RESUMO
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.
Assuntos
Anticorpos Monoclonais/imunologia , Bothrops/classificação , Venenos de Crotalídeos/imunologia , Epitopos/imunologia , Hemorragia/imunologia , Metaloproteases/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/farmacologia , Western Blotting , Bothrops/genética , Bothrops/imunologia , Colubridae/genética , Colubridae/imunologia , Venenos de Crotalídeos/química , Venenos de Crotalídeos/enzimologia , Venenos de Crotalídeos/genética , Venenos de Crotalídeos/toxicidade , Elapidae/genética , Elapidae/imunologia , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Epitopos/genética , Hemorragia/induzido quimicamente , Hemorragia/prevenção & controle , Metaloendopeptidases/química , Metaloendopeptidases/genética , Metaloendopeptidases/imunologia , Metaloproteases/química , Metaloproteases/genética , Camundongos , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Viperidae/genética , Viperidae/imunologia , Veneno de Bothrops jararacaRESUMO
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.
Assuntos
Bothrops , Venenos de Crotalídeos/enzimologia , Venenos de Crotalídeos/farmacologia , Metaloproteases/química , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Colágeno/química , Venenos de Crotalídeos/química , Venenos de Crotalídeos/imunologia , Hemorragia/induzido quimicamente , Metaloendopeptidases/química , Metaloendopeptidases/imunologia , Metaloendopeptidases/farmacologia , Metaloproteases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Relação Estrutura-Atividade , Veneno de Bothrops jararacaRESUMO
Soro de pacientes com doença de Chagas na fase crônica foram submetidos a cromatografia de afinidade com Sepharose 4B conjugada com um extrato antigênico obtido de formas epimastigotas ou tripomastigotas de T. cruzi: os epimastigotas foram obtidos de cultura na fase exponencial de crescimento e os tripomastigotas de sangue de camundongos infectados e imunossuprimidos. Os antígenos de ambas formas parasitárias foram obtidos por tratamento dos parasitas por ultra-som, de centrifugaçäo. A cromatografia de afinidade foi feita passando-se os soros chagásicos através de uma coluna de imunoadsorvente contendo antígenos de pimastigotas ou tripomastigotas. Os anticorpos foram eluídos da coluna com tampäo glicina 0,2 M pH 2,8 a 4-C. Os anticorpos eluidos foram analisados quanto ao seu isotipo e atividade lítica. Os resultados mostraram que os anticorpos anti-T. cruzi com atividade lítica presentes em soros chagásicos estäo localizados no isotipo gG e reconhecem epitopos presentes tanto nos tripomastigotas quanto nos epimastigotas.
