Heterologous expression and biochemical and functional characterization of a recombinant alpha-type myotoxin inhibitor from Bothrops alternatus snake

Author(s):Show less -

Santos-Filho, Norival A. ^{[1, 2]} ; Boldrini-Franca, Johara ^[1] ; Santos-Silva, Ludier K. ^[3] ; Menaldo, Danilo L. ^[1] ; Henrique-Silva, Flavio ^[3] ; Sousa, Tiago S. ^[1] ; Cintra, Adelia C. O. ^[1] ; Mamede, Carla C. N. ^[4] ; Oliveira, Fabio ^[4] ; Arantes, Eliane C. ^[1] ; Greggi Antunes, Lusania M. ^[1] ; Cilli, Eduardo M. ^[2] ; Sampaio, Suely V. ^[1]

Affiliation

^[1] Univ Sao Paulo, Fac Ciencias Farmaceut Ribeirao Preto, Dept Anal Clin Toxicol & Bromatol, FCFRP USP, BR-14049 Ribeirao Preto, SP - Brazil

^[2] Univ Estadual Paulista, UNESP, Inst Quim, Araraquara, SP - Brazil

^[3] Univ Fed Sao Carlos, Dept Genet & Evolucao, UFSCAR, BR-13560 Sao Carlos, SP - Brazil

^[4] Univ Fed Uberlandia, Inst Ciencias Biomed, BR-38400 Uberlandia, MG - Brazil

• Document type: Journal article
• Source: Biochimie; v. 105, p. 119-128, OCT 2014.
• Web of Science Citations: 9

Abstract

Venomous and non-venomous snakes possess phospholipase A(2) (PLA(2)) inhibitory proteins (PLIs) in their blood serum. This study shows the expression and biochemical and functional characterization of a recombinant alpha inhibitor from Bothrops alternatus snake, named rBaltMIP. Its expression was performed in Pichia pastoris heterologous system, resulting in an active recombinant protein. The expressed inhibitor was tested regarding its ability to inhibit the phospholipase activity of different PLA(2)s, showing slight inhibitions especially at the molar ratios of 1:1 and 1:3 (PLA(2):PLI). rBaltMIP was also effective in decreasing the myotoxic activity of the tested toxins at molar ratios greater than 1:0.4 (myotoxin:PLI). The inhibition of the myotoxic activity of different Asp49 (BthTX-II and PrTX-III) and Lys49 (BthTX-I and PrTX-I) myotoxins was also performed without the prior incubation of myotoxins/inhibitor in order to analyze the real possibility of using snake plasma inhibitors or recombinant inhibitors as therapeutic agents for treating envenomations. As a result, rBaltMIP was able to significantly inhibit the myotoxicity of Lys49 myotoxins. Histopathological analysis of the gastrocnemius muscles of mice showed that the myotoxins are able to induce severe damage to the muscle fibers of experimental animals by recruiting a large number of leukocyte infiltrates, besides forming an intense accumulation of intercellular fluid, leading to local edema. When those myotoxins were incubated with rBaltMIP, a reduction of the damage site could be observed. Furthermore, the cytotoxic activity of Asp49 PLA(2)s and Lys49 PLA(2)-like enzymes on C2C12 cell lines was decreased, as shown by the higher cell viabilities after preincubation with rBaltMIP. Heterologous expression would enable large-scale obtainment of rBaltMIP, thus allowing further investigations for the elucidation of possible mechanisms of inhibition of snake PLA(2)s, which have not yet been fully clarified. (C) 2014 Elsevier Masson SAS. All rights reserved. (AU)

• FAPESP's process: 11/23236-4 - Native and recombinant animal toxins: functional, structural and molecular analysis
• Grantee: Suely Vilela
• Support Opportunities: Research Projects - Thematic Grants
• 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