RESUMO
A novel antimicrobial peptide, anoplin, was purified from the venom of the solitary wasp Anoplius samariensis. The sequence was mostly analyzed by mass spectrometry, which was corroborated by solid-phase synthesis. Anoplin, composed of 10 amino acid residues, Gly-Leu-Leu-Lys-Arg-Ile-Lys-Thr-Leu-Leu-NH2, has a high homology to crabrolin and mastoparan-X, the mast cell degranulating peptides from social wasp venoms, and, therefore, can be predicted to adopt an amphipathic alpha-helix secondary structure. In fact, the circular dichroism (CD) spectra of anoplin in the presence of trifluoroethanol or sodium dodecyl sulfate showed a high content, up to 55%, of the alpha-helical conformation. A modeling study of anoplin based on its homology to mastoparan-X supported the CD results. Biological evaluation using the synthetic peptide revealed that this peptide exhibited potent activity in stimulating degranulation from rat peritoneal mast cells and broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria. Therefore, this is the first antimicrobial component to be found in the solitary wasp venom and it may play a key role in preventing potential infection by microorganisms during prey consumption by their larvae. Moreover, this peptide is the smallest among the linear alpha-helical antimicrobial peptides hitherto found in nature, which is advantageous for chemical manipulation and medical application.
Assuntos
Antibacterianos/isolamento & purificação , Oligopeptídeos/química , Oligopeptídeos/isolamento & purificação , Venenos de Vespas/química , Venenos de Vespas/isolamento & purificação , Sequência de Aminoácidos , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos , Degranulação Celular , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Feminino , Mastócitos/efeitos dos fármacos , Mastócitos/fisiologia , Testes de Sensibilidade Microbiana , Modelos Moleculares , Oligopeptídeos/farmacologia , Ratos , Alinhamento de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Venenos de Vespas/farmacologia , VespasRESUMO
The molecular structure of human uropepsin, an aspartic proteinase from the urine produced in the form of pepsinogen A in the gastric mucosa, has been determined by molecular replacement using human pepsin as the search model. Crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 50.99, b = 75.56, c = 89.90 A. Crystallographic refinement led to an R factor of 0.161 at 2.45 A resolution. The positions of 2437 non-H protein atoms in 326 residues have been determined and the model contains 143 water molecules. The structure is bilobal, consisting of two predominantly beta-sheet lobes which, as observed in other aspartic proteinases, are related by a pseudo-twofold axis. A model of the uropepsin-pepstatin complex has been constructed based on the high-resolution crystal structure of pepsin complexed with pepstatin.
Assuntos
Endopeptidases/química , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Cristalização , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Pepstatinas/metabolismo , Conformação Proteica , Controle de Qualidade , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Mastoparans are tetradecapeptides found to be the major component of vespid venoms. These peptides present a wide spectrum of biological activities, such as mast cell degranulation, hemolytic activity and also reveals antimicrobial activity. A mastoparan toxin isolated from the venom of Anterhynchium flavomarginatum micado has been crystallized. At room temperature these crystals diffracted to 2.8 A resolution. However, upon cooling to cryogenic temperature around 85 K, the original resolution limit could be improved to 2.0 A. Crystals were determined to belong to the space group P3(1) (P3(2)). This is the first mastoparan to be crystallized and it will provide further insights in the conformational significance of mastoparan toxins, with respect to their potency and activity in G protein regulation.
Assuntos
Cristalografia por Raios X/métodos , Proteínas de Insetos/química , Venenos de Vespas/química , Vespas/metabolismo , Animais , Temperatura Baixa , Cristalização , Proteínas de Insetos/isolamento & purificação , Venenos de Vespas/isolamento & purificaçãoRESUMO
Mastoparans are tetradecapeptides found to be the major component of vespid venoms. A mastoparan toxin isolated from the venom of Anterhynchium flavomarginatum micado has been crystallized and X-ray diffraction data collected to 2.7 A resolution using a synchrotron-radiation source. Crystals were determined to belong to the space group P6(2)22 (P6(4)22). This is the first mastoparan to be crystallized and will provide further insights into the conformational significance of mastoparan toxins with respect to their potency and activity in G-protein regulation.