Synergism between baltergin metalloproteinase and Ba SPII RP4 PLA(2) from Bothrops alternatus venom on skeletal muscle (C2C12) cells

Acute muscle damage, myonecrosis, is one of the main characteristics of envenoming by Bothrops genus. In this in vitro study we investigated the role of a metalloproteinase (baltergin) and an acidic phospholipase A(2) (Ba SPII RP4) in the cytotoxicity exhibited by Bothrops alternatus venom. Baltergin metalloproteinase purified from the venom exerted a toxic effect on C2C12 myoblast cells (CC50: 583.34 mu g/mL) which involved morphological alterations compatible with apoptosis/anoikis. On the contrary, the most abundant PLA(2) isolated from this venom did not exhibit cytotoxicity at times and doses tested. However, when myoblasts were treated with both enzymes together, synergic activity was demonstrated. Neutralization of the venom with specific antibodies (IgG anti-baltergin and IgG anti-PLA(2)) confirmed this synergism. (C) 2011 Elsevier Ltd. All rights reserved.592338343Secretaria General de Ciencia y Tecnica [PI B013-2010]Agencia de Promocion Cientifica y Tecnologica, FonCyT, y SGCyT UNNE [PICTO 2007-00143]Consejo Nacional de Investigaciones Cientificas y Tecnologicas (CONICET)Secretaria General de Ciencia y Tecnica [PI B013-2010]Agencia de Promocion Cientifica y Tecnologica, FonCyT, y SGCyT UNNE [PICTO 2007-00143

Isolation and functional characterization of a new acidic PLA(2) Ba SpII RP4 of the Bothrops alternatus snake venom from Argentina

An acidic protein with phospholipase A(2) activity was purified to homogeneity from the venom of the Northeast Argentinian viperid Bothrops alternatus by two chromatographic steps: a conventional gel filtration on Sephadex G-75 and reversed phase on C18 HPLC column. A molecular mass of 14185.48 Da was determined by mass spectrometry, displaying a homodimer conformation. The kinetic assay demonstrated a catalytically active phospholipase A(2) in correspondence with Asp49 PLA(2) group. The enzyme designated Ba SpII RP4 contains an amino acid composition of 121 residues and a calculated theoretical pI value of 4.88. Amino acid sequence alignments with other Bothrops PLA(2) revealed a high degree of homology sequence (90-56%). Ba SpII RP4 did not show myotoxic activity upon muscular fibers at doses up to 100 mu g i.m. route injection or lethal response when it was i.p. injected at the hightest dose of 200 mu g. This toxin generates slight biological activities like paw edema inflammation and a delay in the clotting time, although Ba SpII RP4 exhibited catalytic activity. The primary amino acid sequence, determined a quadruple-time of flight (Q-TOF) hybrid mass spectrometer Q-TOF Ultima from Micromass (Manchester, UK) equipped with a nano Zspray source operating in a positive ion mode and tandem mass spectrum, an ESI/MS mass spectrum (TOF MS mode) "de novo amino acid sequencing", also provides more database about the small group of the non-myotoxic PLA(2)s isolated up to the present. (C) 2010 Elsevier Ltd. All rights reserved.5616474Consejo Nacional de Investigaciones Cientificas y Tecnologicas - CONICET [PIP 6298]Secretaria General de Ciencia y Tecnica [PI 051-06]Universidad Nacional del Nordeste (UNNE), ArgentinaConsejo Nacional de Investigaciones Cientificas y Tecnologicas - CONICET [PIP 6298]Secretaria General de Ciencia y Tecnica [PI 051-06