Estudo da variação na composição da Crotapotina no veneno de Crotalus durissus terrificus da região de Botucatu - SP
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2017-07-27
Autores
Oliveira, Laudicéia Alves de [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O veneno de Crotalus durissus terrificus (Cdt), baseado no critério do perfil da cromatografia de exclusão molecular, pode ser descrito como sendo composto de quatro principais toxinas: Giroxina, Crotamina, Crotoxina e Convulxina. A crotoxina é uma neutoroxina, é um heterodímero não covalente, composta por duas subunidades: fosfolipase A2 (PLA2) e crotapotina (Ctp). A crotapotina constitui o componente da crotoxina que apresenta atividade anti-inflamatória além de ser chaperona de PLA2. Esta molécula é constituída por três cadeias peptídicas ligadas por pontes dissulfeto. Além disso, já foram descritas isoformas para a crotapotina. O objetivo deste estudo foi desenvolver um método cromatográfico para isolar a crotapotina, separar suas cadeias e analisá-las por LC/MS e LC-MS/MS para análise proteômica e/ou seqüenciamento de novo e avaliação da possível existência de um padrão de variação nas substituições de aminoácidos. O método C8-RP-HPLC foi capaz de separar os componentes do veneno de Cdt que puderam ser identificados por espectrometria de massas, de acordo com a sua massa molecular. A crotapotina foi isolada, reduzida e alquilada e submetida à outra separação cromatográfica RP-HPLC (C18) para isolamento das subunidades alquiladas. Conforme descrito na literatura, a cadeia α da crotapotina é composta por 40 resíduos de aminoácidos, enquanto que a cadeia β possui 35 resíduos e a cadeia γ 14, pelo que as subunidades podem ser identificadas pelas suas massas moleculares. As análises das cadeias isoladas de crotapotina mostraram a presença de mais de uma molécula por fração, indicando a presença de isoformas no veneno. Análises proteômicas e sequenciamento de novo por LC-MS/MS elucidaram a existência de variações de resíduos de aminoácidos em tais cadeias. Estudos de atividade biológica poderão elucidar as diferenças que tais isoformas apresentam em suas características intrínsecas.
The Crotalus durissus terrificus venom (Cdt), based on molecular size chromatography, can be described as being composed of four main toxins: Giroxin, Crotamine, Crotoxin and Convulxin. Crotoxin is a neutoroxin, a non-covalent heterodimer composed of two subunits: phospholipase A2 (PLA2) and crotapotin (Ctp). Crotapotin is the component of crotaxin that has anti-inflammatory activity besides being a PLA2 chaperone. This molecule consists of three peptide chains linked by disulfide bonds. In addition, isoforms for crotapotin have been described. The objective of this study was to develop a chromatographic methodology to isolate a crotapotin, separate its chains and analyze by LC/MS and LC-MS/MS for proteomic analysis and/or de novo sequencing and evaluation of the possibility of a variation pattern amino acid substitutions. The C8-RPHPLC method was able to separate the Cdt venom components that could be identified by mass spectrometry, according to their molecular mass. The crotapotin was isolated, reduced and alkylated and subjected to another RP-HPLC (C18) chromatographic separation for isolation of the alkylated subunits. As described in the literature, the α chain of crotapotin is composed of 40 amino acid residues, while the β chain has 35 residues and the γ chain, 14, and thus the subunits can be identified by their molecular masses. The analyzes of isolated chains of crotapotin showed the presence of more than one molecule per fraction, indicating the presence of isoforms in the venom. Protein analyzes and de novo sequencing by LC-MS / MS elucidated a existence of variations of amino acid residues in such chains. Studies of biological activity are to elucidate how the differences that such isoforms present in their intrinsic characteristics.
The Crotalus durissus terrificus venom (Cdt), based on molecular size chromatography, can be described as being composed of four main toxins: Giroxin, Crotamine, Crotoxin and Convulxin. Crotoxin is a neutoroxin, a non-covalent heterodimer composed of two subunits: phospholipase A2 (PLA2) and crotapotin (Ctp). Crotapotin is the component of crotaxin that has anti-inflammatory activity besides being a PLA2 chaperone. This molecule consists of three peptide chains linked by disulfide bonds. In addition, isoforms for crotapotin have been described. The objective of this study was to develop a chromatographic methodology to isolate a crotapotin, separate its chains and analyze by LC/MS and LC-MS/MS for proteomic analysis and/or de novo sequencing and evaluation of the possibility of a variation pattern amino acid substitutions. The C8-RPHPLC method was able to separate the Cdt venom components that could be identified by mass spectrometry, according to their molecular mass. The crotapotin was isolated, reduced and alkylated and subjected to another RP-HPLC (C18) chromatographic separation for isolation of the alkylated subunits. As described in the literature, the α chain of crotapotin is composed of 40 amino acid residues, while the β chain has 35 residues and the γ chain, 14, and thus the subunits can be identified by their molecular masses. The analyzes of isolated chains of crotapotin showed the presence of more than one molecule per fraction, indicating the presence of isoforms in the venom. Protein analyzes and de novo sequencing by LC-MS / MS elucidated a existence of variations of amino acid residues in such chains. Studies of biological activity are to elucidate how the differences that such isoforms present in their intrinsic characteristics.
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Palavras-chave
Crotalus durissus terrificus, Veneno, Crotoxina, Crotapotina, Isoformas, Venom, Crotoxin, Crotapotin, Isoform