RESUMO
The lectin from Platypodium elegans seeds (PELa) was purified by affinity chromatography in a mannose-agarose column. The lectin agglutinated rabbit erythrocytes and the agglutinating effect was inhibited by previous incubation with the glycoprotein fetuin, along with N-acetyl-d-glucosamine, D-mannose and its derivatives. The lectin maintained complete activity in temperatures ranging from 40 to 60°C and pH values ranging from 9 to 10. As a glycoprotein, PELa has a carbohydrate content of 2.2%, and its activity requires divalent cations such as Ca2+ and Mn2+. Based on SDS-PAGE, PELa displays a profile similar to that of other Dalbergieae lectins with the main chain of molecular mass around 30kDa and two subunits of 19kDa and 10 kDa each. Two-dimensional (2D) electrophoresis revealed the presence of isoforms with different isoelectric points, and high-performance size exclusion chromatography (HPSEC) was performed to confirm the purity of the sample. The lectin was immobilized in CNBr-activated Sepharose 4B and successfully captured fetuin in solution, demonstrating that this lectin remains active and capable of binding carbohydrates. PELa showed effects different from those of its recombinant form in both pro- and anti-inflammatory tests.
Assuntos
Edema/induzido quimicamente , Fabaceae/química , Proteínas Imobilizadas/farmacologia , Lectinas de Plantas/farmacologia , Proteínas Recombinantes/farmacologia , Sementes/química , Sefarose/química , Animais , Eritrócitos/efeitos dos fármacos , Eritrócitos/imunologia , Hemaglutinação/efeitos dos fármacos , Proteínas Imobilizadas/química , Masculino , Lectinas de Plantas/química , Coelhos , Ratos , Proteínas Recombinantes/químicaRESUMO
The PR-11 peptide corresponds to the N-terminal and active region of the endogenously synthesized PR-39 molecule, of porcine origin. It is known to possess various biological effects including antimicrobial properties, angiogenic and anti-inflammatory activities. Apart from its reported activity as a proteasome inhibitor, a more comprehensive understanding of its function, at the molecular level, is still lacking. In this study, we used a label-free shotgun strategy to evaluate the proteomic alterations caused by exposure of cultured fibroblasts to the peptide PR-11. This approach revealed that more than half of the identified molecules were related to signalling, transcription and translation. Proteins directly associated to regulation of angiogenesis and interaction with the hypoxia-inducible factor 1-α (HIF-1α) were significantly altered. In addition, at least three differentially expressed molecules of the NF-κB pathway were detected, suggesting an anti-inflammatory property of PR-11. At last, we demonstrated novel potential ligands of PR-11, through its immobilization for affinity chromatography. Among the eluted molecules, gC1qR, a known complement receptor, appeared markedly enriched. This provided preliminary evidence of a PR-11 ligand possibly involved in the internalization of this peptide. Altogether, our findings contributed to a better understanding of the cellular pathways affected by PR-39 derived molecules.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Transporte/metabolismo , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Imobilizadas/metabolismo , Proteínas Imobilizadas/farmacologia , Ligantes , Espectrometria de Massas , Proteínas Mitocondriais/metabolismo , NF-kappa B/metabolismo , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Inibidores de Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Proteoma/efeitos dos fármacos , Proteoma/metabolismo , Proteômica , Ratos , Ratos Wistar , SuínosRESUMO
This report details the development of thin films containing an antimicrobial peptide, specifically, dermaseptin 01 (GLWSTIKQKGKEAAIAAA-KAAGQAALGAL-NH2, [DRS 01]), and a natural polysaccharide, for a novel application in detecting the presence of Leishmania cells and maintaining anti-leishmanial activity. The peptide DRS 01 was immobilized in conjunction with natural cashew gum (CG) onto an indium tin oxide (ITO) substrate using the Layer-by-Layer (LbL) deposition technique. The LbL film ITO/CG/DRS 01, containing DRS 01 as the outer layer, was capable of detecting the presence of Leishmania cells and acting as an anti-leishmanial system. Detection was performed using cyclic voltammetry (CV) in phosphate buffer (pH7.2) in the presence of promastigote cells (0-10(7)cells/mL). The results showed a linear and inversely proportional relation between the concentration of Leishmania infantum protozoan cells and the measured current values obtained for the films, which was attributed to the effect of peptide-induced lysis of the cell membrane, and resulted in freed residues that were adsorbed on the electrode surface. With this, the paper shows a method using thin films with this new material to demonstrate the anti-leishmanial activity in vitro models of carpet-like mechanisms.
Assuntos
Anacardium/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Técnicas Eletroquímicas/instrumentação , Leishmania infantum/efeitos dos fármacos , Extratos Vegetais/farmacologia , Tripanossomicidas/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Desenho de Equipamento , Proteínas Imobilizadas/química , Proteínas Imobilizadas/farmacologia , Nanoestruturas/química , Extratos Vegetais/química , Tripanossomicidas/químicaRESUMO
Antimicrobial peptides (AMPs) are essential for the innate immune system of eukaryotes, imparting protection against pathogens and their proliferation in host organisms. The recent interest in AMPs as active materials in bionanostructures is due to the properties shown by these biological molecules, such as the presence of an alpha-helix structure and distribution of positive charges along the chain. In this study the antimicrobial peptide dermaseptin 01 (DS 01), from the skin secretion of Phyllomedusa hypochondrialis frogs was immobilized in nanostructured layered films in conjunction with nickel tetrasulfonated phthalocyanines. The leishmanicidal activity of DS 01 was confirmed using kinetic essays, in which DS 01 promoted death of all metacyclic promastigote cells in 45 minutes. Surprisingly, the immobilized DS 01 molecules displayed electroactivity, as revealed by electrochemical experiments, in which an oxidation peak at about 0.61 V was observed for a DS 01 monolayer deposited on top of a conductive electrode. Such electroactivity was used to investigate the sensing abilities of the nanostructured films toward Leishmania. We observed an increase in the oxidation current as a function of number of Leishmania cells in the electrolytic solution at concentrations down to 10(3) cells/mL. The latter is indicative that the use of AMPs immobilized in electroactive nanostructured films may be of interest for applications in the pharmaceutical industry and diagnosis. FROM THE CLINICAL EDITOR: The recent interest in Antimicrobial peptides (AMPs) as active materials in bionanostructures is due to the properties shown by these biological molecules. Leishmanicidal activity of a particular AMP is demonstrated in this paper.