The structures of four bombesins and their cloned precursor-encoding cDNAs from acid-solvated skin secretion of the European yellow-bellied toad, Bombina variegata

Four different bombesins (bombesin, His(6)-bombesin, Phe(13)-bombesin and Asp(2)-, Phe(4)-SAP-bombesin) have been identified by a systematic sequencing study of peptides in reverse phase HPLC fractions of the skin secretion of the European yellow-bellied toad, Bombina variegata, that had been solvated in 0.1% (v/v) aqueous trifluoroacetic acid (TFA) and stored frozen at -20°C for 12 years. By using a 3'- and 5'-RACE PCR strategy, the corresponding biosynthetic precursor-encoding cDNAs of all four peptides were cloned from a cDNA library made from the same long-term frozen, acid-solvated skin secretion sample following thawing and lyophilization. Canonical bombesin and His(6)-bombesin are classical bombesin sub-family members, whereas Phe(13)-bombesin and Asp(2)-, Phe(4)-SAP-bombesin, belong to the litorin/ranatensin sub-family of bombesin-like peptides (BLPs). Assignment of these peptides to respective sub-families, was based upon both their primary structural similarities and their comparative pharmacological activities. An interesting observation in this study, was that the nucleotide sequences of the open-reading frames of cloned cDNAs encoding bombesin and its His(6)-substituted analog, were identical except for a single base that was responsible for the change observed at the position 6 residue in the mature peptide from Asn to His. In contrast, the precursor cDNA nucleotide sequences encoding the Phe(13)-bombesins, exhibited 53 base differences. The pharmacological activities of synthetic replicates of each bombesin were compared using two different mammalian smooth muscle preparations and all four peptides were found to be active. However, there were significant differences in their relative potencies.     

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.     

Limnonectins: a new class of antimicrobial peptides from the skin secretion of the Fujian large-headed frog (Limnonectes fujianensis)

Amphibian skin secretions are rich sources of biologically-active peptides with antimicrobial peptides predominating in many species. Several studies involving molecular cloning of biosynthetic precursor-encoding cDNAs from skin or skin secretions have revealed that these exhibit highly-conserved domain architectures with an unusually high degree of conserved nucleotide and resultant amino acid sequences within the signal peptides. This high degree of nucleotide sequence conservation has permitted the design of primers complementary to such sites facilitating “shotgun” cloning of skin or skin secretion-derived cDNA libraries from hitherto unstudied species. Here we have used such an approach using a skin secretion-derived cDNA library from an unstudied species of Chinese frog - the Fujian large-headed frog, Limnonectes fujianensis - and have discovered two 16-mer peptides of novel primary structures, named limnonectin-1Fa (SFPFFPPGICKRLKRC) and limnonectin-1Fb (SFHVFPPWMCKSLKKC), that represent the prototypes of a new class of amphibian skin antimicrobial peptide. Unusually these limnonectins display activity only against a Gram-negative bacterium (MICs of 35 and 70 μM) and are devoid of haemolytic activity at concentrations up to 160 μM. Thus the “shotgun” cloning approach described can exploit the unusually high degree of nucleotide conservation in signal peptide-encoding domains of amphibian defensive skin secretion peptide precursor-encoding cDNAs to rapidly expedite the discovery of novel and functional defensive peptides in a manner that circumvents specimen sacrifice without compromising robustness of data.     

Kasstasin: A novel potent vasoconstrictor peptide from the skin secretion of the African red-legged running frog, Kassina maculata

Amphibian skin secretions are established sources of bioactive peptides. Here we describe the isolation, structural and pharmacological characterisation of a novel vasoconstrictor peptide from the skin secretion of the African hyperoliid frog, Kassina maculata, which exhibits no structural similarity to any known class of amphibian skin peptide. The peptide consists of 21 amino acid residues, FIKELLPHLSGIIDSVANAIK, and is C-terminally amidated. The provisional structure was obtained by MS/MS fragmentation using an Orbitrap mass spectrometer and L/I ambiguities were resolved following molecular cloning of biosynthetic precursor-encoding cDNA. A synthetic replicate of the peptide was found to possess weak antimicrobial and haemolytic activities but was exceptionally effective in constricting the smooth muscle of rat tail artery (EC₅₀ of 25pM). In reflection of its exceptional potency in constricting rat arterial smooth muscle, the peptide was named kasstasin, a derivation of Kassina and “stasis” (stoppage of flow). These data illustrate the continuing potential of amphibian skin secretions to provide novel natural peptide templates for biological evaluation.     

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.     

Novel peptide fragments originating from PGLa and the caerulein and xenopsin precursors from Xenopus laevis

Skin secretions from the South African frog Xenopus laevis have been chromatographed by high performance liquid chromatography (HPLC), fractionated, and analyzed by fast atom bombardment-mass spectrometry (FAB-MS). The HPLC chromatograms showed the secretion to be a complex mixture with over 30 components at similar levels to the four peptides previously isolated from X. laevis skin, i.e. xenopsin, caerulein, thyrotropin-releasing hormone, and PGLa. FAB-MS analysis of the HPLC fractions gave numerous protonated molecular ions ranging from m/z 491 to 2662. Preliminary assignments of these components were made by comparing these experimental molecular weights to those predicted for regions within the xenopsin, caerulein, thyrotropin-releasing hormone, and PGLa precursors. These results suggested that many of these skin secretions were peptides originating from additional processing of the xenopsin, caerulein, and PGLa precursors, primarily involving cleavage at single arginine residues, and a novel cleavage at the NH2-terminal side of single lysines. These assignments were subsequently confirmed by Edman degradation, FAB-MS peptide sequencing, and amino acid analysis. All of these peptides contain one or more lysines and would be expected to have amphiphilic structures. As yet, nothing is known about their activity, although they resemble in composition the mast cell degranulating peptides melittin and the bombolitins. These precursor fragments were also found to have limited sequence homology to bombinin, a hemolytic amphibian peptide isolated from the European Bombina toad.     

Medusins: A new class of antimicrobial peptides from the skin secretions of phyllomedusine frogs

Natural drug discovery represents an area of research with vast potential. The investigation into the use of naturally-occurring peptides as potential therapeutic agents provides a new “chemical space” for the procurement of drug leads. Intensive and systematic studies on the broad-spectrum antimicrobial peptides found in amphibian skin secretions are of particular interest in the quest for new antibiotics to treat multiple drug-resistant bacterial infections. Here we report the molecular cloning of the biosynthetic precursor-encoding cDNAs and respective mature peptides representing a novel group of antimicrobial peptides from the skin secretions of representative species of phyllomedusine leaf frogs: the Central American red-eyed leaf frog (Agalychnis callidryas), the South American orange-legged leaf frog (Phyllomedusa hypochondrialis) and the Giant Mexican leaf frog (Pachymedusa dacnicolor). Each novel peptide possessed the highly-conserved sequence, LGMIPL/VAISAISA/SLSKLamide, and each exhibited activity against the Gram-positive bacterium, Staphylococcus aureus and the yeast, Candida albicans, but all were devoid of haemolytic effects at concentrations up to and including the MICs for both organisms. The novel peptide group was named medusins, derived from the name of the hylid frog sub-family, Phyllomedusinae, to which all species investigated belong. These data clearly demonstrate that comparative studies of the skin secretions of phyllomedusine frogs can continue to produce novel peptides that have the potential to be leads in the development of new and effective antimicrobials.     

Cationicity-Enhanced Analogues of the Antimicrobial Peptides,AcrAP1 and AcrAP2, from the Venom of the Scorpion,Androctonus crassicauda,Display Potent Growth Modulation Effects on Human Cancer Cell Lines

The non disulphide-bridged peptides (NDBPs) of scorpion venoms are attracting increased interest due to their structural heterogeneity and broad spectrum of biological activities. Here, two novel peptides, named AcrAP1 and AcrAP2, have been identified in the lyophilised venom of the Arabian scorpion, Androctonus crassicauda, through “shotgun” molecular cloning of their biosynthetic precursor-encoding cDNAs. The respective mature peptides, predicted from these cloned cDNAs, were subsequently isolated from the same venom sample using reverse phase HPLC and their identities were confirmed by use of mass spectrometric techniques. Both were found to belong to a family of highly-conserved scorpion venom antimicrobial peptides - a finding confirmed through the biological investigation of synthetic replicates. Analogues of both peptides designed for enhanced cationicity, displayed enhanced potency and spectra of antimicrobial activity but, unlike the native peptides, these also displayed potent growth modulation effects on a range of human cancer cell lines. Thus natural peptide templates from venom peptidomes can provide the basis for rational analogue design to improve both biological potency and spectrum of action. The diversity of such templates from such natural sources undoubtedly provides the pharmaceutical industry with unique lead compounds for drug discovery.     

Ornithokinin (avian bradykinin) from the skin of the Chinese bamboo odorous frog,Odorrana versabilis

One of the most widespread and abundant families of pharmacologically active peptides in amphibian defensive skin secretions is the bradykinins and related peptides. Despite retaining certain primary structural attributes that assign them to this peptide family, bradykinins and related peptides are unique among amphibian skin peptides in that they exhibit a wide range of primary structural variations, post‐translational modifications and/or N‐terminal or C‐terminal extensions. Initially it was believed that their high degree of primary structural heterogeneity was reflective of random gene mutations within species, but latterly, there is an increasing body of evidence that the spectrum of structural modifications found within this peptide family is reflective of the vertebrate predator spectrum of individual species. Here we report the discovery of ornithokinin (avian bradykinin – Thr⁶, Leu⁸‐bradykinin) in the skin secretion of the Chinese bamboo odorous frog, Odorrana versabilis. Molecular cloning of its biosynthetic precursor‐encoding cDNA from a skin secretion‐derived cDNA library revealed a deduced open‐reading frame of 86 amino acid residues, encoding a single copy of ornithokinin towards its C‐terminus. The domain architecture of this ornithokinin precursor protein was consistent with that of a typical amphibian skin peptide and quite different to that of the ornithokininogen from chicken plasma. Ornithokinin was reported to induce hypotension in the chicken and to contract the chicken oviduct but to have no obvious effect on the rat uterus. However, in this study, synthetic ornithokinin was found to contract the rat ileum (EC₅₀ = 539 nM) and to increase contraction frequency in the rat uterus (EC₅₀ = 1.87 μM). Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Senegalin: a novel antimicrobial/myotropic hexadecapeptide from the skin secretion of the African running frog, Kassina senegalensis

Amphibian skin is a rich and unique source of novel bioactive peptides most of which are endowed with either antimicrobial or pharmacological properties. Here, we report the identification and structural characterization of a novel peptide, named senegalin, which possesses both activities. Senegalin is a hexadecapeptide amide (FLPFLIPALTSLISSL-NH₂) of unique primary structure found in the skin secretion of the African running frog, Kassina senegalensis. The structure of the biosynthetic precursor of senegalin, deduced from cloned skin cDNA, consists of 76 amino acid residues and displays the typical domain organization of an amphibian skin peptide precursor. Both natural senegalin and its synthetic replicate displayed antimicrobial and myotropic activities. Senegalin was active against Staphylococcus aureus (MIC 50 μM) and Candida albicans (MIC 150 μM) but was non-haemolytic at concentrations up to and including 150 μM. In contrast, senegalin induced a dose-dependent contraction of rat urinary bladder smooth muscle (EC₅₀ 2.9 nM) and a dose-dependent relaxation of rat tail artery smooth muscle (EC₅₀ 37.7 nM). Senegalin thus represents a prototype biologically active amphibian skin peptide and illustrates the fact that amphibian skin secretion peptidomes continue to be unique sources of such molecules.     

A novel bradykinin-related peptide from skin secretions of toad Bombina maxima and its precursor containing six identical copies of the final product

Amphibian skin contains rich bradykinin-related peptides, but the mode of biosynthesis of these peptides is unknown. In the present study, a novel bradykinin-related peptide, termed bombinakinin M, was purified from skin secretions of the Chinese red belly toad Bombina maxima. Its primary sequence was established as DLPKINRKGPRPPGFSPFR that comprises bradykinin extended from its N-terminus by a 10-residue segment DLPKINRKGP. The cDNA structure of bombinakinin M was found to contain a coding region of 624 nucleotides. The encoded precursor of bombinakinin M is composed of a signal peptide, an acidic peptide, six 100% identical copies of a 28-amino-acid peptide unit including bombinakinin M plus a spacer peptide. The sequence of bombinakinin M is preceded by a single basic residue (arginine), which represents the site of cleavage for releasing of mature bombinakinin M. This is the first cDNA cloning of bradykinin-related peptides from amphibian skin. The unique cDNA structure encoding bombinakinin M suggests that the generation modes of bradykinin-related peptides in amphibian skin and in mammalian blood system are different.     

Skin secretion of the toad Bombina variegata contains multiple insulin-releasing peptides including bombesin and entirely novel insulinotropic structures

Skin secretions of the toad Bombina variegata were evaluated for the isolation and characterisation of insulinotropic peptides. Crude secretions obtained from young adult toads by mild electrical stimulation of the dorsal skin surface were purified by reverse phase HPLC yielding 44 peaks. In acute incubations with glucose-responsive BRIN-BD11 cells, peaks 21, 22, 23, 24 and 25 showed a 1.5-3.5-fold increase in insulin release compared with 5.6 mM glucose alone (p<0.001; n=3). Structural analyses of the purified insulin-releasing peaks were performed by automated Edman degradation and mass spectrometry. Peptides represented by peaks 21, 22 and 23 had molecular masses of 1641.7 Da, 1662.6 Da and 1619.8 Da respectively. These peptides were unblocked by removal of pyroglutamic acid from the N-terminus prior to Edman degradation, revealing lengths of 14 amino acids. Peak 21 yielded a primary structure of Pyr-QRLGHQWAVGHLM, which a data base search revealed as an analogue of bombesin (His6 bombesin), while peak 23 was an exact match of bombesin (Pyr-QRLGNQWAVGHLM) originally isolated from Bombina bombina. Peak 22 indicated a primary structure of Pyr-DSFGNQWARGHFM (72% homology with bombesin). Peaks 24 and 25 revealed entirely novel insulinotropic peptides with molecular masses and primary structures of 1650.5 Da and 2300.0 Da and GKPFYPPPIYPEDM (GM-14) and IYNAICPCKHCNKCKPGLLAN (IN-21) respectively. Preliminary studies on the mechanisms underlying the insulinotropic actions of peaks 21, 22, 23 and 24 suggest possible involvement of a cAMP-dependent, G protein-insensitive pathway. These data indicate that Bombina variegata skin secretions contain peptides with insulin-releasing activity, which may have mammalian counterparts and prove useful for possible exploitation as antidiabetic agents from natural resources.     

Two novel Bv8-like peptides from skin secretions of the toad Bombina maxima

Two novel bioactive peptides were purified from skin secretions of the toad Bombina maxima. The partial N-terminal sequences of these two peptides were determined by automated Edman degradation. This allowed the cloning of full-length cDNAs encoding these two peptides from a cDNA library prepared from the toad skin. The deduced complete amino acid sequences indicate that both peptides are composed of 77 amino acids. A FASTA search in the databanks revealed that they exhibit 86-91% sequence identity with Bv8, a peptide originally isolated from skin secretions of Bombina variegata. They were thus named as Bv8-like peptide 1 (Bv8-LP1) and Bv8-like peptide 2 (Bv8-LP2), respectively. Sequence differences between Bv8-LP1 and 2 were due to six amino acid substitutions at positions 6, 11, 23, 24, 62 and 63. Bv8-LP1 and 2 differed from Bv8 with eleven and seven amino acid substitutions, respectively. Like Bv8, Bv8-LP1 and 2 possessed contractile activity on isolated guinea pig ileum. Additionally, they stimulated contraction of rabbit aortic rings in a dose-dependent manner at nanomolar concentrations.     

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.     

Molecular cloning of mRNA from toad granular gland secretion and lyophilized skin: identification of Bo8--a novel prokineticin from Bombina orientalis

Prokineticins are small (approximately 8 kDa), biologically active secretory proteins whose primary structures have been highly conserved throughout the Animal Kingdom. Representatives have been identified in the defensive skin secretions of several amphibians reflecting the immense structural/functional diversity of polypeptides in such. Here we describe the identification of a prokineticin homolog (designated Bo8) from the skin secretion of the Oriental fire-bellied toad (Bombina orientalis). Full primary structural characterization was achieved using a combination of direct Edman microsequencing, mass spectrometry and cloning of encoding skin cDNA. The latter approach employed a recently described technique that we developed for the cloning of secretory peptide cDNAs from lyophilized skin secretion, and this was further extended to employ lyophilized skin as the starting material for cDNA library construction. The Bo8 precursor was found to consist of an open-reading frame of 96 amino acid residues consisting of a putative 19-residue signal peptide followed by a single 77-residue prokineticin (Mr=7990 Da). Amino acid substitutions in skin prokineticins from the skin secretions of bombinid toads are confined to discrete sites affording the necessary information for structure/activity studies and analog design.