Bactericidal and neurotoxic activities of two myotoxic phospholipases A2 from Bothrops neuwiedi pauloensis snake venom.
Two basic myotoxic PLA(2)s, namely BnpTX-I and II, were isolated from Bothrops neuwiedi pauloensis snake venom through three chromatographic steps: ion-exchange chromatography on CM-Sepharose, gel filtration on Sephadex G-50 and reverse phase HPLC on a C18 column. Both PLA(2)s showed a M(r) around 14,000 for the monomer and 28,000 for the dimer (as estimated by SDS-PAGE), pI approximately 7.8 and approximately 121 amino acid residues cross-linked by seven disulfide bonds. The N-terminal sequences revealed significant homology with Asp49 basic myotoxic PLA(2)s from other snake venoms. The catalytic and anticoagulant activities of BnpTX-I were higher than those of BnpTX-II. Both were able to induce cytotoxicity in vitro, as well as, myotoxicity, edema and lethality in mice. BnpTX-I also induced neurotoxic effect on mouse neuromuscular preparations and bactericidal activity on Eschericia coli and Staphylococcus aureus. After chemical modification of BnpTX-I with BPB or incubation with EDTA or Mn(2+) ions, the catalytic activity was completely abolished, while the toxic and pharmacological activities were partially reduced. Interaction with heparin inhibited the cytotoxic and bactericidal effects. Anti-BthTX-I, anti-BthTX-II and anti-115-129-C terminal antibodies strongly recognize both BnpTX-I and II. It is shown that the neurotoxic effect induced by B. neuwiedi pauloensis venom is due to the presence of myotoxic PLA(2)s. The data also corroborate the hypothesis of a partial dissociation between toxic and enzymatic domains. In addition, BnpTX-I displays a heparin binding C-terminal region, which is probably responsible for the cytotoxic and bactericidal effects.
Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, UFU, Uberlândia, MG, Brazil.
Veridiana M
VM
Rodrigues
Silvana
S
Marcussi
Rafael S
RS
Cambraia
Ana L
AL
de Araújo
Natael R
NR
Malta-Neto
Amélia
A
Hamaguchi
Eloísa A V
EA
Ferro
Maria I
MI
Homsi-Brandeburgo
José R
JR
Giglio
Andreimar M
AM
Soares
D000595:Amino Acid Sequence; D000704:Analysis of Variance; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D017837:Bothrops; D001938:Brazil; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003402:Creatine Kinase / Q000378:metabolism; D003435:Crotalid Venoms / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D004487:Edema / Q000139:chemically induced; D004492:Edetic Acid / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects; D007118:Immunoassay; D051379:Mice; D008969:Molecular Sequence Data; D009132:Muscles / Q000187:drug effects / Q000473:pathology; D032446:Myoblasts / Q000187:drug effects; D009336:Necrosis; D010741:Phospholipases A / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D010791:Phrenic Nerve / Q000187:drug effects; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013211:Staphylococcus aureus / Q000187:drug effects
D000906:Antibodies; D003435:Crotalid Venoms; D004492:Edetic Acid; D003402:Creatine Kinase; D010741:Phospholipases A