Characterization of the genomic corticotropin-releasing factor (CRF) gene from Xenopus laevis: two members of the CRF family exist in amphibians.

In mammals, the release of pituitary ACTH is stimulated by CRF. Two related peptides exist in nonmammalian vertebrates, sauvagine from frog skin and urotensin-I from the urophysis of teleost fish. Their related structures (approximately 50%) and capacity to stimulate the release of ACTH from mammalian and fish pituitaries has led to the proposal that sauvagine and urotensin-I are homologs of mammalian CRF. However, sauvagine does not appear to stimulate ACTH release in amphibians, although mammalian CRF (ovine) induces a potent response from amphibian pituitaries. This could indicate that the main function of sauvagine does not involve ACTH regulation and suggests that an additional CRF-like peptide exists in Amphibia. We report here the isolation of two highly homologous CRF-like genes from the frog, Xenopus laevis. Analysis of the expression pattern of these CRF-like genes revealed mRNA in splenic tissue and in the preoptic nucleus and paraventricular organ of the brain. The amino acid sequence of the mature peptide regions (1-41) of both X. laevis genes is strikingly conserved, sharing more than 93% homology with mammalian CRFs, yet only 50% homology with sauvagine. In view of the fact that these new amphibian CRF-like genes share far greater homology with mammalian CRF than that exhibited by sauvagine, we propose that the new Xenopus CRF-like genes are the amphibian counterparts to mammalian CRF. Thus, two members of the CRF family have now been identified in the Amphibia, namely CRF and sauvagine.     

Comparison of the biologic actions of corticotropin-releasing factor and sauvagine

Corticotropin-releasing factor (CRF), a peptide isolated from ovine hypothalamus, and sauvagine, a peptide isolated from frog skin, share significant structural homology and elicit a number of similar biological responses. CRF is more potent than sauvagine in stimulating pituitary ACTH secretion. Sauvagine, however, is 5–10 times more potent than CRF to act within the brain to increase plasma levels of catecholamines and glucose and to elevate mean arterial pressure. Sauvagine is likewise more potent than CRF to act outside the brain to increase superior mesenteric artery flow and plasma glucose concentrations and to decrease mean arterial pressure. CRF and sauvagine produce important effects representative of biologically active peptides.     

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.     

Isolation and amino acid sequence of urotensin I, a vasoactive and ACTH-releasing neuropeptide, from the carp (Cyprinus carpio) urophysis

Urotensin I (UI), a 41-residue mammalian hypotensive and fish or mammalian corticotropin-releasing peptide, isolated from 0.1 N HCI extracts of urophyses of the carp (Cyprinus carpio) was purified and the amino acid sequence was determined to be: H-Asn-Asp-Asp-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu- Arg-Asn-Met-Ile-Glu-Met-Ala-Arg-Asn-Glu-Asn-Gln-Arg-Glu-Gln-Ala-Gly-Leu-Asn-Arg-Lys-Tyr-Leu-Asp-Glu-Val-NH2. When the extraction procedure included heating at 100 degrees C for 15 min, UI was cleaved at a highly acid labile Asp-Pro bond to give the fully active UI (4-41). Urotensin I shows close structural and biological homology with the recently isolated ovine hypothalamic corticotropin-releasing factor (CRF) and the frog skin peptide sauvagine and thus may be considered an evolutionary prototype of unique mammalian-hypotensive and vertebrate corticotropin-releasing factors.     

Sauvatide - A novel amidated myotropic decapeptide from the skin secretion of the waxy monkey frog, Phyllomedusa sauvagei

Here we describe the structural and functional characterization of a novel myotropic peptide, sauvatide, from the skin secretion of the waxy monkey frog, Phyllomedusa sauvagei. Sauvatide is a C-terminally amidated decapeptide with the following primary structure - LRPAILVRTKamide - monoisotopic mass 1164.77 Da, which was found to contract the smooth muscle of rat urinary bladder with an EC₅₀ of 2.2 nM. The sauvatide precursor, deduced from cloned skin cDNA, consists of 62 amino acid residues with a single copy of sauvatide located near the C-terminus. The mature peptide is generated from the precursor by cleavage at a classical -KR-cleavage site located proximal to the N-terminus and by removal of a -GKGK sequence at the C-terminus, the first glycyl residue acting as amide donor. Amphibian skin secretions thus continue to be a source of novel and potent biologically active peptides acting through functional targets in mammalian tissues.     

Identification of an urotensin I-like peptide in the pituitary of the lungfish Protopterus annectens: immunocytochemical localization and biochemical characterization

In the present study we have investigated the localization and biochemical characteristics of urotensin I (UI)-like and urotensin II (UII)-like immunoreactive peptides in the central nervous system (CNS) and pituitary of the lungfish, Protopterus annectens, by using antisera raised against UI from the white sucker Catostomus commersoni and against UII from the goby Gillichythys mirabilis. UI-like immunoreactive material was found within the melanotrope cells of the intermediate lobe of the pituitary. By contrast, no UI-immunoreactive structures were found in the brain. No UII-like peptides structurally similar to goby UII were found in the brain and pituitary of P. annectens. The UI-immunoreactive material localized in the pituitary was characterized by combining reversed-phase high-performance liquid chromatography (HPLC) analysis and radioimmunological detection. The UI-like immunoreactivity contained in a pituitary extract eluted as a single peak with a retention time intermediate between those of sucker UI and rat corticotropin-releasing factor (CRF). Control tests on adjacent sections of pituitary showed that the UI antiserum cross-reacted with the frog skin peptide sauvagine, but lungfish UI did not co-elute with synthetic sauvagine on HPLC. On the contrary, no cross-reaction was observed between the UI antiserum and CRF or alpha-melanocyte-stimulating hormone (alpha-MSH). The occurrence of an UI-like peptide in the intermediate lobe of the pituitary of P. annectens suggests that, in lungfish, this peptide may act as a classic pituitary hormone or may be involved in the control of melanotrope cell secretion.     

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.     

Identification of Two Corticotropin-Releasing Factor Receptors from Xenopus laevis with High Ligand Selectivity: Unusual Pharmacology of the Type 1 Receptor

Abstract: Two cDNA clones encoding distinct members of the corticotropin-releasing factor (CRF) receptor family have been isolated from Xenopus laevis with PCR-based approaches. The first full-length cDNA amplified from Xenopus brain encoded a 415-amino acid protein with ∼80% identity to mammalian CRF receptor type 1 (CRF-R1). The second full-length cDNA isolated from Xenopus brain and heart encoded a 413-amino acid protein with ∼81% identity to the α-variant of mammalian CRF receptor, type 2 (CRF-R2). No evidence could be obtained that the β-variant of CRF-R2 existed in Xenopus laevis . Binding studies using human embryonic kidney 293 (HEK 293) cells stably transfected with xenopus CRF-R2 showed that the CRF analogues urotensin I, urocortin, and sauvagine were bound with higher affinities than human/rat CRF, xenopus CRF, and ovine CRF. In contrast to human CRF-R1, xenopus CRF-R1 (xCRF-R1) was very selective for different CRF ligands. Urotensin I, urocortin, human/rat CRF, and xenopus CRF were bound with significantly (10–22-fold) higher affinities than ovine CRF ( K _D = 31.7 n M ) and sauvagine ( K _D = 51.4 n M ). In agreement with these binding data, EC₅₀ values of 39.7 and 1.1 n M were found for sauvagine and for human/rat CRF or xenopus CRF, respectively, when the cyclic AMP production in HEK 293 cells stably transfected with xCRF-R1 was determined.     

Regulation of MSH release from the neurointermediate lobe of Xenopus laevis by CRF-like peptides

Immunocytochemical studies showed the presence of a fiber system containing a CRF-like peptide in the median eminence and in the neural lobe of the pituitary gland of Xenopus laevis. During in vitro superfusion of neurointermediate lobe tissue, CRF, sauvagine and urotensin I induced a rapid and dose-dependent stimulation of secretion of MSH and endorphin. Tissue of white-background adapted animals displayed a remarkably higher sensitivity to CRF and sauvagine than tissue from animals that were adapted to a black background. During superfusion of isolated melanotrope cells in suspension, it was shown that CRF and sauvagine exerted their effect directly on the melanotrope cell. We therefore conclude that there is morphological and biochemical evidence to consider a CRF-like peptide as a physiological MSH-releasing factor.     

A novel myotropic peptide from the skin secretions of the tree frog, Polypedates pingbianensis

A novel myotropic peptide, polypedatein, was purified and characterized from the skin secretions of the tree frog, Polypedates pingbianensis. Its primary structure, TLLCKYFAIC, was determined by Edman degradation and mass spectrometry. Polypedatein was subjected to bioassays including myotropic, antimicrobial, and serine protease inhibitory activities, which are related with many amphibian skin bioactive peptides. It was found to elicit concentration-dependent contractile effects on isolated rat ileum. cDNA clones encoding the precursor of polypedatein were isolated by screening a skin cDNA library of P. pingbianensis and then sequenced. The amino acid sequence deduced from the cDNA sequences matches well with the result from Edman degradation. BLAST search revealed that the sequence of polypedatein did not show similarity to known protein or peptide sequences. Especially, polypedatein does not contain conserved structural motifs of other amphibian myotropic peptides, such as bradykinins, bombesins, cholecystokinin (CCK), and tachykinins, indicating that polypedatein belongs to a novel amphibian myotropic peptide family. The signal peptide of the precursor encoding polypedatein shows significant sequence identity to that of other amphibian skin defensive peptides, such as antimicrobial peptides, bradykinins, lectins, and serine protease inhibitors, suggesting that polypedatein belongs to a novel amphibian myotropic peptide family. Polypedatein is also the first bioactive peptide from the genus of the frog, Polypedates.     

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.     

Cortisol inhibits the ACTH-releasing activity of urotensin I, CRF and sauvagine observed with superfused goldfish pituitary cells

The structurally homologous peptides urotensin I, ovine CRF and sauvagine stimulate the release of immunoreactive ACTH from a superfused dispersed goldfish anterior pituitary cell column. The addition of cortisol to the superfusion buffer resulted, following a latent period, in a decrease in basal release of ACTH from the pituitary cell column and a diminution in the ACTH-releasing activities of urotensin I, CRF and sauvagine. The removal of cortisol from the superfusion buffer resulted in a slow recovery of basal ACTH release and a recovery of the ACTH-releasing activities of urotensin I, CRF and sauvagine. These results are supportive of the view that urotensin I, or a urotensin I-like peptide, serves as a physiological regulator of ACTH release in teleost fishes.     

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.     

Expression of genes encoding antimicrobial and bradykinin-related peptides in skin of the stream brown frog Rana sakuraii

Peptidomic analysis of an extract of the skin of the stream brown frog Rana sakuraii Matsui and Matsui, 1990 led to the isolation of a C-terminally alpha-amidated peptide (VR-23; VIGSILGALASGLPTLISWIKNR x NH2) with broad-spectrum antimicrobial activity that shows structural similarity to the bee venom peptide, melittin together with two peptides belonging to the temporin family (temporin-1SKa; FLPVILPVIGKLLNGIL x NH2 and temporin-1SKb; FLPVILPVIGKLLSGIL x NH2), and peptides whose primary structures identified them as belonging to the brevinin-2 (2 peptides) and ranatuerin-2 (1 peptide) families. Using a forward primer that was designed from a conserved region of the 5'-untranslated regions of Rana temporaria preprotemporins in a 3'-RACE procedure, a cDNA clone encoding preprotemporin-1SKa was prepared from R. sakuraii skin total RNA. Further preprotemporin cDNAs encoding temporin-1SKc (AVDLAKIANIAN KVLSSL F x NH2) and temporin-1SKd (FLPMLAKLLSGFL x NH2) were obtained by RT-PCR. Unexpectedly, the 3'-RACE procedure using the same primer led to amplification of a cDNA encoding a preprobradykinin whose signal peptide region was identical to that of preprotemporin-1SKa except for the substitution Ser18-->Asn. R. sakuraii bradykinin ([Arg0,Leu1,Thr6,Trp8] BK) was 28-fold less potent than mammalian BK in effecting B2 receptor-mediated relaxation of mouse trachea and the des[Arg0] derivative was only a weak partial agonist. The evolutionary history of the Japanese brown frogs is incompletely understood but a comparison of the primary structures of the R. sakuraii dermal peptides with those of Tago's brown frog Rana tagoi provides evidence for a close phylogenetic relationship between these species.     

Amphibian skin peptides and their corresponding cDNAs from single lyophilized secretion samples: identification of novel brevinins from three species of Chinese frogs

Brevinins are peptides of 24 amino acid residues, originally isolated from the skin of the Oriental frog, Rana brevipoda porsa, by nature of their microbicidal activity against a wide range of Gram-positive and Gram-negative bacteria and against strains of pathogenic fungi. cDNA libraries were constructed from lyophilized skin secretion of three, unstudied species of Chinese frog, Odorrana schmackeri, Odorrana versabilis and Pelophylax plancyi fukienensis, using our recently developed technique. In this report, we describe the "shotgun" cloning of novel brevinins by means of 3'-RACE, using a "universal" degenerate primer directed towards a highly conserved nucleic acid sequence domain within the 5'-untranslated region of previously characterized frog skin peptide cDNAs. Novel brevinins, deduced from cloned cDNA open-reading frames, were subsequently identified as mature peptides in the same samples of respective species skin secretions. Bioinformatic analysis of both prepro-brevinin nucleic acid sequences and translated open-reading frame amino acid sequences revealed a highly conserved signal peptide domain and a hypervariable anti-microbial peptide-encoding domain. The experimental approach described here can thus rapidly provide robust structural data on skin anti-microbial peptides without harming the donor amphibians.     

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.     

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.