RESUMO
Microbial proteases are one of the most demanding enzymes for various industries with diverse applications in food, pharmaceutics, and textile industries to name the few. An extracellular alkaline metalloprotease was produced and purified from moderate halophilic bacterial strain, Bacillus cereus TS2, with some unique characteristics required for various industrial applications. The protease was produced in basal medium supplemented with casein and was partially purified by ion exchange chromatography followed by ammonium sulphate precipitation. The alkaline metalloprotease has molecular weight of 35 kDa with specific activity of 535.4 µM/min/mg. It can work at wide range of pH from 3 to 12, while showing optimum activity at pH 10. Similarly, the alkaline metalloprotease is stable till the temperature of 80 °C and works at wide range of temperature from 20 to 90 °C with optimum activity at 60 °C. The turnover rate increases in the presence of NaCl and Co+2 with k cat/KM of 1.42 × 103 and 1.27 × 103 s-1.M-1 respectively, while without NaCl and Co+2 it has a value of 7.58× 102. The alkaline metalloprotease was relatively resistant to thermal and solvent mediated denaturation. Applications revealed that the metalloprotease was efficient to remove hair from goat skin, remove blood stains and degrade milk, thus can be a potential candidate for leather, detergent, and food industry.
Assuntos
Bacillus cereus , Cloreto de Sódio , Cloreto de Sódio/farmacologia , Metaloproteases/química , Peptídeo Hidrolases , Temperatura , Concentração de Íons de HidrogênioRESUMO
Snake venoms have a complex mixture of compounds that are conserved across species and act synergistically, triggering severe local and systemic effects. Identification of the toxin classes that are most damaging to cell homeostasis would be a powerful approach to focus on the main activities that underpin envenomation. Here, we focus on the venom of Bothrops atrox, snake responsible for most of the accidents in Amazon region of South America. We identified the key cytotoxic toxin fractions from B. atrox venom and mapped their biochemical properties, protein composition and cell damage. Five fractions were obtained by mass exclusion chromatography and contained either a single class of enzymatic activity (i.e., L-amino acid oxidases or Hyaluronidases) or different activities co-distributed in two or more protein fractions (e.g., Metalloproteinases, Serine Proteases, or Phospholipases A2). Only three protein fractions reduced cell viability of primary human cells. Strikingly, such activity is accompanied by disruption of cell attachment to substratum and to neighbouring cells. Such strong perturbation of morphological cell features indicates likely defects in tissue integrity in vivo. Mass spectrometry identified the main classes of toxins that contribute to these phenotypes. We provide here a strategy for the selection of key cytotoxic proteins for targeted investigation of their mechanism of action and potential synergism during snakebite envenomation. Our data highlights putative toxins (or combinations of) that may be the focus of future therapeutic interference.
Assuntos
Bothrops , Mordeduras de Serpentes , Animais , Humanos , Antivenenos/análise , Antivenenos/metabolismo , Antivenenos/farmacologia , Bothrops/metabolismo , Mordeduras de Serpentes/terapia , Espectrometria de Massas , Metaloproteases/análise , Metaloproteases/química , Metaloproteases/metabolismoRESUMO
Hemorrhage induced by snake venom metalloproteases (SVMPs) results from proteolysis, capillary disruption, and blood extravasation. HF3, a potent SVMP of Bothrops jararaca, induces hemorrhage at pmol doses in the mouse skin. To gain insight into the hemorrhagic process, the main goal of this study was to analyze changes in the skin peptidome generated by injection of HF3, using approaches of mass spectrometry-based untargeted peptidomics. The results revealed that the sets of peptides found in the control and HF3-treated skin samples were distinct and derived from the cleavage of different proteins. Peptide bond cleavage site identification in the HF3-treated skin showed compatibility with trypsin-like serine proteases and cathepsins, suggesting the activation of host proteinases. Acetylated peptides, which originated from the cleavage at positions in the N-terminal region of proteins in both samples, were identified for the first time in the mouse skin peptidome. The number of peptides acetylated at the residue after the first Met residue, mostly Ser and Ala, was higher than that of peptides acetylated at the initial Met. Proteins cleaved in the hemorrhagic skin participate in cholesterol metabolism, PPAR signaling, and in the complement and coagulation cascades, indicating the impairment of these biological processes. The peptidomic analysis also indicated the emergence of peptides with potential biological activities, including pheromone, cell penetrating, quorum sensing, defense, and cell-cell communication in the mouse skin. Interestingly, peptides generated in the hemorrhagic skin promoted the inhibition of collagen-induced platelet aggregation and could act synergistically in the local tissue damage induced by HF3.
Assuntos
Bothrops , Venenos de Crotalídeos , Camundongos , Animais , Venenos de Crotalídeos/toxicidade , Venenos de Crotalídeos/química , Metaloproteases/química , Metaloproteases/metabolismo , Metaloproteases/farmacologia , Hemorragia/induzido quimicamente , Venenos de Serpentes/toxicidade , Venenos de Serpentes/química , Peptídeos , Bothrops/metabolismoRESUMO
Snakebite envenoming is still a worrying health problem in countries under development, being recognized as a neglected disease by the World Health Organization. In Latin America, snakes from the genus Bothrops are widely spread and in Brazil, the Bothrops moojeni is a medically important species. The pharmacological effects of bothropic snake venoms include pain, blisters, bleeding, necrosis and even amputation of the affected limb. Snake venom metalloproteinases are enzymes abundantly present in venom from Bothrops snakes. These enzymes can cause hemorrhagic effects and lead to myonecrosis due to ischemia. Here, we present BmooMP-I, a new P-I class of metalloproteinase (this class only has the catalytic domain in the mature form) isolated from B. moojeni venom. This protein is able to express fibrinogenolytic and gelatinase activities, which play important roles in the prey's immobilization and digestion, and also induces weak hemorrhagic effect. The primary sequence assignment was done by a novel method, SEQUENCE SLIDER, which combines crystallographic, bioinformatics and mass spectrometry data. The high-resolution crystal structure reveals the monomeric assembly and the conserved metal binding site H141ExxH145xxG148xxH151 with the natural substitution Gly148Asp that does not interfere in the zinc coordination. The presence of a structural calcium ion on the surface of the protein, which can play an important role in the stabilization of hemorrhagic toxins, was observed in the BmooMP-I structure. Due to the relevant local and systemic effects of snake venom metalloproteinases, studies involving these proteins help to better understand the pathological effects of snakebite envenoming.
Assuntos
Bothrops/metabolismo , Venenos de Crotalídeos/enzimologia , Metaloproteases/química , Metaloproteases/farmacologia , Sequência de Aminoácidos , Animais , Cálcio/química , Cátions/química , Biologia Computacional , Venenos de Crotalídeos/química , Venenos de Crotalídeos/isolamento & purificação , Venenos de Crotalídeos/farmacologia , Cristalização , Cristalografia por Raios X , Bases de Dados de Proteínas , Fibrinogênio/metabolismo , Gelatina/metabolismo , Hemorragia/enzimologia , Espectrometria de Massas , Metaloproteases/isolamento & purificação , Camundongos , Modelos Moleculares , Alinhamento de Sequência , Análise de Sequência de Proteína , Pele/enzimologia , Pele/metabolismoRESUMO
Chronic wounds are a major health problem that cause millions of dollars in expenses every year. Among all the treatments used, active wound treatments such as enzymatic treatments represent a cheaper and specific option with a fast growth category in the market. In particular, bacterial and plant proteases have been employed due to their homology to human proteases, which drive the normal wound healing process. However, the use of these proteases has demonstrated results with low reproducibility. Therefore, alternative sources of proteases such as snake venom have been proposed. Here, we performed a functional mining of proteases from rattlesnakes (Crotalus ornatus, C. molossus nigrescens, C. scutulatus, and C. atrox) due to their high protease predominance and similarity to native proteases. To characterize Crotalus spp. Proteases, we performed different protease assays to measure and confirm the presence of metalloproteases and serine proteases, such as the universal protease assay and zymography, using several substrates such as gelatin, casein, hemoglobin, L-TAME, fibrinogen, and fibrin. We found that all our venom extracts degraded casein, gelatin, L-TAME, fibrinogen, and fibrin, but not hemoglobin. Crotalus ornatus and C. m. nigrescens extracts were the most proteolytic venoms among the samples. Particularly, C. ornatus predominantly possessed low molecular weight proteases (P-I metalloproteases). Our results demonstrated the presence of metalloproteases capable of degrading gelatin (a collagen derivative) and fibrin clots, whereas serine proteases were capable of degrading fibrinogen-generating fibrin clots, mimicking thrombin activity. Moreover, we demonstrated that Crotalus spp. are a valuable source of proteases that can aid chronic wound-healing treatments.
Assuntos
Venenos de Crotalídeos/enzimologia , Crotalus/metabolismo , Metaloproteases , Proteínas de Répteis , Serina Proteases , Ferimentos e Lesões/tratamento farmacológico , Animais , Fibrinólise/efeitos dos fármacos , Humanos , Metaloproteases/química , Metaloproteases/farmacologia , Reprodutibilidade dos Testes , Proteínas de Répteis/química , Proteínas de Répteis/farmacologia , Serina Proteases/química , Serina Proteases/farmacologia , Ferimentos e Lesões/metabolismo , Ferimentos e Lesões/patologiaRESUMO
Snake venom metalloproteinases (SVMPs) are key toxins involved in local inflammatory reactions after snakebites. This study aimed to investigate the effect of SVMP domains on the alterations in leukocyte-endothelium interactions in the microcirculation of mouse cremaster muscle. We studied three toxins: BnP1, a PI-toxin isolated from Bothrops neuwiedi venom, which only bears a catalytic domain; Jararhagin (Jar), a PIII-toxin isolated from Bothrops jararaca venom with a catalytic domain, as well as ECD-disintegrin and cysteine-rich domains; and Jar-C, which is produced from the autolysis of Jar and devoid of a catalytic domain. All these toxins induced an increase in the adhesion and migration of leukocytes. By inhibiting the catalytic activity of Jar and BnP1 with 1.10-phenanthroline (oPhe), leukocytes were no longer recruited. Circular dichroism analysis showed structural changes in oPhe-treated Jar, but these changes were not enough to prevent the binding of Jar to collagen, which occurred through the ECD-disintegrin domain. The results showed that the catalytic domain of SVMPs is the principal domain responsible for the induction of leukocyte recruitment and suggest that the other domains could also present inflammatory potential only when devoid of the catalytic domain, as with Jar-C.
Assuntos
Domínio Catalítico/fisiologia , Leucócitos/patologia , Metaloproteases/farmacologia , Venenos de Serpentes/enzimologia , Músculos Abdominais/irrigação sanguínea , Animais , Bothrops , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Endotélio/metabolismo , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Metaloproteases/química , Camundongos , MicrocirculaçãoRESUMO
Atroxlysin-III (Atr-III) was purified from the venom of Bothrops atrox. This 56-kDa protein bears N-linked glycoconjugates and is a P-III hemorrhagic metalloproteinase. Its cDNA-deduced amino acid sequence reveals a multidomain structure including a proprotein, a metalloproteinase, a disintegrin-like and a cysteine-rich domain. Its identity with bothropasin and jararhagin from Bothrops jararaca is 97% and 95%, respectively. Its enzymatic activity is metal ion-dependent. The divalent cations, Mg2+ and Ca2+, enhance its activity, whereas excess Zn2+ inhibits it. Chemical modification of the Zn2+-complexing histidine residues within the active site by using diethylpyrocarbonate (DEPC) inactivates it. Atr-III degrades plasma fibronectin, type I-collagen, and mainly the α-chains of fibrinogen and fibrin. The von Willebrand factor (vWF) A1-domain, which harbors the binding site for GPIb, is not hydrolyzed. Platelets interact with collagen via receptors for collagen, glycoprotein VI (GPVI), and α2ß1 integrin. Neither the α2ß1 integrin nor its collagen-binding A-domain is fragmented by Atr-III. In contrast, Atr-III cleaves glycoprotein VI (GPVI) into a soluble ~55-kDa fragment (sGPVI). Thereby, it inhibits aggregation of platelets which had been stimulated by convulxin, a GPVI agonist. Selectively, Atr-III targets GPVI antagonistically and thus contributes to the antithrombotic effect of envenomation by Bothrops atrox.
Assuntos
Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Venenos de Crotalídeos/enzimologia , Crotalinae , Metaloproteases/farmacologia , Glicoproteínas da Membrana de Plaquetas/biossíntese , Sequência de Aminoácidos , Animais , Crotalinae/metabolismo , Matriz Extracelular , Metaloproteases/química , Metaloproteases/genética , Metaloproteases/isolamento & purificação , Modelos Moleculares , Filogenia , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Glicoproteínas da Membrana de Plaquetas/química , Conformação Proteica , Proteólise , Relação Estrutura-AtividadeRESUMO
Snakebite is a major medical concern in many parts of the world with metalloproteases playing important roles in the pathological effects of Viperidae venoms, including local tissue damage, hemorrhage, and coagulopathy. Hemorrhagic Factor 3 (HF3), a metalloprotease from Bothrops jararaca venom, induces local hemorrhage and targets extracellular matrix (ECM) components, including collagens and proteoglycans, and plasma proteins. However, the full substrate repertoire of this metalloprotease is unknown. We report positional proteomic studies identifying >2000 N-termini, including neo-N-termini of HF3 cleavage sites in mouse embryonic fibroblast secretome proteins. Terminal amine isotopic labeling of substrates (TAILS) analysis identified a preference for Leu at the P1' position among candidate HF3 substrates including proteins of the ECM and focal adhesions and the cysteine protease inhibitor cystatin-C. Interestingly, 190 unique peptides matched to annotated cleavage sites in the TopFIND N-termini database, suggesting that these cleavages occurred at a site prone to cleavage or might have been generated by other proteases activated upon incubation with HF3, including caspases-3 and -7, cathepsins D and E, granzyme B, and MMPs 2 and 9. Using Proteomic identification of cleavage site specificity (PICS), a tryptic library derived from THP-1 monocytic cells was used as HF3 substrates for identifying protease cleavage sites and sequence preferences in peptides. A total of 799 unique cleavage sites were detected and, in accordance with TAILS analysis using native secreted protein substrates of MEF cells, revealed a clear preference for Leu at P1'. Taken together, these results greatly expand the known substrate degradome of HF3 and reveal potential new targets, which may serve as a basis to better elucidate the complex pathophysiology of snake envenomation.
Assuntos
Metaloproteases/genética , Proteoma/genética , Proteômica , Venenos de Serpentes/genética , Sequência de Aminoácidos/genética , Animais , Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/isolamento & purificação , Bothrops/genética , Humanos , Marcação por Isótopo , Metaloproteases/química , Metaloproteases/isolamento & purificação , Camundongos , Biblioteca de Peptídeos , Proteoma/química , Venenos de Serpentes/química , Especificidade por Substrato/genética , Espectrometria de Massas em TandemRESUMO
Variability in snake venom composition has been frequently reported and correlated to the adaptability of snakes to environmental conditions. Previous studies report plasticity for the venom phenotype. However, these observations are not conclusive, as the results were based on pooled venoms, which present high individual variability. Here we tested the hypothesis of plasticity by influence of confinement and single diet type in the venom composition of 13 adult specimens of Bothrops atrox snakes, maintained under captivity for more than three years. Individual variability in venom composition was observed in samples extracted just after the capture of the snakes. However, composition was conserved in venoms periodically extracted from nine specimens, which presented low variability restricted to the less abundant components. In a second group, composed of four snakes, drastic changes were observed in the venom samples extracted at different periods, mostly related to snake venom metalloproteinases (SVMPs), the core function toxins of B. atrox venom, which occurred approximately between 400 and 500 days in captivity. These data show plasticity in the venom phenotype during the lifetime of adult snakes maintained under captive conditions. Causes or functional consequences involved in the phenotype modification require further investigations.
Assuntos
Bothrops , Venenos de Crotalídeos/análise , Animais , Variação Biológica Individual , Venenos de Crotalídeos/enzimologia , Feminino , Metaloproteases/química , Fenótipo , Fosfolipases A2/química , Proteínas de Répteis/química , Serina Proteases/químicaRESUMO
Serratiopeptidase, a metalloprotease produced by Serratia marcescens, is produced through a fermentation process using carbohydrates and proteins as carbon and nitrogen sources. However, some byproducts of the silk industry could be an alternative source for serratiopeptidase production. Therefore, the present work is focused on the purification and characterization of a serratiopeptidase produced from the C8 isolate of Serratia marcescens and obtained from a Colombian silkworm hybrid using casein or silkworm pupae. The protease was purified using ultrafiltration, anion-exchange, and size-exclusion chromatography. The purified enzyme showed a molecular weight of ~50â¯kDa with a purity above 96%, an isoelectric point of ~4.6, optimum pH and temperature of 6 and 50⯰C, and stability at 4⯰C for one month. The kinetic constants using azocasein as substrate were 0.63â¯mM (Km), 2,016⯵M/min (Vmax), 41.41â¯s-1 (Kcat), and 6.56â¯×â¯107â¯M-1â¯s-1 (Kcat/Km). Inhibition by 5â¯mM EDTA or 1,10-phenanthroline was recovered by adding Zn2+ at the same concentration. Mass spectrometry analysis indicated 94% homology with the sequence of serratiopeptidase produced by the E-15 strain. We purified and characterized a serratiopeptidase produced by the C8 isolate of S. marcescens in a culture medium based on a renewable source from the silk industry.