Cloning of a receptor for amphibian [Phe13]bombesin distinct from the receptor for gastrin-releasing peptide: identification of a fourth bombesin receptor subtype (BB4).

Bombesin is a tetradecapeptide originally isolated from frog skin and demonstrated to have a wide range of actions in mammals. Based on structural homology and similar biological activities, gastrin-releasing peptide (GRP) has been considered the mammalian equivalent of bombesin. We previously reported that frogs have both GRP and bombesin, which therefore are distinct peptides. We now report the cloning of a bombesin receptor subtype (BB4) that has higher affinity for bombesin than GRP. PCR was used to amplify cDNAs related to the known bombesin receptors from frog brain. Sequence analysis of the amplified cDNAs revealed 3 classes of receptor subtypes. Based on amino acid homology, two classes were clearly the amphibian homologs of the GRP and neuromedin B receptors. The third class was unusual and a full-length clone was isolated from a Bombina orientalis brain cDNA library. Expression of the receptor in Xenopus oocytes demonstrated that the receptor responded to picomolar concentrations of [Phe13]-bombesin, the form of bombesin most prevalent in frog brain. The relative rank potency of bombesin-like peptides for this receptor was [Phe13]bombesin > [Leu13]bombesin > GRP > neuromedin B. In contrast, the rank potency for the GRP receptor is GRP > [Leu13]bombesin > [Phe13]bombesin > neuromedin B. Transient expression in CHOP cells gave a Ki for [Phe13]bombesin of 0.2 nM versus a Ki of 2.1 nM for GRP. Distribution analysis showed that this receptor was expressed only in brain, consistent with the distribution of [Phe13]-bombesin. Thus, based on distribution and affinity, this bombesin receptor is the receptor for [Phe13]bombesin. Phylogenetic analysis suggests that this receptor separated prior to separation of the GRP and neuromedin B receptors; thus, BB4 receptors and their cognate ligands may also exist in mammals.

Ex Vivo Smooth Muscle Pharmacological Effects of a Novel Bradykinin-Related Peptide, and Its Analogue, from Chinese Large Odorous Frog, Odorrana livida Skin Secretions

Bradykinin-related peptides (BRPs) are one of the most extensively studied frog secretions-derived peptide families identified from many amphibian species. The diverse primary structures of BRPs have been proven essential for providing valuable information in understanding basic mechanisms associated with drug modification. Here, we isolated, identified and characterized a dodeca-BRP (RAP-L1, T6-BK), with primary structure RAPLPPGFTPFR, from the skin secretions of Chinese large odorous frogs, Odorrana livida. This novel peptide exhibited a dose-dependent contractile property on rat bladder and rat ileum, and increased the contraction frequency on rat uterus ex vivo smooth muscle preparations; it also showed vasorelaxant activity on rat tail artery smooth muscle. In addition, the analogue RAP-L1, T6, L8-BK completely abolished these effects on selected rat smooth muscle tissues, whilst it showed inhibition effect on bradykinin-induced rat tail artery relaxation. By using canonical antagonist for bradykinin B1 or B2 type receptors, we found that RAP-L1, T6-BK -induced relaxation of the arterial smooth muscle was very likely to be modulated by B2 receptors. The analogue RAP-L1, T6, L8-BK further enhanced the bradykinin inhibitory activity only under the condition of co-administration with HOE140 on rat tail artery, suggesting a synergistic inhibition mechanism by which targeting B2 type receptors.

A biologically active peptide in the skin of lampreys (Eudontomyzon danfordi vladykovi). [Translation from: Z.Naturforsch. (B), 26(10), 1021-1023, 1971. ]

In recent years interest in the production and description of kinin-type substances has been greatly intensified. So, for example, bradykinin, phyllokinin, physalaemin, ranatensin and caerulein could be extracted from the skin of amphibians as well as. eledoisin out of the salivary glands of Eledon moschata. An examination of lampreys seemed to us particularly profitable in the search for the incidence of further kinins. Ammocoetes of different sizes and also adults of both sexes of the species Eudontomyzon danfordi vladykovi were studied in this research. This species is found in many tributaries of the Danube. Skin extracts were tested on on isolated rat uterus, rat duodenum, guinea pig ileum and rabbit jejunum, further tests were done in order to determine a peptide character of the biologically active substance.

A novel algesic peptide derived from skin secretions of the frog Amolops loloensis

Several algesic agents including bradykinin and tachykinin have been identified from skin secretions of amphibians. They may act in defensive roles against aggressors. In this study, a novel peptide named Amolos with an amino acid sequence of FLPIVGAKL an

Cloning of bradykinin precursor cDNAs from skin of Bombina maxima reveals novel bombinakinin M antagonists and a bradykinin potential peptide

Bombinakinin M (DLPKINRKGP-bradykinin) is a bradykinin-related peptide purified from skin secretions of the frog Bombina maxima. As previously reported, its biosynthesis is characterized by a tandem repeats with various copy numbers of the peptide and sometimes co-expressed with other structure-function distinguishable peptides. At present study, two novel cDNAs encoding bombinakinin M and its variants were cloned from a cDNA library from the skin of the frog. The encoded two precursor proteins are common in that each contains three repeats of a novel 16-amino acid peptide unit and one copy of kinestatin at their N- and C-terminal parts, respectively. They differ in that the first precursor contains two copies of bombinakinin M and the second one contains one copy of a novel bombinakinin M variant. Bombinakinin M was found to elicit concentration-dependent contractile effects on guinea pig ileum, with an EC50 value of 4 nM that is four times higher than that of bradykinin (1 nM). Interestingly, the synthetic peptide (DYTIRTRLH-amide), as deduced from the 16-amino acid peptide repeats in the newly cloned cDNAs, possessed weak inhibitory activity on the contractile effects of bombinakinin M, but not on that of bradykinin. Furthermore, the newly identified bombinakinin M variant (DLSKMSFLHG-Ile(1)-bradykinin), did not show contractile activity on guinea pig ileum, but showed potentiation effect on the myotropic activity of bradykinin. In a molar raito of 1:58, it augmented the activity of bradykinin up to two-fold. (C) 2004 Elsevier B.V. All rights reserved.

Five novel antimicrobial peptides from skin secretions of the frog, Amolops loloensis

While investigating antimicrobial peptide diversity of Amolops loloensis, five novel antimicrobial peptides belonging to two families were identified from skin secretions of this frog. The first family including two members is esculentin-2-AL (esculentin-2-ALa and -ALb): the second family including three members is temporin-AL (temporin-ALd to -ALf). The family of esculentin-2-AL is composed of 37 amino acid residues (aa); the family of temporin-AL is composed of 16, 13 and 10 aa, respectively. All of these antimicrobial peptides showed antimicrobial activities against tested microorganisms. cDNAs encoding precursors of esculentin-2-ALs and temporin-ALs were cloned from the skin cDNA library of A. loloensis. All the precursors share similar overall structures. There is a typical prohormone processing signal (Lys-Arg) located between the acidic propiece and the mature peptide. The antimicrobial peptide family of esculentin-2 is firstly reported in the genus of Amolops. Combined with previous reports, a total of four antimicrobial peptide families have been identified from the genus of Amolops; three of them are also found in the genus of Rana. These results suggest the possible evolutionary connection between the genera Amolops and Rana. (C) 2009 Elsevier Inc. All rights reserved.

Peptide leucine arginine, a potent immunomodulatory peptide isolated and structurally characterized from the skin of the Northern Leopard frog, Rana pipiens

On the basis of histamine release from rat peritoneal mast cells, an octadecapeptide was isolated from the skin extract of the Northern Leopard frog (Rana pipiens), This peptide was purified to homogeneity using reversed-phase high performance liquid chromatography and found to have the following primary structure by Edman degradation and pyridylethylation: LVRGCWTKSYPPKPCFVR, in which Cys(5) and Cys(15) are disulfide bridged. The peptide was named peptide leucine-arginine (pLR), reflecting the N- and C-terminal residues. Molecular modeling predicted that pLR possessed a rigid tertiary loop structure with flexible end regions, pLR was synthesized and elicited rapid, noncytolytic histamine release that had a a-fold greater potency when compared with one of the most active histamine-liberating peptides, namely melittin, pLR was able to permeabilize negatively charged unilamellar lipid vesicles but not neutral vesicles, a finding that was consistent with its nonhemolytic action, pLR inhibited the early development of granulocyte macrophage colonies from bone marrow stem cells but did not induce apoptosis of the end stage granulocytes, i,e. mature neutrophils, pLR therefore displays biological activity with both granulopoietic progenitor cells and mast cells and thus represents a novel bioactive peptide from frog skin.

Novel frenatins from the skin of the Australasian Giant White-lipped tree frog, Litoria infrafrenata: cloning of precursor cDNAs and identification in defensive skin secretion.

The Australasian anuran amphibian genus Litoria, contains many phenotypically-diverse species as a result of radial evolution of an ancestral species into different biotopes much in the manner of the indigenous marsupial mammals. In common with members of the Central/South American genus Phyllomedusa, their specialized skin granular glands are factories for the production of a plethora of biologically-active peptides. Here we report a more detailed study of those present in the defensive skin secretion of the Australasian giant white-lipped tree frog, Litoria infrafrenata, and, for the first time, we have identified three novel frenatins by deduction of primary structures from cDNAs that were cloned from a library constructed from lyophilized skin secretion using a recently-developed technique. All open-reading frames consisted of a putative signal peptide and an acidic pro-region followed by a single copy of a frenatin peptide. Processed peptides corresponding in molecular mass to the deduced molecular masses of frenatins (named 1.1, 3, 3.1 and 4.1) were identified in the same secretion sample using HPLC and mass spectroscopy. The application of this technique thus permits parallel peptidomic and transcriptomic analyzes on the same lyophilized skin secretion sample circumventing sacrifice of specimens from endangered herpetofauna.

The complex array of bradykinin-related peptides (BRPs) in the peptidome of pickerel frog (Rana palustris) skin secretion is the product of transcriptional economy.

Previous peptidomic analyses of the defensive skin secretion from the North American pickerel frog, Rana palustris, have established the presence of canonical bradykinin and multiple bradykinin-related peptides (BRPs). As a consequence of the multiplicity of peptides identified and their diverse primary structures, it was speculated that they must represent the products of expression of multiple genes. Here, we present unequivocal evidence that the majority of BRPs (11/13) identified in skin secretion by the peptidomic approach can be generated by differential site-specific protease cleavage from a single common precursor of 321 amino acid residues, named skin kininogen 1, whose primary structure was deduced from cloned skin secretion-derived cDNA. The organization of skin kininogen 1 consists of a hydrophobic signal peptide followed by eight non-identical domains each encoding a single copy of either canonical bradykinin or a BRP. Two additional splice variants, encoding precursors of 233 (skin kininogen 2) or 189 amino acid residues (skin kininogen 3), were also cloned and were found to lack BRP-encoding domains 5 and 6 or 4, 5 and 6, respectively. Thus, generation of peptidome diversity in amphibian defensive skin secretions can be achieved in part by differential protease cleavage of relatively large and multiple-encoding domain precursors reflecting a high degree of transcriptional economy.