Purification and enzymatic characterization of a novel metalloprotease from Lachesis muta rhombeata snake venom

Author(s)

Cordeiro, Francielle Almeida ^[1] ; Coutinho, Barbara Marques ^[1] ; Wiezel, Gisele Adriano ^[1] ; Figueiredo Bordon, Karla de Castro ^[1] ; Bregge-Silva, Cristiane ^[2] ; Rosa-Garzon, Nathalia Gonsales ^[3] ; Cabral, Hamilton ^[3] ; Ueberheide, Beatrix ^[4] ; Arantes, Eliane Candiani ^[1]

Affiliation

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

^[2] Univ Latina Costa Rica, San Jose - Costa Rica

^[3] Univ Sao Paulo, Sch Pharmaceut Sci Ribeirao Preto, Dept Pharmaceut Sci, Ribeirao Preto, SP - Brazil

^[4] NYU, Prote Resource Ctr, Langone Med Ctr, 430 East 29th St, New York, NY 10016 - USA

• Document type: Journal article
• Source: Journal of Venomous Animals and Toxins including Tropical Diseases; v. 24, NOV 22 2018.
• Web of Science Citations: 0

Abstract

Abstract Background: Lachesis muta rhombeata (Lmr) is the largest venomous snake in Latin America and its venom contains mainly enzymatic components, such as serine and metalloproteases, L-amino acid oxidase and phospholipases A2. Metalloproteases comprise a large group of zinc-dependent proteases that cleave basement membrane components such as fibronectin, laminin and collagen type IV. These enzymes are responsible for local and systemic changes, including haemorrhage, myonecrosis and inflammation. This study aimed the isolation and enzymatic characterization of the first metalloprotease (Lmr-MP) from Lmr venom (LmrV). Methods and results: Lmr-MP was purified through two chromatographic steps and submitted to enzymatic characterization. It showed proteolytic activity on azocasein with maximum activity at pH 7.0-9.0. It was inhibited by EDTA (a metal chelator that removes zinc, which is essential for enzymatic activity) and no effect was observed with PMSF, iodoacetic acid or pepstatin (inhibitors of serine, cysteine and aspartyl proteases, respectively). Ca2+, Mg2+ and Ba2+ ions increased its activity, while Al3+, Cu2+, Ni2+ and Zn2+ inhibited it. Additionally, ZnCl2 showed a dose dependent inhibition of the enzyme. Lmr-MP activity was also evaluated upon chromogenic substrates for plasma kallikrein (S-2302), plasmin and streptokinase-activated plasminogen (S-2251) and Factor Xa (S-2222) showing the highest activity on S-2302. The activity in different solutions (5 mM or 50 mM ammonium bicarbonate, pH 7.8; 0.1% trifluoroacetic acid + 50% acetonitrile; phosphate buffer saline, pH 7.4; 50 mM sodium acetate, pH 4.0 or ammonium acetate pH 4.5) was also evaluated and the results showed that its activity was abolished at acidic pHs. Its molecular mass (22,858 Da) was determined by MALDI-TOF and about 90% of its primary structure was verified by high-resolution mass spectrometry using HCD and ETD fragmentations and database search against the sequence of closely related species. It is a novel enzyme which shared high identity with other snake venom metalloproteases (svMPs) belonging to the P-I group. Conclusion: The purification procedure achieved a novel pure highly active metalloprotease from LmrV. This new molecule can help to understand the metalloproteases mechanisms of action, the Lachesis envenoming, as well as to open new perspectives for its use as therapeutic tools. (AU)

• FAPESP's process: 14/23285-3 - Characterization of post-translation modifications of an L-amino acid oxidase isolated from Crotalus durissus terrificus snake venom
• Grantee: Gisele Adriano Wiezel
• Support Opportunities: Scholarships abroad - Research Internship - Master's degree
• 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