title: Heterologous expression and biochemical and functional characterization of a recombinant alpha-type myotoxin inhibitor from Bothrops alternatus snake.
abstract: Venomous and non-venomous snakes possess phospholipase A2 (PLA2) 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 PLA2s, showing slight inhibitions especially at the molar ratios of 1:1 and 1:3 (PLA2: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 PLA2s and Lys49 PLA2-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 PLA2s, which have not yet been fully clarified.
authors.affiliation: Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, FCFRP-USP, Ribeirão Preto, SP, Brazil; Instituto de Química, Universidade Estadual Paulista, UNESP, Araraquara, SP, Brazil. Electronic address: drnorival@yahoo.com.
authors.forename: Norival A
authors.initials: NA
authors.lastname: Santos-Filho
authors.affiliation: Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, FCFRP-USP, Ribeirão Preto, SP, Brazil.
authors.forename: Johara
authors.initials: J
authors.lastname: Boldrini-França
authors.affiliation: Departamento de Genética e Evolução, Universidade Federal de São Carlos, UFSCAR, São Carlos, SP, Brazil.
authors.forename: Ludier K
authors.initials: LK
authors.lastname: Santos-Silva
authors.affiliation: Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, FCFRP-USP, Ribeirão Preto, SP, Brazil.
authors.forename: Danilo L
authors.initials: DL
authors.lastname: Menaldo
authors.affiliation: Departamento de Genética e Evolução, Universidade Federal de São Carlos, UFSCAR, São Carlos, SP, Brazil.
authors.forename: Flávio
authors.initials: F
authors.lastname: Henrique-Silva
authors.affiliation: Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, FCFRP-USP, Ribeirão Preto, SP, Brazil.
authors.forename: Tiago S
authors.initials: TS
authors.lastname: Sousa
authors.affiliation: Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, FCFRP-USP, Ribeirão Preto, SP, Brazil.
authors.forename: Adélia C O
authors.initials: AC
authors.lastname: Cintra
authors.affiliation: Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, Uberlândia, MG, Brazil.
authors.forename: Carla C N
authors.initials: CC
authors.lastname: Mamede
authors.affiliation: Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, Uberlândia, MG, Brazil.
authors.forename: Fábio
authors.initials: F
authors.lastname: Oliveira
authors.affiliation: Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, FCFRP-USP, Ribeirão Preto, SP, Brazil.
authors.forename: Eliane C
authors.initials: EC
authors.lastname: Arantes
authors.affiliation: Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, FCFRP-USP, Ribeirão Preto, SP, Brazil.
authors.forename: Lusânia M Greggi
authors.initials: LM
authors.lastname: Antunes
authors.affiliation: Instituto de Química, Universidade Estadual Paulista, UNESP, Araraquara, SP, Brazil.
authors.forename: Eduardo M
authors.initials: EM
authors.lastname: Cilli
authors.affiliation: Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, FCFRP-USP, Ribeirão Preto, SP, Brazil. Electronic address: suvilela@usp.br.
authors.forename: Suely V
authors.initials: SV
authors.lastname: Sampaio
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001798:Blood Proteins / Q000096:biosynthesis* / Q000235:genetics; D017837:Bothrops / Q000235:genetics; D003435:Crotalid Venoms / Q000037:antagonists & inhibitors* / Q000737:chemistry; D051379:Mice; D018482:Muscle, Skeletal / Q000187:drug effects; D054467:Phospholipases A2 / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D012910:Snake Venoms / Q000201:enzymology*
chemical_list: D001798:Blood Proteins; D003435:Crotalid Venoms; C400481:PLIalpha; D011994:Recombinant Proteins; D012910:Snake Venoms; C018932:myotoxin A; D054467:Phospholipases A2
keywords: Bothrops alternatus; Myotoxin inhibitor; Phospholipase A(2); Recombinant enzyme; Snake venoms