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| Author(s): |
Carolina Petri Bernardes
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: | 2012-12-07 |
| Examining board members: |
Suely Vilela;
Luciana Simon Pereira Crott;
Silvana Marcussi
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| Advisor: | Suely Vilela |
| Abstract | |
Snake venoms are complex mixtures of bioactive molecules resulting in an efficient biological product with the purpose of promoting immobilization, death and initial digestion of prey. Metalloproteinases and serine proteases are among the major enzymes responsible for affecting the hemostatic system by different mechanisms. The present study aimed at the isolation and biochemical and structural characterization of a P-I class metalloproteinase from Bothrops pirajai snake venom. This enzyme was isolated using three chromatographic steps: molecular exclusion on Sephacryl S-200, ion exchange on CM-Sepharose and affinity on Blue Sepharose, resulting in a metalloproteinase called BpirMP. Its molecular mass was determined by mass spectrometry (23.15 kDa), and as visualized by SDS-PAGE, the molecule showed a single polypeptide chain. The determination of the partial amino acid sequence and multiple alignment with sequences deposited in databases showed high identity (~90%) with other P-I class metalloproteinases from snake venoms. BpirMP has the consensus sequence HELGHNLGMEH and the VCM sequence, which characterize the superfamily of metzincins metalloproteinases. The enzyme showed activity on fibrinogen, degrading Aα and Bβ chains, and was also able to degrade fibrin and blood clots in vitro, completely dissolving the blood clot at the highest dose tested. The enzyme was unable to coagulate blood plasma. The proteolytic activity of the metalloproteinase on azocasein was evaluated under different conditions of pH and temperature, showing that BpirMP has its activity reduced in acidic pHs and temperatures above 60 ºC. The proteolytic activity was inhibited by chelating agents such as EDTA, EGTA, and 1,10- phenanthroline and by reducing agents as β-mercaptoethanol and DTT. Specific inhibitors of serine proteases (benzamidine, leupeptin and aprotinin) had no effect on the proteolytic activity of BpirMP. The enzyme induced hemorrhage, presenting a minimal hemorrhagic dose (MHD) of 50 µg. BpirMP hydrolyzed basement membrane components both in vitro and in vivo, preferably degrading laminin and nidogen. The metalloproteinase showed effects on inflammatory processes such as induction of pain and edema, promoting a pronounced neutrophil recruitment and significant increase in the total number of leukocytes in inflammatory exudate. The evaluation of the contribution of different inflammatory mediators in the induced edema and hyperalgesia indicated the involvement of arachidonic acid metabolites and histamine. The cytotoxic effect induced by BpirMP was evaluated on the gastrocnemius muscle of mice and heart, kidney, lung and spleen tissues, showing degeneration of muscle fibers and leukocyte infiltration. The results demonstrate that BpirMP is a low molecular mass and low hemorrhagic metalloproteinase belonging to the P-I class, described for the B. pirajai species for the first time. The characterization of Bothrops pirajai venom was performed using proteomic techniques to determine its composition. Proteins were separated by RP-HPLC, followed by SDSPAGE, tryptic in-gel digestion and identification by MALDI-TOF/TOF mass spectrometry, assigning family classification by homology to known proteins. Seven protein families were found in the venom of B. pirajai, with an abundance of phospholipases A2 (~40%) and metalloproteinases (~20%). (AU) | |
| FAPESP's process: | 07/08361-1 - Functional and structural characterization of a non-hemorrhagic metalloprotease with antitumor and antimicrobial actvity isolated from Bothrops pirajai snake venom |
| Grantee: | Carolina Petri Bernardes |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |