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| Author(s): |
Norival Alves Santos Filho
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Ribeirão Preto. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC) |
| Defense date: | 2013-01-11 |
| Examining board members: |
Suely Vilela;
Auro Nomizo;
Jose Cesar Rosa;
Antonio Cardozo dos Santos
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| Advisor: | Suely Vilela |
| Abstract | |
Myonecrosis is an important medical complication resulting from snakebites and, in severe cases, local myonecrosis can lead to drastic consequences such as permanent tissue loss, disability or limb amputation. Venomous and non-venomous snakes possess proteins that inhibit phospholipases A2 (PLA2s) in their bloodstream, called PLIs. One hypothesis that could explain the presence of these PLIs in the serum of venomous snakes would be the self-protection against their own venom enzymes, which could eventually reach the circulatory system. In the present work, an inhibitory protein (called ?BaltMIP) that neutralizes the enzymatic and toxic activities of several bothropic PLA2s was isolated from Bothrops alternatus plasma by affinity chromatography using the myotoxin BthTX-I immobilized on a CNBr-activated Sepharose resin. This protein showed relative molecular mass of 24,000 under reducing conditions on SDS-PAGE and greater specificity of inhibition of the cytotoxic and myotoxic activities of Lys49 PLA2-like enzymes compared to the anticoagulant and phospholipase activities of Asp49 PLA2s. In addition, molecular modeling was performed by in silico analysis of both tertiary and quaternary structures of ?BaltMIP. Furthermore, a model of interaction between alpha inhibitors of myotoxins and these toxins was proposed, bringing a new approach to the study of these enzymes. Cloning and expression of the inhibitor (called rBaltMIP in its recombinant form) were also achieved successfully, followed by its partial biochemical characterization. Nterminal sequencing and reverse phase HPLC showed that rBaltMIP was purified in a high purity degree, with 100% sequence identity with the native inhibitor. The inhibitory properties of rBaltMIP were tested against PLA2s and PLA2-like enzymes. rBaltMIP was able to partially inhibit the phospholipase activity of different enzymes, also inhibiting the cytotoxic and myotoxic activity of different Lys49 PLA2-like enzymes. Heterologous expression of rBaltMIP should help obtaining such inhibitor in large-scale, and consequently, the performance of further studies on the mechanism of inhibition of PLA2s, which has not yet been fully clarified. In addition, new information regarding the mechanism of action of these inhibitors may be known, in an attempt to better understand the application of these proteins as new therapeutic models with antiophidian action in neutralizing different types of PLA2s, the supplementation of conventional serum therapy and also in the molecular interaction with PLA2s. (AU) | |
| FAPESP's process: | 08/10760-4 - Functional and structural characterization of alfa-type phospholipase A2 inhibitor from B. alternatus snake plasma: cloning, expression and mapping of the region responsible for its inhibitory activity |
| Grantee: | Norival Alves Santos Filho |
| Support Opportunities: | Scholarships in Brazil - Doctorate |