Frog Peptides Research Articles

In vitro bactericidal activity of the N-terminal fragment of the frog peptide esculentin-1b (Esc 1–18) in combination with conventional antibiotics against Stenotrophomonas maltophilia

In this study the bactericidal effect of the N-terminal fragment of the frog skin peptide esculentin-1b [Esc(1–18)] in combination with clinically used antimicrobial agents was evaluated against Stenotrophomonas maltophilia, either in standard conditions (phosphate buffer) or in the presence of human serum. A synergistic bactericidal effect was observed after a 24 h incubation when combinations of Esc(1–18) and amikacin or colistin were used against clinical strains of S. maltophilia with or without resistance to these antibiotics, both in buffer and in the presence of serum. An indifferent effect was observed when the peptide was combined with levofloxacin or ceftazidime. A synergistic effect was also observed at earlier time points when the peptide was used in combination with colistin. Sequential exposure of bacterial cells to Esc(1–18) and amikacin or colistin, or vice versa, indicated that while Esc(1–18) and colistin cooperated in enhancing the bactericidal effect of their combination, when Esc(1–18) was combined with amikacin, the peptide had a major role in initiating the bactericidal effect, while amikacin was required for the subsequent effector phase. Altogether, the results obtained indicate that exposure of S. maltophilia to sub-bactericidal concentrations of Esc(1–18) increases its susceptibility to amikacin or colistin and may also render resistant strains susceptible to these antibiotics.

Oxidation Versus Carboxamidomethylation of S–S Bond in Ranid Frog Peptides: Pro and Contra for de Novo MALDI-MS Sequencing

Five natural peptides isolated from ranid skin secretions of European frog species of Rana ridibunda and Rana arvalis (molecular masses 3516, 2674, 2636, 1874, and 1810 Da) were studied by MALDI-TOF/TOF to compare two procedures of disulfide bond cleavage: (1) performic oxidation and (2) reduction/carboxamidomethylation. The processes are relevant for the elucidation of the amino acid sequence inside the seven-member cystine ring at the C-terminus. The results clearly demonstrated that oxidation of the disulfide bond led to notably higher abundances of b- and y-ions, corresponding to the C-terminal peptide bonds, than reduction/carboxamidomethylation. This conclusion is true for all five peptides studied. Besides that, the oxidation procedure is simpler than carboxamidomethylation, as it is a one-step process with no purification required. The oxidation is more reproducible. The results were similar each time the peptide was subjected to the process. It was successfully applied to all five peptides while reduction/carboxamidomethylation failed in the case of brevinin-1Ra, despite all variations of reaction conditions.

Melanocortin 4 receptors interact with antimicrobial frog peptide analogues

We have developed fluorescence polarization (FP) assays of human melanocortin 4 receptor (MC4R) in 384-well microtiter plates using TAMRA-NDP-MSH as a tracer. The rank order of potency of agonists and antagonists agrees well relative to the published assays: SHU9119 > MTII > NDP αMSH > αMSH. We have screened libraries of Korean plant extracts and frog peptide analogues in search of MC4R ligands using FP assays and cell-based CRE luciferase reporter assays. We report that FLGFLFKVASK, FLGWLFKVASK, FLGALFKWASK, and FLGWLFKWASK are the peptide analogues, which bind to human MC4R receptor with good affinity in vitro. FLGWLFKVASK and FLGWLFKWASK stimulated CRE-driven reporter gene via MC4R. In luciferase reporter assays, they possess the pharmacological and functional profiles of full agonists. We demonstrate the interaction of MC4R with 11-residue antimicrobial peptides derived from the Korean frog, Rana rugosa. The results suggest that MC4R interacts promiscuously with bioactive analogues of antimicrobial peptide, gaegurin-5.

Cloning from tissue surrogates: Antimicrobial peptide (esculentin) cDNAs from the defensive skin secretions of Chinese ranid frogs

The defensive skin secretions of amphibians are a rich source of bioactive peptides. Here we describe a rapid technique for skin granular gland transcriptome cloning from a surrogate tissue—the secretion itself. cDNA libraries were constructed from lyophilized skin secretion from each of the Chinese frogs ( Rana schmackeri, Rana versabilis, and Rana plancyi fukienensis) using magnetic oligo(dT) bead-captured polyadenylated mRNA as templates. Specific esculentin cDNAs were amplified by 3′-RACE using a degenerate primer designed for a consensus nucleotide sequence in the 5′ untranslated region of previously characterized ranid frog peptide cDNAs. The cloned cDNAs were found to encode the antimicrobial peptides esculentins 1 and 2 from each of the species examined. The presence of predicted peptide structures in skin secretions was confirmed by MALDI-TOF mass spectrometry and automated Edman degradation. This experimental approach can thus rapidly expedite parallel transcriptome and peptidome analysis of amphibian granular gland secretions without harming or sacrificing donor animals.

Frog skin hope for HIV prevention

The discovery that the frog peptides can kill HIV virus even when it is hidden in dendritic cells suggests that they could be developed as mucosal preventives.

Urocortins of the South African Clawed Frog, Xenopus laevis: Conservation of Structure and Function in Tetrapod Evolution

Several corticotropin-releasing factor (CRF) family genes have been identified in vertebrates. Mammals have four paralogous genes that encode CRF or the urocortins 1, 2, and 3. In teleost fishes, a CRF, urotensin I (a fish ortholog of mammalian urocortin 1) and urocortin 3 have been identified, suggesting that at least three of the four mammalian lineages arose in a common ancestor of modern bony fishes and tetrapods. Here we report the isolation of genes orthologous to mammalian urocortin 1 and urocortin 3 from the South African clawed frog, Xenopus laevis. We characterize the pharmacology of the frog peptides and show that X. laevis urocortin 1 binds to and activates the frog CRF1 and CRF2 receptors at picomolar concentrations. Similar to mammals, frog urocortin 3 is selective for the CRF2 receptor. Only frog urocortin 1 binds to the CRF-binding protein, although with significantly lower affinity than frog CRF. Both urocortin genes are expressed in brain, pituitary, heart, and kidney of juvenile frogs; urocortin 1 is also expressed in skin. We also identified novel urocortin sequences in the genomes of pufferfish, zebrafish, chicken, and dog. Phylogenetic analysis supports the view that four paralogous lineages of CRF-like peptides arose before the divergence of the actinopterygian and sarcopterygian fishes. Our findings show that the functional relationships among CRF ligands and binding proteins, and their anorexigenic actions mediated by the CRF2 receptor, arose early in vertebrate evolution.

The bending rigidity of phospholipid monolayers in presence of an antimicrobial frog peptide studied by X-ray grazing incidence diffraction

Peptide secretion by living organisms constitutes an integral response process exploited by natural immune systems. In this work we present a model study and insight into this process reporting the thermodynamic and structural effects induced in phospholipid monolayers due to peptide insertion into the layer. Synchrotron X-ray radiation is combined with the Langmuir technique and exploited to form ‘lipid–peptide’ monolayers and probe the physical characteristics of the fundamental biological process of ‘peptide secretion’. Our experiments show that the insertion of peptides in the phospholipid layer has adverse effects on the elastic properties of the layer manifested through the bending rigidity.

Discovery of Corticotropin Releasing Factor 2 Receptor Selective Sauvagine Analogues for Treatment of Skeletal Muscle Atrophy

The corticotropin release factor 2 receptor (CRF2R) has many biological activities including modulation of the stress response. Recently, we have demonstrated that CRF2R activation functions to prevent skeletal muscle wasting resulting from a variety of physiological stimuli. Thus we are interested in identifying CRF2R selective agonists with optimal pharmacological properties for use in treating muscle wasting diseases. Several CRF2R agonists are known including the frog peptide sauvagine (Svg), which display superior pharmacological properties compared to other CRF2R agonists. Unfortunately sauvagine is a nonselective CRFR agonist, thus making it of less utility due to side effects resulting from corticotropin release factor 1 receptor (CRF1R) activation. Because our initial modifications of Svg at position 11 improved CRF2R selectivity, we investigated the role of amino acids at positions 12 and 13 in Svg. We observed that phenylalanine, leucine, isoleucine, threonine, glutamine, histidine, and tyrosine at the 12th position were the strongest promoters of CRF2R selectivity whereas phenylalanine, glutamine, trytophane, tyrosine, valine, isoleucine, leucine, and 2-naphthylalanine were the preferred residues at the 13th position. Selective sauvagine peptides demonstrated improved antiatrophy effects in a mouse-casting model when compared to sauvagine itself. Thus, we demonstrate that the CRF2R selectivity can be improved by optimizing amino acids at positions 12 and 13 (all with proline at position 11) and that the selective sauvagine analogues demonstrate better in vivo efficacy than sauvagine itself.

Antimicrobial peptides initiate IL-1 beta posttranslational processing: a novel role beyond innate immunity.

Human monocytes stimulated with LPS produce large quantities of prointerleukin-1beta, but little of this cytokine product is released extracellularly as the mature biologically active species. To demonstrate efficient proteolytic cleavage and export, cytokine-producing cells require a secondary effector stimulus. In an attempt to identify agents that may serve as initiators of IL-1beta posttranslational processing in vivo, LPS-activated human monocytes were treated with several individual antimicrobial peptides. Two peptides derived from porcine neutrophils, protegrin (PTG)-1 and PTG-3, promoted rapid and efficient release of mature IL-1beta. The PTG-mediated response engaged a mechanism similar to that initiated by extracellular ATP acting via the P2X(7) receptor. Thus, both processes were disrupted by a caspase inhibitor, both were sensitive to ethacrynic acid and CP-424,174, two pharmacological agents that suppress posttranslational processing, and both were negated by elevation of extracellular potassium. Moreover, the PTGs, like ATP, promoted a dramatic change in monocyte morphology and a loss of membrane latency. The PTG response was concentration dependent and was influenced profoundly by components within the culture medium. In contrast, porcine neutrophil antimicrobial peptides PR-26 and PR-39 did not initiate IL-1beta posttranslational processing. The human defensin HNP-1 and the frog peptide magainin 1 elicited export of 17-kDa IL-1beta, but these agents were less efficient than PTGs. As a result of this ability to promote release of potent proinflammatory cytokines such as IL-1beta, select antimicrobial peptides may possess important immunomodulatory functions that extend beyond innate immunity.

Characterization of benzoquinone-peptide adducts by electrospray mass spectrometry.

Benzoquinone adducts were prepared with model peptides to identify characteristic features of adduct fragmentation in tandem mass spectrometry (MS) experiments. Model peptides contained cysteine and had a molecular mass of less than 2 kDa to facilitate peptide fragmentation in tandem MS analyses. Peptides were adducted with an excess of benzoquinone, and the adducts were analyzed by LC/MS. Adducts were identified by addition of 108 Da to the monoisotopic mass of the peptide, except in the case of oxytocin, which formed a bis adduct with addition of 216 Da. Tandem MS experiments were performed on the [M + 2H](2+) ions and/or the [M + H](+) ions. Sequence information obtained from modified peptides was comparable to that of their unmodified counterparts. A unique ion pair separated by 141 or 142 Da corresponding to beta-elimination of benzoquinol-S or benzoquinol-SH from a b(n) or y(n) series ion indicated attachment at the sulfur of the cysteine residue. An alternate ion pair of 211 Da corresponded to fragmentation at the peptide bond on either side of the adducted cysteine. Enzymatic digestion of BSA and a 2560 Da frog peptide with trypsin yielded tryptic peptides, which were treated with benzoquinone. In addition to ion pairs of 142 and 211 Da, singly and doubly charged tryptic peptide adducts showed a neutral loss of 142 Da from the precursor. Either one or both ion pairs were present in more than half of all the peptides that were examined. The neutral loss of 142 Da was present in all singly charged tryptic peptide adducts and in 11 out of 14 doubly charged tryptic peptide adducts. The data indicate that reliable detection of benzoquinone-cysteinyl peptide adducts requires monitoring of multiple spectral characteristics.

Peptide‐based antibiotics: A potential answer to raging antimicrobial resistance

Antibiotic resistance is an increasing problem worldwide. The emergence and spread of multidrug-resistant bacteria leads to treatment failure and is associated with severe outcomes: increased mortality, morbidity, and expenditure. For some of these organisms, no treatment option is available. In contrast, very few new antibiotics are currently being developed and no new classes of antibiotics which are active against Gram-negative bacilli are under investigation. During the past decade, several peptide-based antimicrobials of vertebrate origin were identified as a novel and promising class of antibiotics. Peptide-based antibiotics are attractive both to fundamental research (a novel mechanism of action) and for their potential therapeutic applications. They are relatively small molecules, their action is fast and lethal to a large spectrum of pathogens, and they seem to escape many of the drug resistance mechanisms. Compared to classical antibiotics, peptides portray a highly modular synthetic antimicrobial system. As shown for the frog peptides, dermaseptins, this system allows a multitude of simple and cost-effective chemical modifications that dramatically affect activity in terms of potency and selectivity. Unlike classical antibiotics that must penetrate the target cell to act on it, antimicrobial peptides are believed to kill target cells by destroying their membrane(s). Theoretically, this mode of action should severely reduce microbial resistance and represents, therefore, a promising alternative in the treatment of raging multidrug-resistant infectious diseases. Drug Dev. Res. 50:440–447, 2000. © 2000 Wiley-Liss, Inc.

Isolation, Structural Characterization, and Bioactivity of a Novel Neuromedin U Analog from the Defensive Skin Secretion of the Australasian Tree Frog, Litoria caerulea

We report the isolation of a novel bioactive peptide, neuromedin U-23 (NmU-23), from the defensive skin secretion of the Australasian tree frog, Litoria caerulea. The primary structure of the peptide was established by a combination of microsequencing, mass spectroscopy and site-directed antiserum immunoreactivity as SDEEVQVPGGVISNGYFLFRPRN-amide (M(r) 2580.6). A synthetic replicate of frog NmU-23 displaced monoradioiodinated rat NmU-23 from uterine membranes in a dose-dependent fashion indistinguishable from nonisotopically labeled rat NmU-23. In a rat uterine smooth muscle strip preparation, synthetic frog NmU-23 produced dose-dependent contractions identical to porcine NmU-25. However, in a preparation of human urinary bladder muscle strip, the synthetic frog peptide was more potent than porcine NmU-25 in eliciting contraction and produced desensitization of the preparation to the latter peptide. This report demonstrates that the defensive skin secretion of a frog contains a novel peptide exhibiting a high degree of primary structural similarity to the endogenous vertebrate peptide, NmU, and that this frog skin analog displays biological activity in mammalian tissues.

Peptide-based antibiotics: A potential answer to raging antimicrobial resistance

Antibiotic resistance is an increasing problem worldwide. The emergence and spread of multidrug-resistant bacteria leads to treatment failure and is associated with severe outcomes: increased mortality, morbidity, and expenditure. For some of these organisms, no treatment option is available. In contrast, very few new antibiotics are currently being developed and no new classes of antibiotics which are active against Gram-negative bacilli are under investigation. During the past decade, several peptide-based antimicrobials of vertebrate origin were identified as a novel and promising class of antibiotics. Peptide-based antibiotics are attractive both to fundamental research (a novel mechanism of action) and for their potential therapeutic applications. They are relatively small molecules, their action is fast and lethal to a large spectrum of pathogens, and they seem to escape many of the drug resistance mechanisms. Compared to classical antibiotics, peptides portray a highly modular synthetic antimicrobial system. As shown for the frog peptides, dermaseptins, this system allows a multitude of simple and cost-effective chemical modifications that dramatically affect activity in terms of potency and selectivity. Unlike classical antibiotics that must penetrate the target cell to act on it, antimicrobial peptides are believed to kill target cells by destroying their membrane(s). Theoretically, this mode of action should severely reduce microbial resistance and represents, therefore, a promising alternative in the treatment of raging multidrug-resistant infectious diseases. Drug Dev. Res. 50:440–447, 2000. © 2000 Wiley-Liss, Inc.

CDNA cloning of Clavanins: antimicrobial peptides of tunicate hemocytes

Clavanins are a family of α-helical antimicrobial peptides found in hemocytes of the tunicate, Styela clava. We examined a cDNA library prepared from pharyngeal tissues of S. clava and sequenced 24 clones that encoded prepropeptides of Clavanins A, C, D or E. These sequences indicated that Clavanins are synthesized as 9.2 kDa prepropeptides which contain a 19-residue signal peptide, followed in turn by a highly polar `pro' region (LEERKSEEEK) with five glutamic acid residues, the 23 residues of the mature Clavanin peptide, the glycine residue needed for its amidation and a 27-residue polar C-terminal extension that is removed in later processing. Although the signal sequence and anionic propiece of Clavanin precursors share features with corresponding regions in precursors of the certain frog peptides, including ranalexin, gaegurins, dermaseptins and deltorphins, their unique multipartite structure suggests that they are not actually homologues of these amphibian peptides.

Inhibition of bacterial growth by synthetic SP-B1-78 peptides.

Residues 12-34 of mature human pulmonary surfactant protein B (SP-B1-78) are 68% homologous to residues 48-72 of the frog peptide antibiotic dermaseptin b I. We examined the effects of SP-B1-78 on the growth of Escherichia coli in order to find whether full length SP-B1-78 might act as a peptide antibiotic. We found that SP-B1-78 peptide inhibited growth of E. coli (MIC = 210 micrograms.ml-1), but that the SP-B variant [R/K-->S]SP-B1-78 was less potent (MIC = 500 micrograms.ml-1).

Structure-activity analysis of thanatin, a 21-residue inducible insect defense peptide with sequence homology to frog skin antimicrobial peptides.

Immune challenge to the insect Podisus maculiventris induces synthesis of a 21-residue peptide with sequence homology to frog skin antimicrobial peptides of the brevinin family. The insect and frog peptides have in common a C-terminally located disulfide bridge delineating a cationic loop. The peptide is bactericidal and fungicidal, exhibiting the largest antimicrobial spectrum observed so far for an insect defense peptide. An all-D-enantiomer is nearly inactive against Gram-negative bacteria and some Gram-positive strains but is fully active against fungi and other Gram-positive bacteria, suggesting that more than one mechanism accounts for the antimicrobial activity of this peptide. Studies with truncated synthetic isoforms underline the role of the C-terminal loop and flanking residues for the activity of this molecule for which we propose the name thanatin.

Isolation and structural characterization of calcitonin gene-related peptide from the brain and intestine of the frog, Rana ridibunda

Calcitonin gene-related peptide (CGRP) was isolated in a single molecular form from an extract of the whole brain and from the small intestine of the European green frog, Rana ridibunda. The primary structure of the peptide was established as: Ala-Cys-Asn-Thr-Ala-Thr-Cys-Val-Thr- His 10-Arg-Leu-Ala-Asp-Phe-Leu-Ser-Arg-Ser-Gly 20-Gly-Met-Ala-Lys-Asn-Asn-Phe-Val-Pro-Thr 30-Asn-Val-Gly-Ser-Lys-Ala-Phe-NH 2. Frog CGRP shows only two amino acid substitutions (Val 22 → Met and Gly 23 å Ala) compared with chicken CGRP, as deduced from the nucleotide sequence of a cloned cDNA. The Asp 14 residue, which leads to enhanced biological potency in chicken CGRP, is also present in the frog peptide. The data show that the structure of CGRP has been strongly conserved during evolution of the vertebrates.

Raman spectroscopy of synthetic antimicrobial frog peptides magainin 2a and PGLa

Magainin and PGLa are 23- and 21-residue peptides isolated from the skin of the African clawed frog Xenopus laevis. They protect the frog from infection and exhibit a broad-spectrum antimicrobial activity in vitro. The mechanism of this activity involves the interaction of magainin with microbial membranes. We have measured the secondary structure and membrane-perturbing ability of these peptides to obtain information about this mechanism. Our results show that mgn2a forms a helix with an average length of less than 20 A upon binding to liposomes. At high concentrations (50 mg/mL) mgn2a spontaneously solubilizes phosphatidylcholine liposomes at temperatures above the gel-liquid-crystalline phase transition. Mgn2a appears to bind to the surface of liposomes made of negatively charged lipids without spontaneously penetrating the bilayer. Finally, mgn2a and PGLa interact together with liposomes in a synergistic way that enhances the helix content of one or both of the peptides and allows the peptides to more easily penetrate the bilayer. PGLa mixed with a small nonperturbing amount of magainin 2 amide is 25-43 times as potent as PGLa alone at inducing the release of carboxyfluorescein from liposomes. The results suggest that the mechanism of antimicrobial activity does not involve a channel formed by transmembrane helical peptides.

A new family of growth factor-like peptides ‘Trefoil’ disulphide loop structures as a common feature in breast cancer associated peptide (pS2), pancreatic spasmolytic polypeptide (PSP), and frog skin peptides (spasmolysins)

Four peptides present in completely different biological sources have been shown to exhibit a large degree of structural similarity. The peptides include: (i) a 60 amino acid residue breast cancer associated pS2 peptide isolated from human gastric juice and the culture media of the human breast cancer cell line MCF-7; (ii) a 106 amino acid residue pancreatic spasmolytic polypeptide (PSP) isolated from porcine pancreas and pancreatic juice; and (iii) a 49 and 50 amino acid residue peptide predicted from a cDNA isolated from the skin of the frog, Xenopus laevis. These peptides are characterized by having one (pS2 and the frog peptides) or two (PSP) domains of a highly conserved 38–39 amino acid residue consensus sequence not found in any other known peptides or proteins. The domain sequences contain 6 cysteine residues in nearly the same positions and it is suggested that these 6 residues are linked by 3 disulphide bonds to form a characteristic ‘trefoil’ disulphide loop structure common in all four peptides. From the sources of which the peptides have been isolated and from experiments showing that PSP has a growth factor stimulatory effect on MCF-7 cells, it is further suggested that these peptides may represent members of a new family of growth factors.

Effect of caerulein on pituitary response to TRH in humans.

The effect of caerulein (100 ng/kg/h X 1 h) on basal as well as on thyrotropin-releasing hormone (TRH)-stimulated prolactin and thyroid-stimulating hormone (TSH) secretion was studied in healthy male volunteers. The peptide did not change the basal levels of prolactin and TSH. However, during the infusion of caerulein, prolactin response to TRH was significantly increased whereas the TSH response was decreased. These data, showing an action of caerulein (a frog peptide which mimics the biological actions of cholecystokinin) on prolactin and TSH release, suggest that cholecystokinin may be involved in the physiological control of human pituitary secretion.