Ontogenetic study of the composition of snake venom inhibitors and evaluation of the neutralizing activity of the gamma-type phospholipase inhibitor (γPLI) present in the plasma of Bothrops jararaca snake

ABSTRACT: Snake venom inhibitors have been studied for over a century. Among them are the plasma proteins of some mammals and of non-venomous and venomous snakes, which have the ability to promote resistance to the venom. These proteins have aroused biotechnological interest, as they are an excellent alternative for the development of new drugs. The main proteins found in animal plasma, capable of neutralizing venom, are metalloproteinase inhibitors and PLA2 inhibitors (PLIs). The PLA2 acts on cell membrane phospholipids resulting in fatty acids, lysophospholipids and cell membrane deconstruction. This protein is commonly responsible for the local effect of envenomation, causing tissue inflammation in the affected area. Based on this information, γBjPLI was recently isolated, which is a PLI present in the blood of the venomous snake Bothrops jararaca. This protein is classified as belonging to the gamma (γ) class, based on its characteristic domains. γBjPLI has approximately 22 kDa and inhibits the enzymatic, edematogenic and myonecrotic activities of PLA2, suggesting a role of this inhibitor in the protection of these snakes against self-envenomation. Knowing that, in this work it is presented a comparative study of the protein composition of the plasma of newborns, young and adults, females and males of B. jararaca, by analysis of SDS-PAGE, Western blotting, affinity chromatography, ELISA and mass spectrometry. Such analysis allowed to identify PLI-like proteins (αPLI, βPLI and γPLI) at all stages of life, however, there was no clear evidence that there is a significant difference in ontogeny. An evaluation of the interaction and neutralization of crotoxin anticoagulant activity by thromboelastometry was also performed, and γBjPLI was able to inhibit up to 43% of the activity. In addition, it showed a greater affinity for PLA2 Asp-49 compared to PLA2 Lys-49, evidenced by an alteration in spectra obtained by circular dichroism, and by the inhibition of myotoxic and edematogenic activities, with values of 19.6% and 38. 5% for PLA2 Asp-49, higher than 16% and 19.6% for PLA2 Lys-49, respectively. Furthermore, to establish new methods for obtaining these inhibitors with greater yield, stability, and activity, γBjPLI was cloned and expressed using the pET28a vector and a SHuffle expression strain of the bacterium species Escherichia coli

INSTRUMENT(S): Synapt MS

ORGANISM(S): Bothrops Jararaca (jararaca) (bothrops Jajaraca)

TISSUE(S): Blood Plasma

SUBMITTER: Alexandre Keiji Tashima  

LAB HEAD: Alexandre Keiji Tashima

PROVIDER: PXD034977 | Pride | 2024-05-21

REPOSITORIES: Pride