Caracterização química e biológica da secreção cutânea do anuro Leptodactylus labyrinthicus: peptídeos antimicrobianos e anticarcinogênicos, fosfolipases e peptidases

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Animal, 2008.Atualmente, o surgimento de resistência às drogas disponíveis no mercado pelos microorganismos e pelas células de câncer tornou o desenvolvimento de novas formas terapêuticas imprescindível. A secreção cutânea dos anfíbios é uma rica fonte natural de peptídeos com atividades anticarcinogênica (PAC) e antimicrobiana (PAM). Entretanto, o veneno desses animais também é constituído de outras macromoléculas, por exemplo, enzimas, as quais têm sido pouco investigadas. O presente estudo teve como objetivo caracterizar, de forma geral, a secreção glandular da rã-pimenta Leptodactylus labyrinthicus. Especificamente, buscou-se identificar e caracterizar química e biologicamente alguns dos peptídeos presentes, além de investigar a presença de atividades enzimáticas. O estudo revelou a ocorrência de inúmeros peptídeos e proteínas, demonstrando a riqueza e o potencial biológico desse veneno. Os dois peptídeos antimicrobianos purificados foram fallaxin e pentadactylin, os quais já haviam sido isolados de outras espécies do gênero Leptodactylus. Ambos apresentaram atividade contra bactérias Gram-positivas e Gram-negativas; e fallaxin foi identificado como fracamente hemolítico. Como vários PAMs de amplo espectro de ação também são citotóxicos para células de câncer, os dois peptídeos isolados foram testados contra as linhagens celulares de câncer HeLa, MCF-7 e B16F10, demonstrando-se efetivos contra todas as linhagens. Observou-se também que os dois PACs são pouco seletivos, pois foram mais tóxicos para células normais de fibroblasto (FHN) do que para as células tumorais. Portanto, o uso terapêutico desses PACs deverá ser via estratégias que aumentem a especificidade dos mesmos. Os estudos de mecanismo de ação com pentadactylin não foram conclusivos, sugerindo a morte de células de MCF-7 via apoptose, podendo ser seguida pelo processo necrótico. Além desses, o fragmento Flx-16 de fallaxin e o peptídeo PGL-Ll, rico em glicina e leucina, foram testados, contudo nenhuma atividade biológica foi detectada. A investigação da atividade enzimática da secreção revelou a presença de enzimas proteolíticas pertencentes aos clãs das metalo- e serino-peptidases e que atuam de maneira ótima em pH neutro a ligeiramente básico; e de fosfolipases do tipo A2. O estudo e caracterização da composição da secreção cutânea de anfíbios são de extrema relevância, podendo ajudar a esclarecer questões taxonômicas, identificar drogas terapêuticas em potencial, além de auxiliar no entendimento da biologia dos anfíbios. _________________________________________________________________________________ ABSTRACTNowadays, the emergence of resistance to the current available chemotherapeutic drugs by microorganisms and cancer cells make the development of new agents imperative. The skin secretion of amphibians is a natural rich source of antimicrobial (PAM) and anticancer peptides (PAC). Nevertheless, the venom of these animals is also composed by other macromolecules, such as enzymes, which have not been studied extensively yet. The aim of this study was to characterize the glandular secretion of the pepper-frog Leptodactylus labyrinthicus, targeting to identify and characterize chemically and biologically some of the peptides present, besides detecting enzymatic activities. The research has found a variety of peptides and proteins, showing the richness and the biological potential of this venom. Two PAMs were purified, fallaxin and pentadactylin, which have already been isolated from other species of the genus Leptodactylus. Both peptides were active against Gram-positive and Gram-negative bacterias; moreover, fallaxin showed a weak hemolytic activity. As some PAMs of broad spectrum of action are also toxic to cancer cells, the two peptides isolated were tested against the cancer cell lines HeLa, MCF-7 and B16F10, being effective against all of them. We noticed that both PACs aren’t specific, since they have been more toxic to normal fibroblast cells (FHN) than to tumor cells. Therefore, it is mandatory that the therapeutic use of these PACs be accompanied by strategies that enhance their selectiveness. The studies of action mechanism with pentadactylin were not conclusive, suggesting that MCF-7 cells die by apoptosis; however it may be followed by necrosis. The peptide PGL-Ll, rich in glycine and leucine, and the fragment Flx-16 from fallaxin were tested, though no biological activity was found. The research of enzyme activity of the secretion revealed the presence of proteolytic enzymes of the clans metallo- and serine-peptidases, and that they act optimally at neutral to slightly basic pH, and of phospholipases A2. The study and characterization of the skin secretion of amphibians is of extreme relevance, helping to elucidate taxonomic issues, identifying putative therapeutic drugs, besides helping to understand the biology of amphibians

The crude skin secretion of the pepper frog Leptodactylus labyrinthicus is rich in metallo and serine peptidases

Peptidases are ubiquitous enzymes involved in diverse biological processes. Fragments from bioactive peptides have been found in skin secretions from frogs, and their presence suggests processing by peptidases. Thus, the aim of this work was to characterize the peptidase activity present in the skin secretion of Leptodactylus labyrinthicus. Zymography revealed the presence of three bands of gelatinase activity of approximately 60 kDa, 66 kDa, and 80 kDa, which the first two were calcium-dependent. These three bands were inhibited either by ethylenediaminetetraacetic acid (EDTA) and phenathroline; thus, they were characterized as metallopeptidases. Furthermore, the proteolytic enzymes identified were active only at pH 6.0–10.0, and their activity increased in the presence of CHAPS or NaCl. Experiments with fluorogenic substrates incubated with skin secretions identified aminopeptidase activity, with cleavage after leucine, proline, and alanine residues. This activity was directly proportional to the protein concentration, and it was inhibited in the presence of metallo and serine peptidase inhibitors. Besides, the optimal pH for substrate cleavage was determined to be 7.0–8.0. The results of the in gel activity assay showed that all substrates were hydrolyzed by a 45 kDa peptidase. Gly-Pro-AMC was also cleaved by a peptidase greater than 97 kDa. The data suggest the presence of dipeptidyl peptidases (DPPs) and metallopeptidases; however, further research is necessary. In conclusion, our work will help to elucidate the implication of these enzymatic activities in the processing of the bioactive peptides present in frog venom, expanding the knowledge of amphibian biology

The pH dependence of the proteolytic activity present in the crude skin secretions of <i>L. labyrinthicus</i> on fluorogenic substrates.

<p>(A) Gly-Pro-AMC; (B) l-Leu-AMC; (C) l-Alanyl-l-Alanyl-l-Phe-Ala-AMC. The assays were performed in AMT buffer (100 mM acetic acid, 100 mM MES, and 100 mM Tris-base). The results are expressed as the percentage of the maximum activity obtained. Each point represents mean ± SD.</p

Proteolytic activity of the crude skin secretions of <i>L. labyrinthicus</i> (40 µg) on 9% SDS-PAGE incubated for 15 min with fluorogenic substrates.

<p>(Lanes 1 and 2) silver stained; (Lanes 3 and 4) Gly-Pro-AMC; (Lanes 5 and 6) l-Leu-AMC; (Lanes 7 and 8) l-Alanyl-l-Alanyl-l-Phe-Ala-AMC. Even numbers are boiled skin secretions incubated with the substrates. Mw: molecular weight markers.</p

Analysis of the pH dependence for the gelatinase activity present in the crude skin secretions of <i>L. labyrinthicus</i>.

<p>The gels (9% SDS-PAGE/gelatin) were incubated for 22 h under the following conditions: 10 mM CaCl₂, 50 mM sodium acetate, pH 4.0–5.0; 10 mM CaCl₂, 50 mM HEPES, pH 6.0–7.0; and 10 mM CaCl₂, 50 mM Tris-HCl, pH 8.0–10.0. The bands were quantified by densitometry and the values were normalized in relation to pH 10.0 gel. The values are shown close to their respective bands. Mw: molecular weight markers.</p

Specificity of the peptidases present in the crude skin secretions of <i>L. labyrinthicus</i> on various fluorescent synthetic substrates.

a<p>Expressed as a percentage of Gly-Pro-AMC hydrolyzing activity, which was taken as 100%.</p

Influence of protein concentration of the crude skin secretions of <i>L. labyrinthicus</i> on the proteolysis (mUF/min) of three fluorogenic substrates: Gly-Pro-AMC; l-Leu-AMC, and l-Alanyl-l-Alanyl-l-Phe-Ala-AMC.

<p>The assay was performed in 25± standard deviation (SD). mUF/min = milli units of fluorescence liberated per minute.</p

Effect of different inhibitors on the peptidases present in the crude skin secretions of <i>L. labyrinthicus</i> (25.6 µg) on the hydrolysis of Gly-Pro-AMC, l-Leu-AMC and l-Alanyl-l-Alanyl-l-Phe-Ala-AMC.

<p>The incubation was performed in 50 mM HEPES buffer, pH 7.5 for 45 min.</p