In vitro and in vivo insulinotropic properties of the multifunctional frog skin peptide hymenochirin-1B: a structure–activity study

Hymenochirin-1b (Hym-1B; IKLSPETKDNLKKVLKGAIKGAIAVAKMV.NH2) is a cationic, α-helical amphibian host-defense peptide with antimicrobial, anticancer, and immunomodulatory properties. This study investigates the abilities of the peptide and nine analogues containing substitutions of Pro5, Glu6, and Asp9 by either l-lysine or d-lysine to stimulate insulin release in vitro using BRIN-BD11 clonal β cells or isolated mouse islets and in vivo using mice fed a high-fat diet to produce obesity and insulin resistance. Hym-1B produced a significant and concentration-dependent increase in the rate of insulin release from BRIN-BD11 cells without cytotoxicity at concentrations up to 1 µM with a threshold concentration of 1 nM. The threshold concentrations for the analogues were: [P5K], [E6K], [D9K], [P5K, E6K] and [E6K, D9k] 0.003 nM, [E6K, D9K] and [D9k] 0.01 nM, [P5K, D9K] 0.1 nM and [E6k] 0.3 nM. All peptides displayed cytotoxicity at concentrations ≥1 µM except the [P5K] and [D9k] analogues which were non-toxic at 3 µM. The potency and maximum rate of insulin release from mouse islets produced by the [P5K] peptide were significantly greater than produced by Hym-1B. Neither Hym-1B nor the [P5K] analogue at 1 µM concentration had an effect on membrane depolarization or intracellular Ca2+. The [P5K] analogue (1 µM) produced a significant increase in cAMP concentration in BRIN-BD11 cells and stimulated GLP-1 secretion from GLUTag cells. Down-regulation of the protein kinase A pathway by overnight incubation with forskolin completely abolished the insulin-releasing effects of [P5K]hym-1B. Intraperitoneal administration of the [P5K] and [D9k] analogues (75 nmol/kg body weight) to high-fat-fed mice with insulin resistance significantly enhanced glucose tolerance with a concomitant increase in insulin secretion. We conclude that [P5K]hym-1B and [D9k]hym-1B show potential for development into anti-diabetic agents.

Effect of cholesterol on the interaction of the amphibian antimicrobial peptide DD K with liposomes

DD K is an antimicrobial peptide previously isolated from the skin of the amphibian Phyllomedusa distincta. The effect of cholesterol on synthetic DD K binding to egg lecithin liposomes was investigated by intrinsic fluorescence of tryptophan residue, measurements of kinetics of 5(6)-carboxyfluorescein (CF) leakage, dynamic light scattering and isothermal titration microcalorimetry. An 8 nm blue shift of tryptophan maximum emission fluorescence was observed when DD K was in the presence of lecithin liposomes compared to the value observed for liposomes containing 43 mol% cholesterol. The rate and the extent of CF release were also significantly reduced by the presence of cholesterol. Dynamic light scattering showed that lecithin liposome size increase from 115 to 140 nm when titrated with DD K but addition of cholesterol reduces the liposome size increments. Isothermal titration microcalorimetry studies showed that DD K binding both to liposomes containing cholesterol as to liposomes devoid of it is more entropically than enthalpically favored. Nevertheless, the peptide concentration necessary to furnish an adjustable titration curve is much higher for liposomes containing cholesterol at 43 mol% (2 mmol L-1) than in its absence (93 mu mol L-1). Apparent binding constant values were 2160 and 10,000 L mol(-1), respectively. The whole data indicate that DD K binding to phosphatidylcholine liposomes is significantly affected by cholesterol, which contributes to explain the low hemolytic activity of the peptide. (C) 2007 Elsevier Inc. All rights reserved.

Anti-infection peptidomics of amphibian skin

Peptidomics and genomics analyses were used to study an anti-infection array of peptides of amphibian skin. 372 cDNA sequences of antimicrobial peptides were characterized from a single individual skin of the frog Odorrana grahami that encode 107 novel an

beta gamma-CAT, a non-lens beta gamma-crystallin and trefoil factor complex from amphibian skin secretions, caused endothelium-dependent myocardial depression in isolated rabbit hearts

Previous in vivo study demonstrated that beta gamma-CAT, a newly identified non-lens beta gamma-crystallin and trefoil factor complex from frog Bombina maxima skin secretions, possessed potent lethal toxicity on mammals resulted from hypotension and cardi

Parallel Peptidome and Transcriptome Analyses of Amphibian Skin Secretions Using Archived Frozen Acid-Solvated Samples

Amphibian skin secretions are unique sources of bioactive peptides and their donor species are currently rapidly disappearing from the biosphere. Here, we report that both peptides and polyadenylated mRNAs from skin granular glands remain amenable to study in samples of stimulated skin secretions following their storage in 0.1 % aqueous trifluoroacetic acid at -20 °C for many years. Frozen acidified solutions of toad (Bombina variegata) skin secretions, stored for 12 years, were thawed and samples removed for direct reverse phase HPLC fractionation. Additional samples were removed, snap frozen and lyophilised for construction of cDNA libraries following polyadenylated mRNA capture using magnetic oligo-dT beads and reverse transcription. Using the bombesin and bradykinin peptides found in bombinid toad skin as models, individual variant peptides of each type were located in reverse phase HPLC fractions and their corresponding biosynthetic precursor-encoding mRNA transcripts were cloned from the cDNA library using a RACE PCR strategy. This study illustrates unequivocally that both amphibian skin secretion peptides and their biosynthetic precursor-encoding polyadenylated mRNAs are stable in frozen acid-solvated skin secretion samples for considerable periods of time-a finding that may have fundamental implications in the study of archived materials but also in the wider field of molecular biology.

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.

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 50µM) and Candida albicans (MIC 150µM) but was nonhaemolytic at concentrations up to and including 150µM. In contrast, senegalin induced a dose-dependent contraction of rat urinary bladder smooth muscle (EC50 2.9nM) and a dosedependent relaxation of rat tail artery smooth muscle (EC50 37.7nM). Senegalin thus represents a prototype biologically-active amphibian skin peptide and illustrates the fact thatamphibian skin secretion peptidomes continue to be unique sources of such molecules.

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).

Trypsin inhibitory loop is an excellent lead structure to design serine protease inhibitors and antimicrobial peptides

The disulfide-bridged hendecapeptide ( CWTKSIPPKPC) loop, derived from an amphibian skin peptide, is found to have strong trypsin inhibitory capability. This loop, called the trypsin inhibitory loop ( TIL), appears to be the smallest serine protease inhib

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 bell

Bombinakinin M gene associated peptide, a novel bioactive peptide from skin secretions of the toad Bombina maxima

A novel 28-amino acid peptide, termed bombinakinin-GAP, was purified and characterized from skin secretions of the toad Bombina maxima. Its primary structure was established as DMYEIKQYKTAHGRPPICAPGEQCPIWV-NH2, in which two cysteines form a disulfide bond. A FASTA search of SWISS-PROT databank detected a 32% sequence identity between the sequences of the peptide and a segment of rat cocaine- and amphetamine-regulated transcript (CART). Intracerebroventricular (i.c.v.) administration of the peptide induced a significant decrease in food intake in rats, suggesting that it played a role in the control of feeding by brain. Analysis of its cDNA structure revealed that this peptide is coexpressed with bombinakinin M, a bradykinin-related peptide from the same toad. Bombinakinin-GAP appears to be the first example of a novel class of bioactive peptides from amphibian skin, which may be implicated in feeding behavior. (C) 2003 Elsevier Science Inc. All rights reserved.

The first antimicrobial peptide from sea amphibian

The crab-eating frog, Rana cancrivora, is one of only a handful of amphibians worldwide that tolerates saline waters. It typically inhabits brackish water of mangrove forests of Southeast Asia. A large amount of antimicrobial peptides belonging to different families have been identified from skins of amphibians inhabiting freshwater. No antimicrobial peptide from sea amphibians has been reported. In this paper, we firstly reported the antimicrobial peptide and its cDNA cloning from skin secretions of the crab-eating frog R. cancrivora. The antimicrobial peptide was named cancrin with an amino acid sequence of GSAQPYKQLHKVVNWDPYG. By BLAST search, cancrin had no significant similarity to any known peptides. The cDNA encoding cancrin was cloned from the cDNA library of the skin of R. cancrivora. The cancrin precursor is composed of 68 amino acid residues including a signal peptide, acidic spacer peptide, which are similar to other antimicrobial peptide precursors from Ranid amphibians and mature cancrin. The overall structure is similar to other amphibian antimicrobial peptide precursors although mature cancrin is different from known peptides. The current results reported a new family of amphibian antimicrobial peptide and the first antimicrobial peptide from sea amphibian. (c) 2007 Elsevier Ltd. All rights reserved.

Pelophylaxins: novel antimicrobial peptide homologs from the skin secretion of the Fukien gold-striped pond frog, Pelophylax plancyi fukienensis: identification by “shotgun” cDNA cloning and sequence analysis.

Amphibian skin secretions are rich in antimicrobial peptides that act as important components of an innate immune system. Here, we describe a novel “shotgun” skin peptide precursor cloning technique that facilitates rapid access to these genetically encoded molecules and effects their subsequent identification and structural characterization from the secretory peptidome. Adopting this approach on a skin secretion-derived library from a hitherto unstudied Chinese species of frog, we identified a family of novel antimicrobial peptide homologs, named pelophylaxins, that belong to previously identified families (ranatuerins, brevinins and temporins) found predominantly in the skin secretions from frogs of the genus Rana. These data further substantiate the scientifically robust nature of applying parallel transcriptome and peptidome analyses on frog defensive skin secretions that can be obtained in a non-invasive, non-destructive manner. In addition, the present data illustrate that rapid structural characterization of frog skin secretion peptides can be achieved from an unstudied species without prior knowledge of primary structures of endogenous peptides.