Full text |
|
Research article - Vol. 25, 2019 |
Subproteome of Lachesis muta rhombeatavenom and preliminary studies on LmrSP-4, a novel snake venom serine proteinase
1 Department of Physics and Chemistry, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Av. do Café s/n, 14040-903, Ribeirão Preto, SP, Brazil.
2 Institute of Biosciences, Letters and Exact Sciences, Universidade Estadual Paulista, Rua Cristóvão Colombo, 2265, 15054-000, São José do Rio Preto, SP, Brazil.
3 Institute of Genetics and Biochemistry, Federal University of Uberlândia, Av. Pará, 1720, 38400-902, Uberlândia, MG, Brazil.
4 Department of Chemical and Physical, State University of Southwest Bahia, Rua José Moreira Sobrinho, até 873 874, 45506-210, Jequié, BA, Brazil.
5 Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Av. do Café s/n, 14040-903, Ribeirão Preto, SP, Brazil.
6 Proteomics Resource Center, New York University Langone Medical Center, 430 East 29th St., 10016, New York City, USA.
ABSTRACT
Background:
Lachesis muta rhombeata is one of the venomous snakes of medical importance in Brazil whose envenoming is characterized by local and systemic effects which may produce even shock and death. Its venom is mainly comprised of serine and metalloproteinases, phospholipases A2 and bradykinin-potentiating peptides. Based on a previously reported fractionation of L. m. rhombeata venom (LmrV), we decided to perform a subproteome analysis of its major fraction and investigated a novel component present in this venom.
Methods:
LmrV was fractionated through molecular exclusion chromatography and the main fraction (S5) was submitted to fibrinogenolytic activity assay and fractionated by reversed-phase chromatography. The N-terminal sequences of the subfractions eluted from reversed-phase chromatography were determined by automated Edman degradation. Enzyme activity of LmrSP-4 was evaluated upon chromogenic substrates for thrombin (S-2238), plasma kallikrein (S-2302), plasmin and streptokinase-activated plasminogen (S-2251) and Factor Xa (S-2222) and upon fibrinogen. All assays were carried out in the presence or absence of possible inhibitors. The fluorescence resonance energy transfer substrate Abz-KLRSSKQ-EDDnp was used to determine the optimal conditions for LmrSP-4 activity. Molecular mass of LmrSP-4 was determined by MALDI-TOF and digested peptides after trypsin and Glu-C treatments were analyzed by high resolution MS/MS using different fragmentation modes.
Results:
Fraction S5 showed strong proteolytic activity upon fibrinogen. Its fractionation by reversed-phase chromatography gave rise to 6 main fractions (S5C1-S5C6). S5C1-S5C5 fractions correspond to serine proteinases whereas S5C6 represents a C-type lectin. S5C4 (named LmrSP-4) had its N-terminal determined by Edman degradation up to the 53rd amino acid residue and was chosen for characterization studies. LmrSP-4 is a fibrinogenolytic serine proteinase with high activity against S-2302, being inhibited by PMSF and benzamidine, but not by 1,10-phenantroline. In addition, this enzyme exhibited maximum activity within the pH range from neutral to basic and between 40 and 50 °C. About 68% of the LmrSP-4 primary structure was covered, and its molecular mass is 28,190 Da.
Conclusions:
Novel serine proteinase isoforms and a lectin were identified in LmrV. Additionally, a kallikrein-like serine proteinase that might be useful as molecular tool for investigating bradykinin-involving process was isolated and partially characterized.
Keywords: bushmaster; snake venom; SVSP; kallikrein-like; plasminogen activator; kininogenase; lectin; protease; envenomation
Received: June 29, 2018.
Accepted: October 02, 2018.
Correspondence: ecabraga@fcfrp.usp.br
Competing interests The authors declare that there are no competing interests.
Authors' contributions GAW performed the experiments related to the venom fractionation, structural and biochemical characterizations of LmrSP-4, including MS/MS analyses, analyzed data from all experiments and drafted the manuscript. KCFB conducted the N-terminal sequencing and helped to draft the manuscript. RRS and MSRG performed the enzyme activity assays using FRET substrate and chromogenic substrates, respectively. HC and VMR participated in research design and supervised the enzyme activity assays using FRET substrate and chromogenic substrates, respectively. BU participated in research design and supervised the MS/MS analysis. ECA participated in research design, supervised experiments related to the venom fractionation, structural and biochemical characterizations of LmrSP-4 and participated in manuscript writing. All authors read and approved the final manuscript.