RESUMO
Myonecrosis, in addition to edema and other biological manifestations, are conspicuous effects of Bothrops snake venoms, some of them caused by phospholipases A(2) (PLA(2)s). Asp49-PLA(2)s are catalytically active, whereas Lys49-PLA(2)s, although highly toxic, have little or no enzymatic activity upon artificial substrates, due to a substitution of lysine for aspartic acid at position 49. Crotapotin (CA), the acidic counterpart of crotoxin PLA(2) (CB), is a PLA(2)-like protein from Crotalus durissus terrificus snake venom, and is considered a chaperone protein for CB, able to increase its lethality about ten fold, but to inhibit the formation of the rat paw edema induced by carrageenin and by snake venoms. In this study, we demonstrate that CA significantly inhibits the edema induced by BthTX-I (23% inhibition), BthTX-II (27%), PrTX-I (25%), PrTX-III (35%) and MjTX-II (10%) on the mouse paw. CK levels evoked by isolated Asp49 or Lys49-PLA(2)s were reduced by 40% to 54% in the presence of CA and, in all cases, the membrane damaging activity of the toxins was also reduced. Circular dichroism spectra of the PLA(2)s in the presence and absence of CA showed that there was not any detectable secondary structural modification due to association between CA and the myotoxins. However, Fourier Transformed Infrared (FT-IR) analysis indicated that ionic and hydrophobic contacts contributed to stabilize this interaction.
Assuntos
Bothrops , Venenos de Crotalídeos/enzimologia , Crotoxina/toxicidade , Inibidores Enzimáticos/toxicidade , Músculo Esquelético/efeitos dos fármacos , Fosfolipases A/antagonistas & inibidores , Animais , Dicroísmo Circular/métodos , Creatina Quinase/metabolismo , Crotoxina/análise , Relação Dose-Resposta a Droga , Edema/induzido quimicamente , Edema/patologia , Inibidores Enzimáticos/análise , Membro Posterior , Masculino , Camundongos , Músculo Esquelético/enzimologia , Músculo Esquelético/patologia , Fosfolipases A/análise , Fosfolipases A/classificação , Isoformas de Proteínas , Espectroscopia de Infravermelho com Transformada de Fourier/métodosRESUMO
Considerable progress has been made in characterizing the individual participant enzymes and their relative contributions in the generation of eicosanoids, lipid mediators derived from arachidonic acid, such as prostaglandins and leukotrienes. However, the role of individual phospholipase (PL) A(2) enzymes in providing arachidonic acid to the downstream enzymes for eicosanoid generation in biologic processes has not been fully elucidated. In this review, we will provide an overview of the classification of the families of PLA(2) enzymes, their putative mechanisms of action, and their role(s) in eicosanoid generation and inflammation.
Assuntos
Fosfolipases A/fisiologia , Animais , Araquidonato 5-Lipoxigenase/metabolismo , Ácidos Araquidônicos/biossíntese , Ácidos Araquidônicos/metabolismo , Membrana Celular/enzimologia , Inflamação/enzimologia , Inflamação/prevenção & controle , Camundongos , Camundongos Knockout , Neoplasias/enzimologia , Neoplasias/prevenção & controle , Fosfolipases A/classificação , Fosfolipases A/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismoRESUMO
In order to analyze its structure-function relationships, the complete amino acid sequence of myotoxin II from Atropoides (Bothrops) nummifer from Costa Rica was determined. This toxin is a Lys49-type phospholipase A(2) (PLA(2)) homologue, devoid of catalytic activity, structurally belonging to class IIA. In addition to the Asp49 --> Lys change in the (inactive) catalytic center, substitutions in the calcium-binding loop suggest that its lack of enzymatic activity is due to the loss of ability to bind Ca(2+). The toxin occurs as a homodimer of basic subunits of 121 residues. Its sequence has highest similarity to Lys49 PLA(2)s from Cerrophidion, Trimeresurus, Bothrops and Agkistrodon species, which form a subfamily of proteins that diverged early from Asp49 PLA(2)s present in the same species, as shown by phylogenetic analysis. The tertiary structure of the toxin was modeled, based on the coordinates of Cerrophidion godmani myotoxin II. Its exposed C-terminal region 115-129 shows several differences in comparison to the homologous sequences of other Lys49 PLA(2)s, i.e. from Agkistrodon p. piscivorus and Bothrops asper. Region 115-129 of the latter two proteins has been implicated in myotoxic activity, on the basis of the direct membrane-damaging of their corresponding synthetic peptides. However, peptide 115-129 of A. nummifer myotoxin II did not exert toxicity upon cultured skeletal muscle cells or mature muscle in vivo. Differences in several amino acid residues, either critical for toxicity, or influencing the conformation of free peptide 115-129 from A. nummifer myotoxin II, may account for its lack of direct membrane-damaging properties.
Assuntos
Venenos de Crotalídeos/química , Lisina/química , Fosfolipases A/química , Sequência de Aminoácidos , Animais , Bothrops , Cromatografia por Troca Iônica , Fosfolipases A2 do Grupo II , Modelos Moleculares , Dados de Sequência Molecular , Fosfolipases A/classificação , Fosfolipases A/isolamento & purificação , Filogenia , Conformação Proteica , Proteínas de Répteis , Homologia de Sequência de AminoácidosRESUMO
Nigroxins A and B, two myotoxic phospholipases A2 (PLA2s) from the venom of the American elapid Micrurus nigrocinctus, belong to a new PLA2 subclass. Their primary structures were established and compared with those of PLA2s that have already been studied with respect to myotoxic activity. The combination of amino acid residues Arg15, Ala100, Asn108 and a hydrophobic residue at position 109 is present exclusively in class I PLA2s that display myotoxic activity. These residues cluster within a surface region rich in positive charges and are suggested to play a role in the interaction with the target membrane of the muscle fibers. It is concluded that the myotoxic PLA2s resulted from recruitment of an ancient scaffold. Dendrotoxins and alpha-neurotoxins are similarly derived from other old structures, which are, however, now also present in nontoxic proteins that are widely distributed throughout the animal kingdom. The evolutionary pathways by which elapid PLA2s acquired myotoxicity and dendrotoxins acquired K+-channel blocker activity are traced. They demonstrate how existing scaffolds were adapted stepwise to serve toxic functions by exchange of a few surface-exposed residues.