A novel bradykinin-like peptide from skin secretions of the frog, Rana nigrovittata.

A bradykinin-like peptide has been isolated from the skin secretions of the frog Rana nigrovittata. This peptide was named ranakinin-N. Its primary structure, RAEAVPPGFTPFR, was determined by Edman degradation and mass spectrometry. It is structurally related to bradykinin-like peptides identified from skin secretions of other amphibians. Ranakinin-N is composed of 13 amino acid residues and is related to the bradykinin identified from the skin secretions of Odorrana schmackeri, which is composed of 9 amino acid residues. Ranakinin-N was found to exert concentration-dependent contractile effects on isolated guinea pig ileum. cDNA sequence encoding the precursor of ranakinin-N was isolated from a skin cDNA library of R. nigrovittata. The amino acid sequences deduced from the cDNA sequences match well with the results from Edman degradation. Analysis of different amphibian bradykinin cDNA structures revealed that the deficiency of a 15-nucleotide fragment (agaatgatcagacgc in the cDNA encoding bradykinin from O. schmackeri) in the peptide-coding region resulted in the absence of a dibasic site for trypsin-like proteinases and an unusual -AEVA- insertion in the N-terminal part of ranakinin-N. The -AEAV- insertion resulted in neutral net charge at the N-terminus of ranakinin-N. Ranakinin-N is the first reported bradykinin-like peptide with a neutral net charge at the N-terminus.     

A novel family of antimicrobial peptides from the skin of Amolops loloensis

While conducting experiments to investigate antimicrobial peptides of amphibians living in the Yunnan-Sichuan region of southwest China, a new family of antimicrobial peptides was identified from skin secretions of the rufous-spotted torrent frog, Amolops loloensis. Members of the new peptide family named amolopins are composed of 18 amino acids with a unique sequence, for example, NILSSIVNGINRALSFFG. By BLAST search, amolopins did no show similarity to any known peptides. Among the tested microorganisms, native and synthetic peptides only showed antimicrobial activities against Staphylococcus aureus ATCC2592 and Bacillus pumilus, no effects on other microorganisms. The CD spectroscopy showed that it adopted a structure of random combined with beta-sheet in water, Tris-HCl or Tris-HCl-SDS. Several cDNAs encoding amolopins were cloned from the skin cDNA library of A. loloensis. The precursors of amolopin are composed of 62 amino acid residues including predicted signal peptides, acidic propieces, and mature antimicrobial peptides. The preproregion of amolopin precursor comprises a hydrophobic signal peptide of 22 residues followed by an 18 residue acidic propiece which terminates by a typical prohormone processing signal Lys-Arg. The preproregions of precursors are very similar to other amphibian antimicrobial peptide precursors but the mature amolopins are different from other antimicrobial peptide families. The remarkable similarity of preproregions of precursors that give rise to very different antimicrobial peptides in distantly related frog species suggests that the corresponding genes form a multigene family originating from a common ancestor.     

Amphibian cathelicidin fills the evolutionary gap of cathelicidin in vertebrate

Cathelicidins comprise a family of antimicrobial peptides (AMPs) sharing a highly conserved cathelin domain, and play a central role in the innate defense against infection in most of vertebrates. But so far it has not yet been found in amphibians although a large number of other groups of AMPs have been identified. In the current work, the first amphibian cathelicidin (cathelicidin-AL) has been characterized from the frog skin of Amolops loloensis. Cathelicidin-AL (RRSRRGRGGGRRGGSGGRGGRGGGGRSGAGSSIAGVGSRGGGGGRHYA) is a cationic peptide containing 48 amino acid residues (aa) with 12 basic aa and no acidic aa. The chemical synthesized peptide efficiently killed bacteria and some fungal species including clinically isolated drug-resistance microorganisms. The cDNA encoding cathelicidin-AL precursor was cloned from the skin cDNA library of A. loloensis. As other cathelicidins, the precursor of cathelicidin-AL also contains highly conserved anionic cathelin domain of cysteine proteinase inhibitor followed by the AMP fragment at C-terminus. Phylogenetic analysis revealed that as connecting link, the amphibian cathelicidin predates reptilia but postdates fish cathelicidin. The peptide purification combined with gene cloning results confirms the presence of cathelicidin in amphibians and filled the evolutionary gap of cathelicidin in vertebrate, considering amphibians' special niche as the animals bridging the evolutionary land-water gap.     

Gene cloning and characterization of novel antinociceptive peptide from the brain of the frog, Odorrana grahami

Amphibian opiate peptides including dermorphins and deltorpins have been recently found only in the skin of South American frogs belonging to the subfamily Phyllomedusinae (Phyllomedusa, Agalychnis and Pachymedusa species). No opiate peptides have ever been identified from other amphibians or organs except skin. Here we report the purification and characterization of a novel antinociceptive peptide named odorranaopin from the homogenates of the frog brains, Odorrana grahami, which is also the first antinociceptive peptide found in Ranidae amphibian. Odorranaopin comprises 17 amino acid residues with the sequence of DYTIRTRLHQESSRKVL (Mr 2102 Da). The cDNA encoding odorranaopin was cloned from the frog brain cDNA library, and it was confirmed to be a specific gene. The odorranaopin precursor deduced is composed of 61 amino acid residues including the predicted signal peptide, acidic spacer peptide and mature odorranaopin positioned at the C-terminus. Odorranaopin could inhibit nociceptive responses induced by formalin and acetic acid. It also inhibited the contractile responses of ileum smooth muscle induced by bradykinin, implying that the antinociceptive activity of odorranaopin possibly results from its blockade on bradykinin or bradykinin receptor functions. Odorranaopin is the first antinociceptive peptide found in Ranidae amphibian.     

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 new family of antimicrobial peptides from skin secretions of Rana pleuraden

While conducting experiments to investigate antimicrobial peptides of amphibians living in the Yunnan-Guizhou region of southwest China, a new family of antimicrobial peptides was identified from skin secretions of the Yunnan frog, Rana pleuraden. Members of the new peptide family named pleurain-As are composed of 26 amino acids with a unique N-terminal sequence (SIIT) and a disulfide-bridged heptapeptide sequence (CRLYNTC). By BLAST search, pleurain-As had no significant similarity to any known peptides. Native and synthetic peptides showed antimicrobial activities against tested microorganisms including Gram-negative and Gram-positive bacteria and fungi. Twenty different cDNAs encoding pleurain-As were cloned from the skin cDNA library of R. pleuraden. The precursors of pleurain-As are composed of 69 amino acid residues including predicted signal peptides, acidic propieces, and cationic mature antimicrobial peptides. The preproregion of pleurain-A precursor comprises a hydrophobic signal peptide of 22 residues followed by an 18 residue acidic propiece which terminates by a typical prohormone processing signal Lys-Arg. The preproregions of precursors are very similar to other amphibian antimicrobial peptide precursors but the mature pleurain-As are different from other antimicrobial peptide families. The remarkable similarity of preproregions of precursors that give rise to very different antimicrobial peptides in distantly related frog species suggests that the corresponding genes form a multigene family originating from a common ancestor. Furthermore, pleurain-As could exert antimicrobial capability against Helicobacter pylori. This is the first report of naturally occurring peptides with anti-H. pylori activity from Rana amphibians.     

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.     

Five novel antimicrobial peptides from the Kuhl’s wart frog skin secretions, Limnonectes kuhlii

Five novel antimicrobial peptides (temporin-LK1, rugosin-LK1, rugosin-LK2, gaegurin-LK1, and gaegurin-LK2) are purified and characterized from Kuhl’s wart frog skin secretions, Limnonectes kuhlii. They share obvious similarity to temporin, rugosin, and gaegurin antimicrobial peptide family, respectively. Their amino acid sequences were determined by Edman degradation and mass spectrometry, and further confirmed by cDNA cloning. Nine cDNA sequences encoding precursors of these five purified antimicrobial peptides and other four hypothetical antimicrobial peptides were cloned from the skin cDNA library of L. kuhlii. The deduced precursors are composed of a predicted signal peptide, an acidic spacer peptide, and a mature antimicrobial peptide. Most of them showed strong antimicrobial activities against Gram-positive and Gram-negative bacteria and fungi. The current work identified and characterized three families of antimicrobial peptides from L. kuhlii skins and confirmed that the genus of Limnonectes amphibians share similar antimicrobial peptide families with the genus of Rana amphibians. In addition, a unique antimicrobial peptide (temporin-LK1) with 17 residues including four phenylalanines, which is significantly different from other temporins (16 residues, one or two phenylalanines), was identified in this work. Such unique structure might provide novel template or leading structure to design antimicrobial agents.     

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 and isolated from skin secretion of the frog Amolops loloensis, is shown to strengthen nociceptive responses induced by inflammatory factors and strongly inhibit the contraction of isolated ileum. A synthetic peptide based on the sequence obtained showed characterization data identical to those of the isolated material, confirming its structure. These two types of responses seem to be a part of the defensive functions against predators or aggressors. The current results suggest that pharmacological molecules in amphibian skins not only act as innate defense mechanisms against microorganisms but also exert other defensive physiological functions against other aggressors.     

Amphibian tachykinin precursor

The precursor of amphibian tachykinin has not been found although more than 30 tachykinins have been isolated from amphibians since 1964. In this report, two tachykinin-like peptides are identified from the skin secretions of the frog, Odorrana grahami. Their amino acid sequences are DDTEDLANKFIGLM-NH(2) (named tachykinin OG1) and DDASDRAKKFYGLM-NH(2) that is the same with ranamargarin found in Rana margaretae, respectively, with a conserved FXGLM-NH(2) C-terminal consensus motif. By cDNA cloning, their precursors were screened from the skin cDNA library of O. grahami. The precursors are composed of 61 amino acid (aa) residues including a signal peptide followed by an acidic spacer peptide and one copy of mature tachykinin-like peptide. Their overall structure is different from structures of other tachykinin precursors such as human protachykinin 1 precursor containing 143 aa including one copy of substance P (SP) and neurokinin A (NKA), and ascidian tachykinin 1 precursor containing 164 aa including two copies of tachykinin-like peptides. The current results demonstrate that the biosynthesis mode of tachykinins in amphibians is different from other animals.     

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.     

BSTI, a trypsin inhibitor from skin secretions of Bombina bombina related to protease inhibitors of nematodes.

From skin secretions of the European frog Bombina bombina, a new peptide has been isolated that contains 60 amino acids, including 10 cysteine residues. Its sequence was determined by automated Edman degradation and confirmed by analysis of the cDNA encoding the precursor. A search in the databanks demonstrated that the pattern of cysteine residues in this skin peptide is similar to the ones found in protease inhibitors from Ascaris and in a segment of human von Willebrand factor. The 3D structure of the trypsin inhibitor from Ascaris suum could be used as a template to build a model of the amphibian peptide. In addition, we have demonstrated that this constituent of skin secretion is indeed an inhibitor of trypsin and thrombin, with K(i) values in the range of 0.1 to 1 microM. The new peptide was thus named BSTI for Bombina skin trypsin/thrombin inhibitor.     

棕点湍蛙皮肤分泌液中促胰岛素释放肽的分离纯化、基因克隆及活性分析

通过分离纯化棕点湍蛙(Amolops loloensis)皮肤分泌液中的生物活性物质,得到有促胰岛素释放活性的分离峰,并鉴定其结构.采用葡聚糖Sephadex G-50凝胶层析和反相高效液相(RP-HPLC)等手段对棕点湍蛙皮肤分泌液进行分离纯化,利用胰岛素释放实验进行活性检测,Edman降解法测定活性峰的氨基酸序列,反转录法构建cDNA文库并克隆其基因.得到一个具有显著的促胰岛素释放活性的十六肽,测得其氨基酸序列为:FMPIvGKsMSGLSGKL-NH2,命名为amolopin-1.由cDNA(开放阅读框为192bp)推导的氨基酸一级结构显示,其前体由64个氨基酸残基(aa)组成,包括高度保守的信号肽(22aa),酸性肽以及成熟肽.经过数据库序列比对,从棕点湍蛙皮肤中得到一个新的促胰岛素释放肽,进一步分析其作用机理和药代动力学,极有可能得到一个新的治疗糖尿病的降糖药物.     

Bi-functional peptides with both trypsin-inhibitory and antimicrobial activities are frequent defensive molecules in Ranidae amphibian skins

Amphibian skins act as the first line against noxious aggression by microorganisms, parasites, and predators. Anti-microorganism activity is an important task of amphibian skins. A large amount of gene-encoded antimicrobial peptides (AMPs) has been identified from amphibian skins. Only a few of small protease inhibitors have been found in amphibian skins. From skin secretions of 5 species (Odorrana livida, Hylarana nigrovittata, Limnonectes kuhlii, Odorrana grahami, and Amolops loloensis) of Ranidae frogs, 16 small serine protease inhibitor peptides have been purified and characterized. They have lengths of 17-20 amino acid residues (aa). All of them are encoded by precursors with length of 65-70 aa. These small peptides show strong trypsin-inhibitory abilities. Some of them can exert antimicrobial activities. They share the conserved GCWTKSXXPKPC fragment in their primary structures, suggesting they belong to the same families of peptide. Signal peptides of precursors encoding these serine protease inhibitors share obvious sequence similarity with those of precursors encoding AMPs from Ranidae frogs. The current results suggest that these small serine protease inhibitors are the common defensive compounds in frog skin of Ranidae as amphibian skin AMPs.     

Three novel antimicrobial peptides from the skin of Rana shuchinae

Intensive studies have demonstrated that there are many antimicrobial peptides in amphibian skins. Three novel antimicrobial peptides were identified from the skin of the frog, Rana shuchinae. They are named shuchins 3–5. Their sequences were determined as KAYSMPRCKGGFRAVMCWL-NH₂, KAYSTPRCKGLFRALMCWL-NH₂, and KAYSMPRCKYLFRAVLCWL-NH₂ by Edman degradation and mass spectrometry analysis, respectively. They are composed of 19 amino acids (aa) with unique sequences. BLAST search indicated that they showed no similarity to any known peptides or proteins. They are a novel family of antimicrobial peptide. These peptides showed antimicrobial activities against all of tested microorganisms including Gram-positive bacteria, Gram-negative bacteria and fungi. The cDNAs encoding precursors of these peptides were cloned from the skin cDNA library of R. shuchinae. The precursors are composed of 64 amino acid residues including predicted signal peptides, acidic spacer peptides, and mature antimicrobial peptides. The current work identified a novel antimicrobial peptide family.     

A novel bombesin-like peptide from skin of <Emphasis Type="Italic">Rana shuchinae</Emphasis>

Bombesin and its receptors have been demonstrated to be involved in a larger array of physiological and pathophysiological conditions including memory and fear behavior, lung development and injury, and tumor growth. A bombesin-like peptide named bombesin-RS was purified and characterized from the skin secretions of the frog, Rana shuchinae. Its amino acid sequence (pETSFMAPSWALGHLM-NH₂) was determined by auto Edman degradation and mass spectrometry. The cDNA encoding bombesin-RS precursor was cloned from skin cDNA library of this frog. The precursor is composed of 67 amino acid residues including a copy of mature bombesin-RS. Two predicted enzymatic processing sites (-Lys-Lys- and -Lys-Arg-) are flanked at the mature bombesin-RS sequence. In the in vitro myotropic contraction experiment, the synthesized bombesin-RS displayed stimulating effect toward rat stomach strips, suggesting that bombesin-RS acts as agonist as most other bombesin-related peptides do.     

Cloning and characterization of the first amphibian bradykinin gene

More than ten bradykinin-related peptides and their cDNAs have been identified from amphibians, but their genes are unknown. In present study, four cDNAs encoding one, two, four and six copies of bradykinin-related peptides were cloned from the frog (Odorrana grahami) skin cDNA library, respectively. Three bradykinin-related peptides (bradykinin, Thr6-bradykinin, Leu5Thr6-bradykinin) were deduced from these four cDNA sequences. Based on the cDNA sequence, the gene sequence encoding an amphibian bradykinin-related peptide from O. grahami was determined. It is composed of 7481 base pairs including two exons and two introns. The first exon codes signal peptide and the second exon codes acidic spacer peptide and Thr6-bradykinin. The promoter region of the bradykinin gene contains several putative recognition sites for nuclear factors, such as SRY, GATA-1, LYF-1, DeltaE, CDXA, NKX-2.5, MIF1 and S8. The current work may facilitate to understand the regulation and possible functions of amphibian skin bradykinin-related peptides.