RESUMO
Jararhagin is a PIII class SVMP with hemorrhagic activity, found in Bothrops jararaca venom. By the process of autolysis, Jararhagin gives rise to Jararhagin-C (JarC), a protein with disintegrin-like and cysteine-rich domains, containing the ECD motif in the disintegrin domain, which selectively recognizes the α2β1 integrin present in platelets and endothelial cells. Several studies have shown the ability of JarC to increase leukocyte adhesion and migration and to promote angiogenesis in experimental models in vivo. However, obtaining JarC from the venom is a limiting factor for the study of this toxin, as it represents about 1% of the crude venom, and expressing this protein in the recombinant form has solved this problem. Escherichia coli was successfully used to express the protein of interest in fusion with the SUMOUlp1 protein. In this work, we used native and recombinant JarC to investigate and compare, in vitro, its action on the migration of endothelial cells CRL-1730 in a Boyden chamber model using type I collagen as substrate and Fetal Bovine Serum (FBS) as cell migration promoting agent. We also verified the gene expression of the IL-8 chemokine by cells incubated with the proteins at 3h, 6h and 24h. Our results showed the effect of both JarC molecules in inhibiting FBS-stimulated cell migration. In the presence of FBS, JarC down regulated the expression of IL-8 at 3h and 6h, however, at 24h there was a slight increase in the expression of this chemokine. Regarding rJarC there was down regulation with 3h, stability with 6h and up regulation with 24h of treatment. However, the behavior of JarC (both in the native and recombinant form) in a culture of cells grown in the absence of FBS was completely different, being possible to observe an up regulation of IL-8 with 3 and 24 hours of stimulation, and a down regulation with 6 hours of stimulation. Our results clearly demonstrate a role for JarC, an ECD-disintegrin, in inhibiting endothelial cell migration. This effect was reproduced by the recombinant protein, confirming that its biological activity is preserved. A protein with an inhibitory action on endothelial cell migration may be a promising tool to treat pathologies that have angiogenesis as a central mechanism, such as age-related macular degeneration or tumor growth.
A Jararagina é uma SVMP de classe PIII com ação hemorrágica encontrada no veneno de Bothrops jararaca. Pelo processo de autólise, a Jararagina dá origem a Jararagina-C (JarC), uma proteína com os domínios tipo-disintegrina e rico em cisteína, contendo o motivo ECD no domínio disintegrina, que reconhece seletivamente a integrina α2β1 presente em plaquetas e células endoteliais. Diversos estudos têm mostrado a capacidade da JarC em aumentar a adesão e migração de leucócitos e promover a angiogênese em modelos experimentais in vivo. Entretanto, a obtenção da JarC a partir do veneno é um fator limitante para o estudo desta toxina, pois ela representa cerca de 1% da massa do veneno seco, e expressar essa proteína de forma recombinante tem solucionado esse problema. A Escherichia coli foi utilizada com sucesso para expressar a proteína de interesse em fusão com a proteína SUMOUlp1. Neste trabalho, utilizamos a JarC nativa e recombinante a fim de investigar e comparar in vitro, sua ação na migração de células endoteliais da linhagem CRL-1730 em modelo de câmara de Boyden usando colágeno tipo I como substrato e Soro Fetal Bovino (SFB) como agente promotor da migração celular. Verificamos também a expressão gênica da quimiocina IL-8 pelas células incubadas com as proteínas em 3h, 6h e 24h. Nossos resultados evidenciaram o efeito de ambas as moléculas de JarC em inibir a migração celular estimulada pelo SFB, sendo a JarC nativa mais eficiente. Na presença de SFB, a JarC regulou negativamente a expressão da IL-8 com 3h e 6h, no entanto, com 24h houve um aumento discreto na expressão desta quimiocina. Com relação a rJarC houve regulação negativa de IL-8 com 3h, estabilidade com 6h e regulação positiva com 24h de tratamento. Porém, o comportamento da JarC (tanto na forma nativa, quanto recombinante) em uma cultura de células cultivadas na ausência de SFB foi completamente diferente, sendo possível observar uma regulação positiva de IL-8 com 3 e 24 horas de estimulo, e uma regulação negativa com 6 horas de estímulo. Nossos resultados demonstram claramente o papel da JarC, uma ECD-disintegrina, inibindo a migração de células endoteliais. Este efeito foi reproduzido pela proteína recombinante, confirmando a sua atividade biológica preservada. Uma proteína com ação inibitória da migração de células endoteliais pode ser uma ferramenta promissora para tratar patologias que tenham a angiogênese como mecanismo central, como por exemplo a degeneração macular relacionada à idade ou ainda o crescimento de tumores.
RESUMO
Since the first record of the five founder members of the group of Natterin proteins in the venom of the medically significant fish Thalassophryne nattereri, new sequences have been identified in other species. In this work, we performed a detailed screening using available genome databases across a wide range of species to identify sequence members of the Natterin group, sequence similarities, conserved domains, and evolutionary relationships. The high-throughput tools have enabled us to dramatically expand the number of members within this group of proteins, which has a remote origin (around 400 million years ago) and is spread across Eukarya organisms, even in plants and primitive Agnathans jawless fish. Overall, the survey resulted in 331 species presenting Natterin-like proteins, mainly fish, and 859 putative genes. Besides fish, the groups with more species included in our analysis were insects and birds. The number and variety of annotations increased the knowledge of the obtained sequences in detail, such as the conserved motif AGIP in the pore-forming loop involved in the transmembrane barrel insertion, allowing us to classify them as important constituents of the innate immune defense system as effector molecules activating immune cells by interacting with conserved intracellular signaling mechanisms in the hosts.
