title: Synergism between basic Asp49 and Lys49 phospholipase A2 myotoxins of viperid snake venom in vitro and in vivo.
abstract: Two subtypes of phospholipases A2 (PLA2s) with the ability to induce myonecrosis, 'Asp49' and 'Lys49' myotoxins, often coexist in viperid snake venoms. Since the latter lack catalytic activity, two different mechanisms are involved in their myotoxicity. A synergism between Asp49 and Lys49 myotoxins from Bothrops asper was previously observed in vitro, enhancing Ca2+ entry and cell death when acting together upon C2C12 myotubes. These observations are extended for the first time in vivo, by demonstrating a clear enhancement of myonecrosis by the combined action of these two toxins in mice. In addition, novel aspects of their synergism were revealed using myotubes. Proportions of Asp49 myotoxin as low as 0.1% of the Lys49 myotoxin are sufficient to enhance cytotoxicity of the latter, but not the opposite. Sublytic amounts of Asp49 myotoxin also enhanced cytotoxicity of a synthetic peptide encompassing the toxic region of Lys49 myotoxin. Asp49 myotoxin rendered myotubes more susceptible to osmotic lysis, whereas Lys49 myotoxin did not. In contrast to myotoxic Asp49 PLA2, an acidic non-toxic PLA2 from the same venom did not markedly synergize with Lys49 myotoxin, revealing a functional difference between basic and acidic PLA2 enzymes. It is suggested that Asp49 myotoxins synergize with Lys49 myotoxins by virtue of their PLA2 activity. In addition to the membrane-destabilizing effect of this activity, Asp49 myotoxins may generate anionic patches of hydrolytic reaction products, facilitating electrostatic interactions with Lys49 myotoxins. These data provide new evidence for the evolutionary adaptive value of the two subtypes of PLA2 myotoxins acting synergistically in viperid venoms.
authors.affiliation: Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica.
authors.forename: Diana
authors.initials: D
authors.lastname: Mora-Obando
authors.affiliation: Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica.
authors.forename: Julián
authors.initials: J
authors.lastname: Fernández
authors.affiliation: Department of Biomedical Sciences, University of Padova, Padova, Italy.
authors.forename: Cesare
authors.initials: C
authors.lastname: Montecucco
authors.affiliation: Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica.
authors.forename: José María
authors.initials: JM
authors.lastname: Gutiérrez
authors.affiliation: Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica.
authors.forename: Bruno
authors.initials: B
authors.lastname: Lomonte
mesh_terms: D000098:Acetophenones / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D017837:Bothrops / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D003402:Creatine Kinase / Q000097:blood; D003435:Crotalid Venoms / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000633:toxicity*; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000737:chemistry; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000633:toxicity; D008239:Lysine / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D018485:Muscle Fibers, Skeletal / Q000187:drug effects* / Q000473:pathology; D018482:Muscle, Skeletal / Q000187:drug effects / Q000201:enzymology / Q000473:pathology; D009498:Neurotoxins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000633:toxicity*; D054467:Phospholipases A2 / Q000737:chemistry / Q000302:isolation & purification / Q000633:toxicity*; D030162:Reptilian Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000633:toxicity*; D013329:Structure-Activity Relationship
chemical_list: D000098:Acetophenones; D003435:Crotalid Venoms; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009498:Neurotoxins; D030162:Reptilian Proteins; D001224:Aspartic Acid; C018932:myotoxin A; D003402:Creatine Kinase; D054467:Phospholipases A2; D008239:Lysine; C006821:4-bromophenacyl bromide