, BnP1 was isolated from B. neuwiedi venom and used as a weakly hemorrhagic P-I SVMPs and jararhagin was used as a model of potently hemorrhagic P-III SVMP. BnP1 was isolated by size exclusion and anion-exchange chromatographies, showing apparent molecular mass of approximately 24kDa and sequence similarity with other members of SVMPs, which allowed its classification as a group P-I SVMP. The comparison of local effects induced by SVMPs showed that BnP1 was devoid of significant myotoxic and hemorrhagic activities and jararhagin presented only hemorrhagic activity. BnP1 and jararhagin were able to hydrolyze fibrinogen and fibrin, although the latter displayed higher activity in both systems. Using HUVEC primary cultures, we observed that BnP1 induced cell detachment and a decrease in the number of viable endothelial cells in levels comparable to those observed by treatment with jararhagin. Moreover, both BnP1 and jararhagin induced apoptosis in HUVECs while only a small increase in LDH supernatant levels was observed after treatment with jararhagin, suggesting that the major mechanism involved in endothelial cell death is apoptosis. Jararhagin and BnP1 induced little effects on C2C12 muscle cell cultures, characterized by a partial detachment 24h after treatment and a mild necrotic effect as evidenced by a small increase in the supernatants LDH levels. Taken together, our data show that P-I and P-III SVMPs presented comparable effects except for the hemorrhagic activity, suggesting that hydrolysis of coagulation factors or damage to endothelial cells are not sufficient for induction of local bleeding.     

Characterization of inflammatory reaction induced by neuwiedase, a P-I metalloproteinase isolated from Bothrops neuwiedi venom

The Snake Venom Metalloproteinases (SVMPs) play a relevant role in the multifactorial inflammatory response induced by Bothrops envenomations. Neuwiedase, an SVMP isolated from Bothrops neuwiedi venom, is devoid of hemorrhagic activity on skin tests, but is able to induce myonecrosis and degrade fibrinogen, fibrin, type I collagen, fibronectin and laminin. In this study, we analyzed the inflammatory reaction induced by neuwiedase in gastrocnemius muscle, with special focus on cytokines release. Our results showed clear evidence of inflammatory infiltrate in the gastrocnemius muscle and an increase of MMP-9, and the cytokines KC, IL-1β and IL-6 in the early periods after toxin injection. The cytokine release was also evaluated in inflammatory and muscular cell culture. Both murine peritoneal adherent cells (MPACs) and muscle cells (C2C12) released pro-inflammatory cytokines after stimulus with neuwiedase. MPACs showed increased production of KC, IL-1β and IL-6 in the cell culture supernatant while in C2C12, the predominant chemokine expressed was KC. These data reinforce the importance of SVMPs in the inflammatory response caused by envenomation and point out the role of muscle cells in this event by releasing pro-inflammatory mediators able to attract leukocytes to the muscle, thus starting and amplifying the setting of the inflammatory reaction.     

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).     

Pulmonary hemorrhage induced by jararhagin, a metalloproteinase from Bothrops jararaca snake venom

Jararhagin is the most important hemorrhagic component in the venom of the snake Bothrops jararaca, a species of medical importance in South America. It is a P-III zinc-dependent metalloproteinase comprising catalytic, disintegrin-like, and cysteine-rich domains. Jararhagin injected intravenously into mice induced rapid and prominent bleeding in the lungs, whereas other organs were devoid of overt hemorrhagic manifestations. This action depends on the proteolytic activity of jararhagin, since it was abrogated by the synthetic inhibitor batimastat. There were conspicuous ultrastructural alterations in cells at the alveolo-capillary unit, i.e., capillary endothelial cells and type I pneumocytes, with a characteristic pattern of "regional alveolar damage" associated with extravasation. These pathological effects were observed under conditions in which the whole blood clotting time, bleeding time, and fibrinogen levels were not affected. 125I-labeled jararhagin is concentrated mainly in liver and kidneys after iv injection, with little radioactivity observed in the lungs, thereby indicating that the predominance of pulmonary microvascular damage is not due to a preferential concentration of this enzyme in the lungs. Despite the fact that jararhagin is complexed by plasma proteins after iv injection, its hemorrhagic activity was not inhibited by the plasma proteinase inhibitor alpha(2)-macroglobulin, and was only partially reduced by normal mouse serum, suggesting that resistance to inhibition may contribute to its ability to cause pulmonary hemorrhage.     

Batroxase, a new metalloproteinase from B. atrox snake venom with strong fibrinolytic activity

The structures and functional activities of metalloproteinases from snake venoms have been widely studied because of the importance of these molecules in envenomation. Batroxase, which is a metalloproteinase isolated from Bothrops atrox (Pará) snake venom, was obtained by gel filtration and anion exchange chromatography. The enzyme is a single protein chain composed of 202 amino acid residues with a molecular mass of 22.9 kDa, as determined by mass spectrometry analysis, showing an isoelectric point of 7.5. The primary sequence analysis indicates that the proteinase contains a zinc ligand motif (HELGHNLGISH) and a sequence C₁₆₄I₁₆₅M₁₆₆ motif that is associated with a “Met-turn” structure. The protein lacks N-glycosylation sites and contains seven half cystine residues, six of which are conserved as pairs to form disulfide bridges. The three-dimensional structure of Batroxase was modeled based on the crystal structure of BmooMPα-I from Bothrops moojeni. The model revealed that the zinc binding site has a high structural similarity to the binding site of other metalloproteinases. Batroxase presented weak hemorrhagic activity, with a MHD of 10 μg, and was able to hydrolyze extracellular matrix components, such as type IV collagen and fibronectin. The toxin cleaves both α and β-chains of the fibrinogen molecule, and it can be inhibited by EDTA, EGTA and β-mercaptoethanol. Batroxase was able to dissolve fibrin clots independently of plasminogen activation. These results demonstrate that Batroxase is a zinc-dependent hemorrhagic metalloproteinase with fibrin(ogen)olytic and thrombolytic activity.     

BthMP: a new weakly hemorrhagic metalloproteinase from Bothrops moojeni snake venom

In this work, a new weakly hemorrhagic metalloproteinase (BthMP) was purified from Bothrops moojeni snake venom. This enzyme was homogeneous by native and SDS-PAGE. It showed a polypeptide chain of 23.5 kDa, pI = 7.1, and N-terminal blocked. BthMP is comprised of high proteolytic activity on casein, fibrin and bovine fibrinogen, with no coagulating, esterase or phospholipase A₂ activities; it was inhibited by EDTA, EGTA and 1,10-phenanthroline and maintained its activity on pH from 7.0 to 9.0 and temperature from 5-40 °C. Assays with metal ions showed that Ca²⁺ is an activator, whereas Zn²⁺ and Hg²⁺ inhibited about 50 and 80% of its activity, respectively. The edema evidenced the important role of the toxin in the inflammatory activity of the venom. BthMP also caused unclotting, and provoked histological alterations in the gastrocnemius muscle of mice inducing hemorrhage, necrosis and leukocytic infiltrate. The molecular mass and the inhibition assays suggest that the metalloproteinase BthMP belongs to class P-I of SVMPs.     

Free energy calculations on snake venom metalloproteinase BaP1

BaP1 is a snake venom metalloproteinase from the venom of Bothrops asper, showing high structural homology with the catalytic domain of human adamalysins and matrix metalloproteinases. It induces the release of cytokines, like interleukin-1 and tumor necrosis factor alpha. Recently, the high-resolution crystal structure of BaP1 with a bound inhibitor became available, representing an interesting model concerning inhibitor design for medicinally important metalloproteinases such as tumor necrosis factor alpha-converting enzyme and MMP13. We here use computational modeling to gain a better understanding about the binding properties of various ligands to BaP1, with a focus on computing ligand binding free energies. The obtained results should be of general significance for future research on medicinally important metalloproteinases. We have investigated the binding of the original inhibitor in detail and calculated its binding strength using MMP/GBSA free energy calculations. Additionally, the binding strengths of alternative ligands have been computed, and two of them are predicted and experimentally verified to strongly inhibit the enzyme. A suggestion for chemical modifications of BaP1 inhibitors could be made to guide future synthesis efforts. Furthermore, a contribution to the proteolytic reaction mechanism of metzincins is given. The pK value of the catalytically active glutamic acid residue 143 has been found to be significantly raised when compared with a free glutamate side chain. Calculations on other matrix metalloproteinases confirmed that this is not confined to BaP1, but seems to be a common feature of metzincins.     

烙铁头蛇毒中一个具有抗补体活性金属蛋白酶的纯化和性质研究

目的从烙铁头蛇毒中筛选分离抗补体活性蛋白,并对其部分生物学活性开展研究,以了解其在蛇伤中的病理生理作用和潜在的应用价值。方法采用蛋白层析技术分离纯化抗补体活性蛋白,测定其分子量、等电点、抗补体作用、多种蛋白水解酶活性、水肿活性及出血活性。结果从烙铁头蛇毒中分离纯化出一个抗补体活性蛋白TMAC-1,表观分子量约为25ku,等电点为9.0。TMAC-1能够抑制补体经典途径和替代途径的溶血,预孵条件下,其IC50分别为62、29mg·L-1;不预孵条件下,其IC50为263、246mg·L-1。TMAC-1能够裂解C3、C5,但裂解产物不能诱导内皮细胞P-selectin的表达。TMAC-1能依次降解纤维蛋白原的Aα、Bβ、γ链,该活性能被EDTA、1,10-phenanthroline、EGTA完全抑制,不受PMSF、SBTI的抑制。TMAC-1具有水肿活性和微弱的偶氮酪蛋白水解活性,没有精氨酸酯酶水解活性和皮下出血活性。结论 TMAC-1是一个非出血性的金属蛋白酶,它可通过酶切补体特定成分抑制补体激活。     

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。     

PO41, a snake venom metalloproteinase inhibitor isolated from Philander opossum serum.

PO41 was isolated from Philander opossum serum by DEAE-Sephacel, Phenyl Superose and Superdex 200 chromatographies and showed a molecular mass of 41,330 Da by MALDI-TOF MS. Molecular masses of 81.5 and 84.5 kDa were obtained by size exclusion chromatography and dynamic laser light scattering, respectively, suggesting that PO41 is dimeric. Its isoelectric point was estimated to be lower than 3.5. PO41 presented similar amino terminal sequence to those of DM40 and DM43, two antihaemorrhagins previously isolated from Didelphis marsupialis serum and was recognized by polyclonal antibodies raised against D. marsupialis antibothropic fraction. To study the inhibitory properties of this protein, the metalloproteinases bothrolysin and jararhagin were isolated from Bothrops jararaca venom by chromatographies on Superdex 200 and Phenyl Superose. Jararhagin was further submitted to a Mono Q column. The proteolytic and haemorrhagic effects of these haemorrhagins were neutralized by PO41. Both snake venom metalloproteinases formed stable complexes with PO41. The stoichiometry of the complex PO41-jararhagin was one inhibitor subunit to one molecule of the enzyme. These results show that PO41 has physicochemical, structural, immunoreactive and biological properties similar to other metalloproteinase inhibitors belonging to the supergene family of immunoglobulins.     

蛇毒抗肿瘤成分的研究进展

目的探讨蛇毒抗肿瘤成分的作用及其在医药领域的应用,为蛇毒成分在抗肿瘤领域的研发提供参考。方法查阅并总结资料文献,对国内外蛇毒抗肿瘤成分的研究进行综述。结果蛇毒具有多种抗肿瘤成分,对多种肿瘤均有抑制作用,可抑制肿瘤相关的基因表达、抑制肿瘤细胞增殖、诱导肿瘤细胞凋亡和抑制肿瘤血管再生与转移等。结论对蛇毒抗肿瘤成分进行深入研究,开发其在抗肿瘤药物研究领域的价值。     

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.     

蛇毒抗肿瘤转移的研究现状

蛇毒含有多种酶和多种不同生理、药理活性蛋白。人们对纯化的蛇毒制品和非纯化的蛇毒制品已有了较深入的研究。本针对血小板、解离素、粘附分子、纤维蛋白以及血管生成等方面，并首重对蛇毒抗肿瘤转移作了简要综述。     

Immunochemical and biological characterization of monoclonal antibodies against BaP1, a metalloproteinase from Bothrops asper snake venom

BaP1 is a P-I class of Snake Venom Metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomations by Bothrops asper, a medically-important species in Central America and parts of South America. Six monoclonal antibodies (MoAb) against BaP1 (MABaP1) were produced and characterized regarding their isotype, dissociation constant (K_d), specificity and ability to neutralize BaP1-induced hemorrhagic and proteolytic activity. Two MABaP1 are IgM, three are IgG1 and one is IgG2b. The K_ds of IgG MoAbs were in the nM range. All IgG MoAbs recognized conformational epitopes of BaP1 and B. asper venom components but failed to recognize venoms from 27 species of Viperidae, Colubridae and Elapidae families. Clone 7 cross-reacted with three P-I SVMPs tested (moojeni protease, insularinase and neuwiedase). BaP1-induced hemorrhage was totally neutralized by clones 3, 6 and 8 but not by clone 7. Inhibition of BaP1 enzymatic activity on a synthetic substrate by MABaP1 was totally achieved by clones 3 and 6, and partially by clone 8, but not by clone 7. In conclusion, these neutralizing MoAbs against BaP1 may become important tools to understand structure-function relationships of BaP1 and the role of P-I class SVMP in snakebite envenomation.     

Inflammatory effects of BaP1 a metalloproteinase isolated from Bothrops asper snake venom: leukocyte recruitment and release of cytokines.

The inflammatory events induced by BaP1, a 22.7 kDa metalloproteinase isolated from Bothrops asper snake venom, were studied. BaP1 i.p. injection in mice induced a marked inflammatory cell infiltrate into peritoneal cavity of animals with predominance of neutrophils in the early phase followed by mononuclear cells in the late period. Inhibition of enzymatic activity of BaP1 by chelation with EDTA resulted in a drastic reduction of this effect. In addition, BaP1 induced a significant increase of blood neutrophil numbers before its accumulation in peritoneal cavity, thus suggesting a stimulatory action of BaP1 on mechanisms of cell mobilization from bone marrow reserve compartments. A reduction in the number of neutrophils was observed in the exudate when antibodies against LECAM-1, CD18 and LFA-1 were used, suggesting the involvement of these adhesion molecules in the effects of BaP1. In contrast, there was no effect with antibodies against ICAM-1 and PECAM-1. Moreover, a conspicuous increment in the levels of IL-1 and TNF-alpha, but not of LTB4, was observed in peritoneal washes collected from mice injected with BaP1. It is concluded that BaP1 induces in vivo a marked leukocyte influx, which parallels an increased number of these cells in the blood, and is associated to the expression of specific leukocyte adhesion molecules and release of chemotactic inflammatory cytokines. Since BaP1 is a P-I class metalloproteinase, these results indicate that the proteolytic domain of metalloproteinases per se can trigger specific inflammatory events.     

蛇毒纤溶酶的分离纯化及其溶栓作用

目的获得大量高纯度的蛇毒纤溶酶并进行药效实验。方法用离子交换柱色谱和单克隆抗体亲和色谱技术进行纯化 ,用体外和半体内血栓模型研究其抗血栓作用。结果得到纯度为 95 %以上的纤溶酶。结论纯化后的蛇毒纤溶酶具有显著的抑制血栓形成的作用     

Development of process to produce polyvalent IgY antibodies anti-African snake venom

Polyvalent anti-Bitis and anti-Naja antivenom IgY antibodies were prepared using B. arietans, B. nasicornis, B. rhinoceros, N. melanoleuca, and N. mossambica venoms to immunize chickens. Blood and eggs were collected before and during the 10-month immunization period; the sera and yolk extracts were then prepared and assayed for the presence of antivenom antibodies by ELISA and Western blot methods. ELISA Antivenom antibody titers, referred to as U-ELISA/ml of serum or egg yolk extracts, absent in pre-immunization sera or yolk, increased sharply during the 4weeks after immunization, reaching a plateau thereafter. Yolk extracts with high antivenom titers, as detected by ELISA were used to isolate and purify IgY. Purified IgY preparations recognized venom protein bands from 10 to 20kDa to 60 and 70kDa, as shown by Western blot. Recovery of antivenom antibodies from the whole yolk was over 80%. Final preparations exhibited high antivenom activity (>100,000 U-ELISA/ml) as well as efficacy in neutralizing venom lethality (1440mug of IgY neutralize 62.2 LD(50) of venom), and were free of toxic products, pyrogen or bacterial and fungal contaminations.