Subproteome of Lachesis muta rhombeata venom and preliminary studies on LmrSP-4, a novel snake venom serine proteinase

Autor(es)

Wiezel, Gisele A. ^[1] ; Bordon, Karla C. F. ^[1] ; Silva, Ronivaldo R. ^[2] ; Gomes, Mario S. R. ^{[3, 4]} ; Cabral, Hamilton ^[5] ; Rodrigues, Veridiana M. ^[3] ; Ueberheide, Beatrix ^[6] ; Arantes, Eliane C. ^[1]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Sch Pharmaceut Sci Ribeirao Preto, Dept Phys & Chem, Av Cafe S-N, BR-14040903 Ribeirao Preto, SP - Brazil

^[2] Univ Estadual Paulista, Inst Biosci Letters & Exact Sci, Rua Cristovao Colombo 2265, BR-15054000 Sao Jose Do Rio Preto, SP - Brazil

^[3] Univ Fed Uberlandia, Inst Genet & Biochem, Av Para 1720, BR-38400902 Uberlandia, MG - Brazil

^[4] State Univ Southwest Bahia, Dept Chem & Phys, Rua Jose Moreira Sobrinho, Ate 873 874, BR-45506210 Jequie, BA - Brazil

^[5] Univ Sao Paulo, Sch Pharmaceut Sci Ribeirao Preto, Dept Pharmaceut Sci, Av Cafe S-N, BR-14040903 Ribeirao Preto, SP - Brazil

^[6] NYU, Langone Med Ctr, Prote Resource Ctr, 430 East 29th St, New York, NY 10016 - USA

• Tipo de documento: Artigo Científico
• Fonte: Journal of Venomous Animals and Toxins including Tropical Diseases; v. 25, APR 15 2019.
• Citações Web of Science: 0

Resumo

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. (AU)

• Processo FAPESP: 10/06199-5 - "Purificação e caracterização bioquímica da hialuronidase presente na peçonha de Lachesis muta"
• Beneficiário: Gisele Adriano Wiezel
• Modalidade de apoio: Bolsas no Brasil - Iniciação Científica
• Processo FAPESP: 14/23285-3 - Caracterização de modificações pós-traducionais de uma L-aminoácido oxidase isolada da peçonha da serpente Crotalus durissus terrificus
• Beneficiário: Gisele Adriano Wiezel
• Modalidade de apoio: Bolsas no Exterior - Estágio de Pesquisa - Mestrado
• Processo FAPESP: 15/18432-0 - Bioprospecção de toxinas animais com interesse biotecnológico a partir de ferramentas ômicas
• Beneficiário: Eliane Candiani Arantes Braga
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 11/23236-4 - Toxinas animais nativas e recombinantes: análise funcional, estrutural e molecular
• Beneficiário: Suely Vilela
• Modalidade de apoio: Auxílio à Pesquisa - Temático