RESUMEN
The Imbabura treefrog (Boana picturata) is an underexplored source of bioactive peptides. The combination of molecular cloning and mass spectrometry allowed us to identify three new peptide families, named "Picturins" (PTR), "Pictuseptins" (PTS), and "Boanins" (BNS). PTR is composed of three 25-mer peptides, characterized by the N-terminal sequence: GVFKDALKQ and the C-terminal sequence: AANALKPK. The sequences of PTR-1, -2 and - 3 are highly conserved only showing two divergent sites: (L/F) in position 10 and (K/Q) in position 17. PTS gathers six peptides. PTS -1, -2 and - 4 have 22 amino acid residues in length, while PTS -3, -5 and - 6 are composed of 26 residues. Whereas BNS are four 28-37 mer peptides, showing two conserved regions: the N-terminal sequence FLGAL and the C-terminal sequence KALNP. PTR-1 to 3 and PTS -1 to -3 were chemically synthetized and their antimicrobial and haemolytic activity was assessed. PTR displayed moderate activity against Escherichia coli (MIC 24.80 to 48.95 µM), while PTS showed a broad antimicrobial and antifungal effect. PTS-1 was the most active peptide against E. coli (6.8 µM) followed by PTS-3 (11.7 µM) and PTS-2 (14.24 µM). These peptides also showed low haemolytic activity, pointing to a favorable selectivity. Overall, new unique non-hemolytic and cationic peptide sequences were characterized that could be valuable for the next-generation of anti-infective drugs. Future functional studies should explore the pharmacological potential of Boanins to include them as antimicrobial scaffolds. BIOLOGICAL SIGNIFICANCE: Nature-inspired solutions have shown their importance mainly for the development of the pharmaceutical industry. Frog skin peptides are excellent examples of the biomedical potential of naturally evolved molecules for specific targets, including multi-resistant bacteria. The characterization of new chemical entities from poorly studied skin secretions of Ecuadorian biodiversity, such as B. picturata, represents an unprecedented opportunity to identify candidates to tackle global concerns, for instance, antibiotic resistance.
Asunto(s)
Antiinfecciosos , Péptidos Catiónicos Antimicrobianos , Animales , Antibacterianos/farmacología , Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Antimicrobianos , Anuros , Escherichia coli , Hemólisis , Humanos , Pruebas de Sensibilidad Microbiana , PielRESUMEN
Here we report two novel 17-mer amidated linear peptides (TsAP-1 and TsAP-2) whose structures were deduced from cDNAs cloned from a venom-derived cDNA library of the Brazilian yellow scorpion, Tityus serrulatus. Both mature peptides were structurally-characterised following their location in chromatographic fractions of venom and synthetic replicates of each were subjected to a range of biological assays. The peptides were each active against model test micro-organisms but with different potencies. TsAP-1 was of low potency against all three test organisms (MICs 120-160 µM), whereas TsAP-2 was of high potency against the Gram-positive bacterium, Staphylococcus aureus (MIC 5 µM) and the yeast, Candida albicans (10 µM). Haemolytic activity of TsAP-1 was low (4% at 160 µM) and in contrast, that of TsAP-2 was considerably higher (18% at 20 µM). Substitution of four neutral amino acid residues with Lys residues in each peptide had dramatic effects on their antimicrobial potencies and haemolytic activities, particularly those of TsAP-1. The MICs of the enhanced cationic analogue (TsAP-S1) were 2.5 µM for S. aureus/C. albicans and 5 µM for E. coli but with an associated large increase in haemolytic activity (30% at 5 µM). The same Lys residue substitutions in TsAP-2 produced a dramatic effect on its MIC for E. coli lowering this from >320 µM to 5 µM. TsAP-1 was ineffective against three of the five human cancer cell lines tested while TsAP-2 inhibited the growth of all five. Lys residue substitution of both peptides enhanced their potency against all five cell lines with TsAp-S2 being the most potent with IC50 values ranging between 0.83 and 2.0 µM. TsAP-1 and TsAP-2 are novel scorpion venom peptides with broad spectrum antimicrobial and anticancer cell activities the potencies of which can be significantly enhanced by increasing their cationicity.
Asunto(s)
Antiinfecciosos/farmacología , Antineoplásicos/farmacología , Péptidos/farmacología , Venenos de Escorpión/química , Escorpiones , Secuencia de Aminoácidos , Animales , Antiinfecciosos/química , Antiinfecciosos/aislamiento & purificación , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Secuencia de Bases , Candida albicans/efectos de los fármacos , Línea Celular Tumoral , Clonación Molecular , ADN Complementario/genética , Disulfuros/química , Humanos , Datos de Secuencia Molecular , Péptidos/química , Péptidos/genética , Péptidos/aislamiento & purificación , Staphylococcus aureus/efectos de los fármacosRESUMEN
Sauvagine is a potent and broad-spectrum biologically active peptide of 40 amino acid residues originally isolated from the skin of the South American frog, Phyllomedusa sauvagei. Since its discovery, no additional sauvagine structures have been reported. Following the discovery of sauvagine, peptides with similar primary structures/activities were identified in mammalian brain [corticotropin-releasing factor (CRF) and urocortin]. Here, we report the identification of a second sauvagine from the Mexican giant leaf frog, Pachymedusa dacnicolor, which displays primary structural features of both sauvagine and CRF. A cDNA encoding the peptide precursor was "shotgun" cloned from a cDNA library constructed from lyophilised skin secretion by 3'- and 5'-RACE reactions. From this, the primary structure of a 38-mer peptide was deduced and this was located in reverse phase HPLC fractions of skin secretion and both its mass and structure were confirmed by mass spectrometry. The biological activities of synthetic replicates of PD-sauvagine and sauvagine were compared using two different mammalian smooth muscle preparations and the novel peptide was found to be more potent in both. Bioinformatic analyses of PD-sauvagine revealed that it shared different regional sequence identities with both sauvagine and CRF.
Asunto(s)
Proteínas Anfibias/metabolismo , Anuros/metabolismo , Colon/efectos de los fármacos , Precursores de Proteínas/metabolismo , Piel/metabolismo , Vejiga Urinaria/efectos de los fármacos , Secuencia de Aminoácidos , Proteínas Anfibias/genética , Proteínas Anfibias/farmacología , Animales , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Clonación Molecular , Relación Dosis-Respuesta a Droga , Cobayas , Técnicas In Vitro , Masculino , Datos de Secuencia Molecular , Contracción Muscular/efectos de los fármacos , Precursores de Proteínas/genética , Precursores de Proteínas/farmacología , Señales de Clasificación de Proteína , Estructura Secundaria de Proteína , Ratas , Ratas Wistar , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Natriuretic peptides are common components of reptile venoms and molecular cloning of their biosynthetic precursors has revealed that in snakes, they co-encode bradykinin-potentiating peptides and in venomous lizards, some co-encode bradykinin inhibitory peptides such as the helokinestatins. The common natriuretic peptide/helokinestatin precursor of the Gila Monster, Heloderma suspectum, encodes five helokinestatins of differing primary structures. Here we report the molecular cloning of a natriuretic peptide/helokinestatin precursor cDNA from a venom-derived cDNA library of the Mexican beaded lizard (Heloderma horridum). Deduction of the primary structure of the encoded precursor protein from this cloned cDNA template revealed that it consisted of 196 amino acid residues encoding a single natriuretic peptide and five helokinestatins. While the natriuretic peptide was of identical primary structure to its Gila Monster (H. suspectum) homolog, the encoded helokinestatins were not, with this region of the common precursor displaying some significant differences to its H. suspectum homolog. The helokinestatin-encoding region contained a single copy of helokinestatin-1, 2 copies of helokinestatin-3 and single copies of 2 novel peptides, (Phe)(5)-helokinestatin-2 (VPPAFVPLVPR) and helokinestatin-6 (GPPFNPPPFVDYEPR). All predicted peptides were found in reverse phase HPLC fractions of the same venom. Synthetic replicates of both novel helokinestatins were found to antagonize the relaxing effect of bradykinin on rat tail artery smooth muscle. Thus lizard venom continues to provide a source of novel biologically active peptides.
Asunto(s)
Bradiquinina/antagonistas & inhibidores , Lagartos/metabolismo , Péptidos Natriuréticos/química , Precursores de Proteínas/química , Ponzoñas/química , Secuencia de Aminoácidos , Animales , Arterias/efectos de los fármacos , Bradiquinina/genética , Bradiquinina/metabolismo , Cromatografía de Fase Inversa , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Biblioteca de Genes , Lagartos/genética , Masculino , México , Datos de Secuencia Molecular , Relajación Muscular/efectos de los fármacos , Péptidos Natriuréticos/genética , Péptidos Natriuréticos/metabolismo , Péptidos Natriuréticos/farmacología , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Ratas , Ratas Wistar , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Técnicas de Cultivo de Tejidos , Ponzoñas/genética , Ponzoñas/metabolismo , Ponzoñas/farmacologíaRESUMEN
Amphibian skin secretions represent a unique resource for the discovery of new bioactive peptides. Here we report the isolation, structural and functional characterization of a novel heptapeptide amide, DMSPPWHamide, from the defensive skin secretion of the Mexican giant leaf frog, Pachymedusa dacnicolor. This peptide is of unique primary structure and has been classified as a member of the rather heterogenous tryptophyllin-2 (T-2) family of amphibian skin peptides and named P. dacnicolor Tryptophyllin-2 (PdT-2) in accordance. PdT-2 is the first Type 2-tryptophyllin to possess discrete bioactivity. Both natural and synthetic replicates of the peptide were found to contract the smooth muscle of rat urinary bladder, the latter displaying an EC50 of 4 nM.
Asunto(s)
Anuros/metabolismo , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Piel/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Masculino , Datos de Secuencia Molecular , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Oligopéptidos/química , Oligopéptidos/aislamiento & purificación , Ratas , Ratas Wistar , Alineación de SecuenciaRESUMEN
By integrating systematic peptidome and transcriptome studies of the defensive skin secretion of the Central American red-eyed leaf frog, Agalychnis callidryas, we have identified novel members of three previously described antimicrobial peptide families, a 27-mer dermaseptin-related peptide (designated DRP-AC4), a 33-mer adenoregulin-related peptide (designated ARP-AC1) and most unusually, a 27-mer caerin-related peptide (designated CRP-AC1). While dermaseptin and adenoregulin were originally isolated from phyllomedusine leaf frogs, the caerins, until now, had only been described in Australian frogs of the genus, Litoria. Both the dermaseptin and adenoregulin were C-terminally amidated and lacked the C-terminal tripeptide of the biosynthetic precursor sequence. In contrast, the caerin-related peptide, unlike the majority of Litoria analogs, was not C-terminally amidated. The present data emphasize the need for structural characterization of mature peptides to ensure that unexpected precursor cleavages and/or post-translational modifications do not produce mature peptides that differ in structure to those predicted from cloned biosynthetic precursor cDNA. Additionally, systematic study of the secretory peptidome can produce unexpected results such as the CRP described here that may have phylogenetic implications. It is thus of the utmost importance in the functional evaluation of novel peptides that the primary structure of the mature peptide is unequivocally established -- something that is often facilitated by cloning biosynthetic precursor cDNAs but obviously not reliable using such data alone.
Asunto(s)
Proteínas Anfibias/química , Péptidos Catiónicos Antimicrobianos/química , Anuros/fisiología , Genómica , Homología de Secuencia de Aminoácido , Piel/metabolismo , Secuencia de Aminoácidos , Proteínas Anfibias/análisis , Proteínas Anfibias/aislamiento & purificación , Proteínas Anfibias/metabolismo , Animales , Péptidos Catiónicos Antimicrobianos/análisis , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos/metabolismo , Anuros/anatomía & histología , Anuros/genética , Secuencia de Bases , América Central , Clonación Molecular , ADN Complementario/genética , Femenino , Masculino , Datos de Secuencia MolecularRESUMEN
The defensive strategy of amphibians against predator attack relies heavily on the secretion of noxious/toxic chemical cocktails from specialized skin granular glands. Bioactive peptides constitute a major component of secretions in many species and the most complex are produced by neotropical leaf frogs of the sub-family Phyllomedusinae. We recently reported that these skin secretions contain elements of both the granular gland peptidome and transcriptome and that polyadenylated mRNAs constituting the latter are protected from degradation by interactions with endogenous amphipathic peptides. This thus permits parallel amino acid sequencing of peptides and nucleic acid sequencing of cloned precursor transcripts from single lyophilized samples of secretion. Here we report that the protection afforded is sufficiently robust to permit transcriptome studies by cloning of full-length polyadenylated peptide precursor encoding mRNAs from libraries constructed using ambient temperature air-dried skin from recently deceased specimens as source material. The technique was sufficiently sensitive to permit the identification of cDNAs encoding antimicrobial peptides constituted by six different isoforms of phylloseptin and two dermaseptins. Also, for the first time, establishment of the nucleic acid and amino acid sequence of the precursor encoding the phyllomedusine frog skin bradykinin-related peptide, phyllokinin, from cloned cDNA, was achieved. These data unequivocally demonstrate that the granular gland transcriptome persists in air-dried amphibian skin--a finding that may have fundamental implications in the study of archived materials but also in the wider field of molecular biology.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/genética , Anuros/genética , Piel/metabolismo , Secuencia de Aminoácidos , Proteínas Anfibias/química , Proteínas Anfibias/genética , Proteínas Anfibias/metabolismo , Animales , Péptidos Catiónicos Antimicrobianos/metabolismo , Anuros/clasificación , Anuros/metabolismo , Secuencia de Bases , Bradiquinina/química , Bradiquinina/genética , Bradiquinina/metabolismo , Biblioteca de Genes , Cininas/química , Cininas/genética , Cininas/metabolismo , Datos de Secuencia Molecular , Preservación Biológica , Proteoma/genética , Precursores del ARN/química , Precursores del ARN/metabolismo , ARN Mensajero/metabolismo , Piel/química , América del Sur , Transcripción GenéticaRESUMEN
Reptile venoms are complex cocktails of bioactive molecules, including peptides. While the drug discovery potential of most species remains unrealized, many are endangered and afforded protection under international treaties. In this study, we describe how potential clinically important bioactive peptides and their corresponding mRNAs can be structurally characterized from single, small samples of reptile venom. The potential type-2 diabetes therapeutics, exendin-3 and exendin-4, from the Mexican beaded lizard (Heloderma horridum) and the Gila monster (Heloderma suspectum), respectively, have been characterized at both protein and nucleic acid levels to illustrate the efficacy of the technique and its contribution to biodiversity conservation.
Asunto(s)
Lagartos , Péptidos/química , Ponzoñas/química , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/química , Exenatida , Biblioteca de Genes , Péptido 1 Similar al Glucagón/química , Péptido 1 Similar al Glucagón/genética , Péptido 1 Similar al Glucagón/aislamiento & purificación , Datos de Secuencia Molecular , Péptidos/genética , Péptidos/aislamiento & purificación , Ponzoñas/genética , Ponzoñas/aislamiento & purificaciónRESUMEN
The defensive skin secretions of many amphibians contain a wide spectrum of biologically active compounds, particularly antimicrobial peptides that act as a first line of defence against bacterial infection. Here we describe for the first time the identification of three novel dermaseptin-related peptides (dermaseptins sVI-sVIII) whose primary structures were deduced from cDNAs cloned from a library constructed from lyophilised skin secretion of the South American hylid frog, Phyllomedusa sauvagei. The molecular masses of each were subsequently confirmed by interrogation of archived LC/MS files of fractionated skin secretion followed by automated Edman degradation sequencing. The heterogeneity of primary structures encountered in amphibian skin antimicrobial peptides may in part be explained by individual variation-a factor essential for selective functional molecular evolution and perhaps, ultimately in speciation.