RESUMO
Proteomic analysis of wound exudates represents a valuable tool to investigate tissue pathology and to assess the therapeutic success of various interventions. In this study, the ability of horse-derived IgG and F(ab')(2) antivenoms to neutralize local pathological effects induced by the venom of the snake Bothrops asper in mouse muscle was investigated by the proteomic analysis of exudates collected in the vicinity of affected tissue. In experiments involving the incubation of venom and antivenom prior to injection in mice, hemorrhagic activity was completely abolished and local muscle-damaging activity was significantly reduced by the antivenoms. In these conditions, the relative amounts of several intracellular and extracellular matrix proteins were reduced by the action of antivenoms, whereas the relative amounts of various plasma proteins were not modified. Because not all intracellular proteins were reduced, it is likely that there is a residual cytotoxicity not neutralized by antivenoms. In experiments designed to more closely reproduce the actual circumstances of envenoming, that is, when antivenom is administered after envenomation, the number of proteins whose amounts in exudates were reduced by antivenoms decreased, underscoring the difficulty in neutralizing local pathology due to the very rapid onset of venom-induced pathology. In these experiments, IgG antivenom was more efficient than F(ab')(2) antivenom when administered after envenomation, probably as a consequence of differences in their pharmacokinetic profiles.
Assuntos
Antivenenos/farmacologia , Bothrops , Venenos de Crotalídeos/imunologia , Exsudatos e Transudatos/metabolismo , Fragmentos Fab das Imunoglobulinas/farmacologia , Proteoma/metabolismo , Animais , Antivenenos/uso terapêutico , Proteínas Sanguíneas/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Hemorragia/induzido quimicamente , Hemorragia/prevenção & controle , Cavalos , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Imunoglobulina G , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , ProteômicaRESUMO
Six novel peptides (named bactridines) were isolated from Tityus discrepans scorpion venom. From mass spectrometry molecular masses were 6916, 7362, 7226, 7011, 7101 and 7173 Da (bactridines 1-6). Bactridines 1 and 2 were sequenced by Edman degradation. The sequences and in silico analysis, indicated that they are positively charged polypeptides comprised of 61 and 64 amino acids (AA), respectively, bactridine 1 and bactridine 2 containing 4 disulfide bridges. Bactridine 1 was only toxic to cockroaches and crabs, and bactridine 2-6 were only toxic to mice. Bactridine 1 has a 78% sequence identity with ardiscretin. Ardisctretin is an insect specific sodium toxin which also produces a small depolarization and induces repetitive firing in squid axons resembling those of DDT [1,10(pchlorobenzyl) 2-trichloretane] in its ability to slow down action potential, to induce repetitive firing. Measured as the minimal inhibitory concentration, bactridines had high antibacterial activity against a wide range of gram positive and gram negative bacteria. Complete bacterial growth inhibition occurred at concentrations from 20 to 80 microM depending on the bacteria and peptide tested. Effects on membrane Na(+) permeability induced by bactridines were observed on Yersinia enterocolitica loaded with 1 microM CoroNa Red. CoroNa Red fluorescence leakage from bacteria was observed after exposure to 0.3 microM of any bactridine tested, indicating that they modified Na(+) membrane permeability. This effect was blocked by 10 microM amiloride and by 25 microM mibefradil drugs that affect Na(+) and Ca(2+) channels respectively. We found no evidence of changes of K(+) or Ca(2+) concentrations neither inside nor outside the bacteria in experiments using the fluorescent dyes Fluo 4AM (10 microM) and PBFI (20 microM).
Assuntos
Antibacterianos/farmacologia , Membrana Celular/metabolismo , Corantes Fluorescentes/química , Peptídeos/farmacologia , Venenos de Escorpião/química , Sódio/metabolismo , Potenciais de Ação , Sequência de Aminoácidos , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Decapodiformes , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/isolamento & purificação , Permeabilidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Snake venom metalloproteinases (SVMPs) are members of the Reprolysin family of metalloproteinases to which the ADAM (a disintegrin and metalloproteinase) proteins also belong. The disintegrin-like/cysteine-rich domains of the ADAMs have been implicated in their function. In the case of the SVMPs, we hypothesized that these domains could function to target the metalloproteinases to key extracellular matrix proteins or cell surface proteins. Initially we detected interaction of collagen XIV, a fibril-associated collagen with interrupted triple helices containing von Willebrand factor A (VWA) domains, with the PIII SVMP catrocollastatin. Next we investigated whether other VWA domain-containing matrix proteins could support the binding of PIII SVMPs. Using surface plasmon resonance, the PIII SVMP jararhagin and a recombinant cysteine-rich domain from a PIII SVMP were demonstrated to bind to collagen XIV, collagen XII, and matrilins 1, 3, and 4. Jararhagin was shown to cleave these proteins predominantly at sites localized at or near the VWA domains suggesting that it is the VWA domains to which the PIII SVMPs are binding via their cysteine-rich domain. In light of the fact that these extracellular matrix proteins function to stabilize matrix, targeting the SVMPs to these proteins followed by their specific cleavage could promote the destabilization of extracellular matrix and cell-matrix interactions and in the case of capillaries could contribute to their disruption and hemorrhage. Although there is only limited structural homology shared by the cysteine-rich domains of the PIII SVMPs and the ADAMs our results suggest an analogous function for the cysteine-rich domains in certain members of the expanded ADAM family of proteins to target them to VWA domain-containing proteins.
Assuntos
Bothrops , Venenos de Crotalídeos/metabolismo , Cisteína/metabolismo , Metaloproteases/metabolismo , Fator de von Willebrand/metabolismo , Animais , Linhagem Celular , Colágeno/metabolismo , Venenos de Crotalídeos/química , Crotalus , Cisteína/fisiologia , Proteínas da Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Glicoproteínas/metabolismo , Humanos , Ligantes , Proteínas Matrilinas , Metaloendopeptidases/metabolismo , Metaloproteases/química , Metaloproteases/fisiologia , Ligação Proteica , Estrutura Terciária de Proteína/fisiologia , Especificidade por Substrato , Fator de von Willebrand/química , Veneno de Bothrops jararacaRESUMO
The cysteine-rich domain of the haemorrhagic metalloproteinase atrolysin A was shown to inhibit collagen-stimulated platelet aggregation and to interact with MG-63 osteosarcoma cells via integrin alpha2beta1 to inhibit adhesion to collagen I. In addition, we demonstrate by solid-phase binding assays that atrolysin A binds to collagen I and to vWF (von Willebrand factor) via exosites in the cysteine-rich domain. Interestingly, the binding site of the cysteine-rich domain on collagen I is distinct from the cell adhesion site, since the incubation of collagen-I-coated plates with the cysteine-rich domain did not prevent the adhesion of MG-63 cells to collagen. Finally, we show by surface plasmon resonance (BIAcore) analyses that the cysteine-rich domain can block vWF binding to collagen I as well as the binding of collagen I to vWF. Taken together, these results indicate that this domain may function as a cell-surface-receptor-binding site and/or a substrate recognition exosite and may thus play a role in the pathologies associated with atrolysin A.
Assuntos
Colágeno Tipo I/metabolismo , Cisteína/metabolismo , Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Fator de von Willebrand/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Metaloendopeptidases/farmacologia , Dados de Sequência Molecular , Agregação Plaquetária/efeitos dos fármacos , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes , Venenos de Serpentes/química , Ressonância de Plasmônio de SuperfícieRESUMO
The complexity of Viperid venoms has long been appreciated by investigators in the fields of toxinology and medicine. However, it is only recently that the depth of that complexity has become somewhat quantitatively and qualitatively appreciated. With the resurgence of two-dimensional gel electrophoresis (2-DE) and the advances in mass spectrometry virtually all venom components can be visualized and identified given sufficient effort and resources. Here we present the use of 2-DE for examining venom complexity as well as demonstrating interesting approaches to selectively delineate subpopulations of venom proteins based on particular characteristics of the proteins such as antibody cross-reactivity or enzymatic activities. 2-DE comparisons between venoms from different species of the same genus (Bothrops) of snake clearly demonstrated both the similarity as well as the apparent diversity among these venoms. Using liquid chromatography/tandem mass spectrometry we were able to identify regions of the two-dimensional gels from each venom in which certain classes of proteins were found. 2-DE was also used to compare venoms from Crotalus atrox and Bothrops jararaca. For these venoms a variety of staining/detection protocols was utilized to compare and contrast the venoms. Specifically, we used various stains to visualize subpopulations of the venom proteomes of these snakes, including Coomassie, Silver, Sypro Ruby and Pro-Q-Emerald. Using specific antibodies in Western blot analyses of 2-DE of the venoms we have examined subpopulations of proteins in these venoms including the serine proteinase proteome, the metalloproteinase proteome, and the phospholipases A2 proteome. A functional assessment of the gelatinolytic activity of these venoms was also performed by zymography. These approaches have given rise to a more thorough understanding of venom complexity and the toxins comprising these venoms and provide insights to investigators who wish to focus on these venom subpopulations of proteins in future studies.
Assuntos
Bothrops/genética , Venenos de Crotalídeos/análise , Eletroforese em Gel Bidimensional , Proteoma/análise , Proteômica , Animais , Western Blotting , Cromatografia Líquida , Venenos de Crotalídeos/enzimologia , Venenos de Crotalídeos/genética , Eletroforese em Gel de Poliacrilamida , Variação Genética , Processamento de Imagem Assistida por Computador , Espectrometria de Massas , Metaloendopeptidases/análise , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Fosfolipases A/análise , Fosfolipases A/genética , Fosfolipases A/metabolismo , Proteoma/genética , Serina Endopeptidases/análise , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Tripsina/metabolismoRESUMO
BaP1 is a 22.7-kD P-I-type zinc-dependent metalloproteinase isolated from the venom of the snake Bothrops asper, a medically relevant species in Central America. This enzyme exerts multiple tissue-damaging activities, including hemorrhage, myonecrosis, dermonecrosis, blistering, and edema. BaP1 is a single chain of 202 amino acids that shows highest sequence identity with metalloproteinases isolated from the venoms of snakes of the subfamily Crotalinae. It has six Cys residues involved in three disulfide bridges (Cys 117-Cys 197, Cys 159-Cys 181, Cys 157-Cys 164). It has the consensus sequence H(142)E(143)XXH(146)XXGXXH(152), as well as the sequence C(164)I(165)M(166), which characterize the "metzincin" superfamily of metalloproteinases. The active-site cleft separates a major subdomain (residues 1-152), comprising four alpha-helices and a five-stranded beta-sheet, from the minor subdomain, which is formed by a single alpha-helix and several loops. The catalytic zinc ion is coordinated by the N(epsilon 2) nitrogen atoms of His 142, His 146, and His 152, in addition to a solvent water molecule, which in turn is bound to Glu 143. Several conserved residues contribute to the formation of the hydrophobic pocket, and Met 166 serves as a hydrophobic base for the active-site groups. Sequence and structural comparisons of hemorrhagic and nonhemorrhagic P-I metalloproteinases from snake venoms revealed differences in several regions. In particular, the loop comprising residues 153 to 176 has marked structural differences between metalloproteinases with very different hemorrhagic activities. Because this region lies in close proximity to the active-site microenvironment, it may influence the interaction of these enzymes with physiologically relevant substrates in the extracellular matrix.
Assuntos
Bothrops , Venenos de Crotalídeos/enzimologia , Metaloendopeptidases/química , Sequência de Aminoácidos , Animais , Cálcio/química , Domínio Catalítico/fisiologia , Venenos de Crotalídeos/química , Venenos de Crotalídeos/isolamento & purificação , Cristalografia por Raios X , Hemorragia/etiologia , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Zinco/químicaRESUMO
DM43, an opossum serum protein inhibitor of snake venom metalloproteinases, has been completely sequenced, and its disulfide bond pattern has been experimentally determined. It shows homology to human alpha(1)B-glycoprotein, a plasma protein of unknown function and a member of the immunoglobulin supergene family. Size exclusion and dynamic laser light scattering data indicated that two monomers of DM43, each composed of three immunoglobulin-like domains, associated to form a homodimer in solution. Analysis of its glycan moiety showed the presence of N-acetylglucosamine, mannose, galactose, and sialic acid, most probably forming four biantennary N-linked chains. DM43 inhibited the fibrinogenolytic activities of bothrolysin and jararhagin and formed 1:1 stoichiometric stable complexes with both metalloproteinases. DM43 was ineffective against atrolysin C or A. No complex formation was detected between DM43 and jararhagin C, indicating the essential role of the metalloproteinase domain for interaction. Homology modeling based on the crystal structure of a killer cell inhibitory receptor suggested the existence of an I-type Ig fold, a hydrophobic dimerization surface and six surface loops potentially forming the metalloproteinase-binding surface on DM43.