Identification and Functional Analysis of Novel Bradykinin-Related Peptides (BRPs) from Skin Secretions of Five Asian Frogs

In recent decades, various types of bioactive substances have been identified from amphibian skin and its secretions. Bradykinin-related peptides (BRPs) are among these compounds that make up the host defence system of amphibians. In the present study, we identified six novel BRPs, amolopkinin-GN1, amolopkinin-RK1, amolopkinin-TR1, amolopkinin-LF1, ranakinin-MS1, and ranakinin-MS2, from five East Asian amphibians, Amolops granulosus, Amolops ricketti, Amolops torrentis, Amolops lifanensis, and Hylarana maosonensis. This is the first report on BRPs in the skin of these species. Physiological assays reveal that these peptides have a contractive effect on the smooth muscle of rat ileum.     

Novel dermaseptins from Phyllomedusa hypochondrialis (Amphibia)

Six new antimicrobial peptides structurally related to the dermaseptin family have been isolated from the skin secretion of the amphibian Phyllomedusa hypochondrialis. The primary structures of these molecules named as DShypo 01, 02, 03, 04, 06, and 07 were determined by de novo MS/MS experiments, Edman degradation, and cDNA sequencing. The fifth peptide was found to be precisely the same DS 01 from Phyllomedusa oreades previously described by our group. The majority of the peptides purified from the crude skin secretion could be directly localized and mapped onto a freshly dissected dorsal skin fragment using mass spectrometry-imaging techniques. Comparisons between peptides and commercial drugs on their antibacterial and anti-Leishmania amazonensis efficiencies, associated with peptide lytic effects on mammalian blood cells and surface plasmon resonance interaction studies on immobilized DMPC vesicles, were also performed.     

A fish bradykinin (Arg0, Trp5, Leu8-bradykinin) from the defensive skin secretion of the European edible frog, Pelophylax kl. esculentus: structural characterization; molecular cloning of skin kininogen cDNA and pharmacological effects on mammalian smooth muscle

Extensive studies on bradykinin-related peptides (BRPs) generated from plasma kininogens in representative species of various vertebrate taxa, have confirmed that many amphibian skin BRPs reflect those present in putative vertebrate predators. For example, the (Val¹, Thr⁶)-bradykinin, present in the defensive skin secretions of many ranids and phyllomedusines, can be generated from plasma kininogens in colubrid snakes—common predators of these frogs. Here, we report the presence of (Arg⁰, Trp⁵, Leu⁸)-bradykinin in the skin secretion of the European edible frog, Pelophylax kl. esculentus, and have found it to be encoded in single copy by a kininogen with an open-reading frame of 68 amino acid residues. This peptide is the archetypal bony fish bradykinin that has been generated from plasma kininogens of the bowfin (Amia calva), the long-nosed gar (Lepisosteus oseus) and the rainbow trout (Onchorhynchus mykiss). More recently, this peptide has been shown to be encoded within cloned kininogens of the Atlantic cod (Gadus morhua) spotted wolf-fish (Anarichas minor), zebrafish (Danio rerio), pufferfish (Tetraodon nigroviridis) and Northern pike (Esox lucius). The latter species is regarded as a major predator of P. kl. esculentus. Synthetic (Arg⁰, Trp⁵, Leu⁸)-bradykinin was previously reported as having multiphasic effects on arterial blood pressure in conscious trout and here we have demonstrated that it can antagonize the relaxation in rat arterial smooth muscle induced by canonical mammalian bradykinin. The discovery of (Arg⁰, Trp⁵, Leu⁸)-bradykinin in the defensive skin secretion of this amphibian completes the spectrum of vertebrate taxon-specific BRPs identified from this source.     

Dermatoxin and phylloxin from the waxy monkey frog, Phyllomedusa sauvagei: cloning of precursor cDNAs and structural characterization from lyophilized skin secretion

Amphibian skin is a morphologically, biochemically and physiologically complex organ that performs the wide range of functions necessary for amphibian survival. Here we describe the primary structures of representatives of two novel classes of amphibian skin antimicrobials, dermatoxin and phylloxin, from the skin secretion of Phyllomedusa sauvagei, deduced from their respective precursor encoding cDNAs cloned from a lyophilized skin secretion library. A degenerate primer, designed to a highly conserved domain in the 5'-untranslated region of analogous peptide precursor cDNAs from Phyllomedusa bicolor, was employed in a 3'-RACE reaction. Peptides with molecular masses coincident with precursor-deduced mature toxin peptides were identified in LC/MS fractions of skin secretion and primary structures were confirmed by MS/MS fragmentation. This integrated experimental approach can thus rapidly expedite the primary structural characterization of amphibian skin peptides in a manner that circumvents specimen sacrifice whilst preserving robustness of scientific data.     

Identification of three novel Phyllomedusa sauvagei dermaseptins (sVI-sVIII) by cloning from a skin secretion-derived cDNA library

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.     

Identification of SepCRP analogues in the cuttlefish Sepia officinalis: a novel family of ovarian regulatory peptides

In the cuttlefish, Sepia officinalis, the ovary appears to be one of the main sources of regulatory peptides involved in the successive steps of egg-laying. Following the identification of the SepCRP-1, which is a peptide extracted from ovary and involved in egg capsule secretion, investigations were focused on the identification of related peptides. Seven related-Sepia Capsule Releasing Peptides (R-SepCRPs) were identified by means of mass spectrometry and characterized using MS/MS spectra and/or Edman degradation. Finally, primary structures were verified by the comparison of MS/MS spectra from endogenic and synthetic peptides. This new ovarian peptide family exhibits a conserved SLXKD tag involved in the biological activity. LC-MS/MS screening clearly demonstrates that R-SepCRPs are restricted to the female genital tract. Expressed during vitellogenesis, they are released by vitellogenic follicles and full-grown oocytes (FGO) in the genital coelom. Biological activities suggest that R-SepCRPs would be responsible for the storage of FGO before mating and would take part in the mechanical secretion of egg capsule products, as previously described for SepCRP-1.     

Identification of highly acidic peptides from processing of the skin prepropeptides of Xenopus laevis

The skin secretion of the frog Xenopus laevis has been fractionated by reverse-phase HPLC and the most polar components studied by fast-atom-bombardment mass spectrometry (FAB/MS). Esterification of the hydrophilic peptides with methanol and ethanol was employed to improve the sensitivity of the technique. A number of small, highly acidic peptides have been identified, and alcoholysis of the peptide bonds within a number of these permitted their sequencing by FAB/MS. The sequences confirmed that they originate from acidic spacer regions found in the precursors to peptide hormones, such as caerulein, which have already been found in the secretion. In addition, acidic peptides derived from the spaces of the precursor to the antimicrobial peptides, PGS (or the magainins) have been isolated. The release of these from the preprotein cannot be fully accounted for by documented processing mechanisms, suggesting that a novel type of cleavage site has been identified.     

Hylaranins: prototypes of a new class of amphibian antimicrobial peptide from the skin secretion of the oriental broad-folded frog,Hylarana latouchii

Amphibian skin secretions contain a broad spectrum of biologically active compounds, particularly antimicrobial peptides, which are considered to constitute a first line of defence against bacterial infection. Here we describe the identification of two prototype peptides representing a novel structural class of antimicrobial peptide from the skin secretion of the oriental broad-folded frog, Hylarana latouchii. Named hylaranin-L1 (GVLSAFKNALPGIMKIIVamide) and hylaranin-L2 (GVLSVIKNALPGIMRFIAamide), both peptides consist of 18 amino acid residues, are C-terminally amidated and are of unique primary structures. Their primary structures were initially deduced by MS/MS fragmentation sequencing from reverse-phase HPLC fractions of skin secretion that demonstrated antimicrobial activity. Subsequently, their precursor-encoding cDNAs were cloned from a skin secretion-derived cDNA library and their primary structures were confirmed unequivocally. Synthetic replicates of both peptides exhibited broad-spectrum antimicrobial activity with mean inhibitory concentrations (MICs) of 34 μM against Gram-negative Escherichia coli, 4.3 μM against Gram-positive Staphylococcus aureus and 4–9 μM against the yeast, Candida albicans. Both peptides exhibited little haemolytic activity (<6 %) at the MICs for S. aureus and C. albicans. Amphibian skin secretions thus continue to provide novel antimicrobial peptide structures that may prove to be lead compounds in the design of new classes of anti-infection therapeutics.     

Host defence peptides from the skin glands of the Australian blue mountains tree-frog Litoria citropa. Solution structure of the antibacterial peptide citropin 1.1.

Nineteen citropin peptides are present in the secretion from the granular dorsal glands of the Blue Mountains tree-frog Litoria citropa; 15 of these peptides are also present in the secretion from the submental gland. Two major peptides, citropin 1.1 (GLFDVIKKVASVIGGL-NH2), citropin 1.2 (GLFDIIKKVASVVGGL-NH2) and a minor peptide, citropin 1.3 (GLFDIIKKVASVIGGL-NH2) are wide-spectrum antibacterial peptides. The amphibian has an endoprotease which deactivates these membrane-active peptides by removing residues from the N-terminal end: loss of three residues gives the most abundant degradation products. The solution structure of the basic peptide citropin 1.1 has been determined by NMR spectroscopy [in a solvent mixture of trifluoroethanol/water (1 : 1)] to be an amphipathic alpha-helix with well-defined hydrophobic and hydrophilic regions. The additional four peptides produced by the dorsal glands are structurally related to the antibacterial citropin 1 peptides but contain three more residues at their C-terminus [e.g. citropin 1.1.3 (GLFDVIKKVASVIGLASP-OH)]. These peptides show minimal antibacterial activity; their role in the amphibian skin is not known.     

Amphibian skin secretomics: application of parallel quadrupole time-of-flight mass spectrometry and peptide precursor cDNA cloning to rapidly characterize the skin secretory peptidome of Phyllomedusa hypochondrialis azurea: discovery of a novel peptide family, the hyposins

This study reports the variety of peptides present in the skin secretory peptidome of Phyllomedusa hypochondrialis azurea. Peptide structures, along with post-translational modifications, were elucidated by QTOF MS/MS analysis, cDNA sequencing, or a combination of both. Twenty-two peptides, including 19 novel structures, were identified from six different structural classes, including tryptophyllins, dermorphins, and a novel group of peptides termed hyposins. The study demonstrates the power of this combined approach to mine the rich peptidome compliment of the amphibian defensive skin secretome.     

A combined mass spectrometric and cDNA sequencing approach to the isolation and characterization of novel antimicrobial peptides from the skin secretions of Phyllomedusa hypochondrialis azurea

Studies conducted on amphibian skin secretions over the past 40 years have isolated and identified huge arrays of bioactive peptides, many of which have demonstrated potent anti-microbial activity. Such peptides are attracting increasing attention due to the growing problem of pathogenic microorganisms resistant to conventional antibiotics. The current study utilized a combined proteomic/genomic approach to facilitate the high throughput sequencing of five novel dermaseptins and four novel phylloseptins from the skin secretions of Phyllomedusa hypochondrialis azurea. Peptides were partially identified using Q-TOF MS/MS fragmentation and de novo sequencing, while a cDNA library was constructed from the lyophilized skin secretion. 3'-RACE reactions used primers designed for the highly conserved 5'-signal regions of previously deduced dermaseptin precursors. cDNA sequenced peptides were attributed to their respective fragmentation spectra to confirm the structure of the final processed peptides. Such an approach identified post-translational modifications in addition to deciphering isobaric amino acids. Several of the peptides were purified to homogeneity and displayed potent antimicrobial activity with minimum inhibitory concentrations starting at 0.4 microM when tested against and range of Gram-positive and Gram-negative bacteria including Escherichia coli, Staphylococcus aureus and Micrococcus luteus.     

Identification and Functional Analysis of a Novel Tryptophyllin Peptide from the Skin of the Red-eye Leaf Frog,Agalychnis callidryas

Amphibian skin has proved repeatedly to be a largely untapped source of bioactive peptides and this is especially true of members of the Phyllomedusinae subfamily of frogs native to South and Central America. Tryptophyllins are a group of peptides mainly found in the skin of members of this genus. In this study, a novel tryptophyllin (TPH) type 3 peptide, named AcT-3, has been isolated and structurally-characterised from the skin secretion and lyophilised skin extract of the red-eye leaf frog, Agalychnis callidryas. The peptide was identified in and purified from the skin secretion by reverse-phase HPLC. MALDI-TOF mass spectrometry and MS/MS fragmentation sequencing established its primary structure as: pGlu-Gly-Lys-Pro-Tyr-Trp-Pro-Pro-Pro-Phe-Leu-Pro-Glu, with a non-protonated molecular mass of 1538.19Da. The mature peptide possessed the canonical N-terminal pGlu residue that arises from post-translational modification of a Gln residue. The deduced open-reading frame consisted of 63 amino acid residues encoding a highly-conserved signal peptide of approximately 22 amino acid residues, an intervening acidic spacer peptide domain, a single AcT-3 encoding domain and a C terminal processing site. A synthetic replicate of AcT-3 was found to antagonise the effect of BK on rat tail artery smooth muscle and to contract the intestinal smooth muscle preparations. It was also found that AcT-3 could dose-dependently inhibit the proliferation of human prostate cancer cell lines after 72h incubation.     

Peptidomic analyses of mouse astrocytic cell lines and rat primary cultured astrocytes

Astrocytes play an active role in the modulation of synaptic transmission by releasing cell-cell signaling molecules in response to various stimuli that evoke a Ca(2+) increase. We expand on recent studies of astrocyte intracellular and secreted proteins by examining the astrocyte peptidome in mouse astrocytic cell lines and rat primary cultured astrocytes, as well as those peptides secreted from mouse astrocytic cell lines in response to Ca(2+)-dependent stimulations. We identified 57 peptides derived from 24 proteins with LC-MS/MS and CE-MS/MS in the astrocytes. Among the secreted peptides, four peptides derived from elongation factor 1, macrophage migration inhibitory factor, peroxiredoxin-5, and galectin-1 were putatively identified by mass-matching to peptides confirmed to be found in astrocytes. Other peptides in the secretion study were mass-matched to those found in prior peptidomics analyses on mouse brain tissue. Complex peptide profiles were observed after stimulation, suggesting that astrocytes are actively involved in peptide secretion. Twenty-six peptides were observed in multiple stimulation experiments but not in controls and thus appear to be released in a Ca(2+)-dependent manner. These results can be used in future investigations to better understand stimulus-dependent mechanisms of astrocyte peptide secretion.     

LC-MS/MS with 2D mass mapping of skin secretions' peptides as a reliable tool for interspecies identification inside Rana esculenta complex

Identification of species constituting Rana esculenta complex represents a certain problem as two parental species Rana ridibunda and Rana lessonae form their hybrid R. esculenta, while external signs and sizes of the members of this complex are intersected. However the composition of skin secretion consisting mainly of peptides is different for the species of the complex. LC-MS/MS is an ideal analytical tool for the quantitative and qualitative analysis of these peptides. The results covering elemental composition of these peptides, their levels in the secretion, as well as their belonging to a certain family of peptides may be visualized by means of 2D mass maps. The proposed approach proved itself to be a perspective tool for the reliable identification of all 3 species constituting R. esculenta complex. Easy distinguishing between the species may be achieved using 2D maps as fingerprints. Besides this approach may be used to study hybridogenesis and mechanisms of hemiclonal transfer of genetic information, when rapid and reliable identification of species involved in the process is required.     

Lividins: novel antimicrobial peptide homologs from the skin secretion of the Chinese Large Odorous frog, Rana (Odorrana) livida. Identification by "shotgun" cDNA cloning and sequence analysis

Odorous frogs of the sub-genus Odorrana are of oriental distribution, and are so called due to the foul smell of their defensive skin secretions released from specialized skin glands following stress or predator attack. Here we report the application of a "shotgun" skin secretion cDNA library cloning technique which can rapidly expedite identification of secretion bioactive peptides. From a library constructed from the skin secretion of the Large Chinese Odorous frog, Rana (Odorrana) livida, we have identified four novel peptides whose primary structures were deduced initially from cloned precursors. Subsequently, mature peptides were located in and structurally characterized from reverse phase HPLC fractions of skin secretion. Named lividins 1-4, these were found to be structural homologs of known antimicrobial peptide families from Rana frogs. Rapid identification of novel peptides can thus be rapidly achieved using this non-invasive, non-destructive technology and the extensive similarities revealed between antimicrobial peptide precursor organization and nucleic acid sequences would lend support to the hypothesis that they have a common ancestral origin.     

Caerulein secretion by dermal glands in xenopus laevis

Since gastrin and its related peptides are secreted by a minority population of widely dispersed cells in mamamalian tissues it has, in the past, been difficult to study the subcellular aspects of their secretion. From published reports (1, 2) it seemed possible that a satisfactory system for such studies might be provided by the skin of certain amphibians such as Xenopus laevis since in these tissues high concentrations of peptides such as caerulein exist, and there is some indication (3) that this, or a similar gastrin-like peptide, may be a dermal gland secretory product. We have therefore explored this possibility by studying the structure, secretory process, and secretory product of the most prominent non mucous type of gland in the skin of X. laevis. These studies clearly demonstrate that most, if not all, of the caerulein in which the skin is contained in secretion granules within the dermal glands and that its release can be specifically evoked by adrenergic stimulation. The release process by a holocrine mechanism expels all of the stored secretion onto the skin surface and thus for biosynthetic studies it should now be possible to synchronize the processes which lead to the replenishment of the peptide.     

Non-invasive proteomic analysis of human skin keratins: screening of methionine oxidation in keratins by mass spectrometry

Keratins are the main constituent of human skin and have been identified as major oxidative target proteins. However, there has been a lack of studies aimed at identifying the oxidation sites of keratins because of the difficulties associated with their insolubility and handling. Here, we introduce a mass spectrometry (MS)-based proteomic methodology to screen oxidative modifications in human skin keratins. Human skin proteins were obtained non-invasively by tape stripping and solubilized in SDS buffer, followed by purification and digestion using the modified filter-aided sample preparation method. The tryptic peptides were then analyzed by MALDI-TOF/MS, LC-ESI/MS, and MS/MS. PMF analyses have identified keratins K1 and K10 as the major proteins of human skin. Met(259), Met(262), Met(296), and Met(469), located in the α-helical rod domain of K1, were the most susceptible sites to oxidation induced by hydrogen peroxide in vitro and in vivo. Our results indicate a potential use of the identified methionine residues as biomarkers of oxidative skin damage. The present methodology is the first MS-based approach to detecting oxidative modifications in keratins obtained directly from human skin and can be easily applied to the monitoring of other keratin modifications in various skin conditions.