Characterization of naturally occurring peptides in the skin secretion of Rana pipiens frog reveal pipinin-1 as the novel insulin-releasing agent.

Naturally occurring insulinotropic peptides were isolated from the skin secretions of Rana pipiens frogs. Crude secretions (50 mg; 5-10 frogs) obtained by mild electrical stimulation of the dorsal skin surface were purified by reversed-phase high-performance liquid chromatography (HPLC) yielding 80 fractions. In acute incubations with glucose-responsive BRIN-BD11 cells, fractions 40-47 (band 1) and fractions 60-65 (band 2) showed significant 1.7-6.7-fold increases in insulin-releasing activity (P < 0.001) compared with 5.6 mm glucose alone. Pooled fractions in bands 1 and 2 were rechromatographed yielding a total of seven peaks capable of subsequent 1.2-1.8-fold stimulation of insulin release. Final purification by HPLC to single homogenous peaks revealed one prominent peptide (peak 4.1) with insulin-releasing activity which lacked effects on cell viability. Electrospray mass spectrometric analysis of this peptide indicated molecular mass of 2562.6 Da. Determination of the primary amino acid sequence of this peptide revealed a 24-amino acid sequence: FLPIIAGVAAKVFPKIFCAISKKC. Database search showed a 100% homology to histamine-releasing pipinin-1. In conclusion, this study revealed the skin secretions of Rana pipiens to be a rich source of insulin-releasing peptides. The discovery of insulinotropic activity for pipinin-1, initially characterized as an antimicrobial is interesting and merits further investigation.     

Isolation and structural characterization of novel Rugosin A-like insulinotropic peptide from the skin secretions of Rana saharica frog

Skin secretions of Rana saharica were evaluated for the isolation and characterization of novel insulinotropic peptides. Crude secretions obtained from young adult frogs by mild electrical stimulation of the dorsal skin surface were purified by reversed-phase HPLC yielding 80 fractions. In acute incubations with glucose-responsive BRIN-BD11cells, fractions 36-43, 46-54 and 57-63 showed the significant 2-8-fold increase in insulin-releasing activity (P<0.001) compared with 5.6mM of glucose alone. A pool of fractions 36-43 was subsequently rechromatographed to 28 homogenous peaks out of which 7 were capable of subsequent 1.5-3-fold increase in insulin release (P<0.001). Structural analysis of the non-toxic peptides with greatest insulin-releasing activity was performed by mass spectrometry and Edman degradation. Mass spectrometry analysis of two peaks indicated the molecular masses of 1892.6 and 2930.8Da. The sequence of the 1892.6-Da peptide was determined as KGAAKGLLEVASCKLSKSC, which has 68% homology with Rugosin A originally isolated from the skin secretion of Rana rugosa. A partial N-terminal sequence was determined for the 2930.8-Da peptide as AVITGACERDVQCGGGTCCAVSLI.... These data indicate that the skin secretions of Rana saharica frogs contain novel peptides with insulin-releasing activity.     

Isolation and structural characterisation of a novel 13-amino acid insulin-releasing peptide from the skin secretion of Agalychnis calcarifer

We describe the isolation and characterisation of an insulinotropic peptide from the skin secretions of Agalychnis calcarifer frogs. Peptides in crude secretions obtained by mild electrical stimulation from the dorsal skin surface were purified by reversed-phase HPLC, yielding fractions in two zones with insulin-releasing activity ( p <0.001). The peaks showing greatest in vitro insulin-releasing activity were subsequently purified to homogeneity, revealing a novel insulinotropic 13-amino-acid (1653.2 Da) peptide with the primary structure RRKPLFPFIPRPK [corrected] (RK-13). A database search for RK-13 showed 53.8% similarity with the N-terminal region of proline-arginine-rich antimicrobial peptide (PR-39). Synthetic RK-13 stimulated insulin release in a dose-dependent, glucose-sensitive manner, exerting its effects through a cyclic AMP-protein kinase A pathway independent of pertussis toxin-sensitive G proteins. Unlike PR-39, RK-13 lacks antimicrobial effects on the growth of yeast, and Gram-positive and Gram-negative bacteria. Our data indicate that skin secretions of Agalychnis calcarifer frogs contain insulin-releasing peptides, including RK-13, which merit further investigation as insulin secretagogues.     

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.     

蛙皮抗菌肽促胰岛素分泌功能研究进展

随着对蛙皮抗菌肽功能研究的不断深入,陆续发现部分肽具有促胰岛素分泌活性,该活性对于2型糖尿病治疗具有较好的应用前景。蛙皮抗菌肽即可以通过克服注射胰岛素产生的低血糖反应,又能改善2型糖尿病胰岛素抵抗的问题,这使其有希望成为安全、高效治疗2型糖尿病药物的新药物。本文综述了具有促胰岛素分泌功能的蛙皮抗菌肽的序列特征和工作机制的研究进展,为进一步开展相关研究提供参考。     

Comparison of the insulinotropic activity of glucagon-superfamily peptides in rat pancreas perfusion.

Previous studies have demonstrated that glucagon-superfamily peptides stimulate insulin release from the pancreatic islets in a glucose dependent manner. In this study we have carried out a structure-activity study of their insulinotropic activity using a rat pancreas perfusion with 5.5 mM glucose concentration. The following peptides were examined: glucagon-like peptide-1(7-36)amide (tGLP-1), glucagon, gastric inhibitory peptide (GIP), peptide having an amino-terminal histidine and carboxy-terminal isoleucine amide (PHI), vasoactive intestinal polypeptide (VIP), growth hormone releasing factor(1-29)amide (GRF), GRF(1-27)amide and synthetic hybrid-peptides of PHI-GRF, PHI(1-11)-GRF(12-27) and PHI(1-20)-GRF(21-27). Their potencies were evaluated as: tGLP-1 = GIP > glucagon > PHI = VIP > PHI(1-20)-GRF(21-27) > PHI(1-11)-GRF(12-27) > GRF(1-29) = GRF(1-27). It is clear that 0.1 nM tGLP-1 stimulated insulin release, whereas 1 microM GRF(1-29) did not. These results indicate that 1) in addition to N-terminal amino acid (histidine or tyrosine), position 4 (glycine), position 9 (aspartic acid) and position 11 (serine) in the amino acid sequence are important for their insulinotropic activity, 2) not only the N-terminal portion but also the C-terminal portion of these peptides contribute to their insulinotropic activity.     

Evidence from peptidomic analysis of skin secretions that the red-legged frogs, Rana aurora draytonii and Rana aurora aurora, are distinct species

The northern red-legged frog Rana aurora aurora and the California red-legged frog Rana aurora draytonii are traditionally classified together in the same species group. Ten peptides with antimicrobial activity were isolated from norepinephrine-stimulated skin secretions of R. aurora draytonii and purified to near homogeneity. The peptides were identified as belonging to the ranatuerin-2 family (two peptides), brevinin-1 family (four peptides), temporin family (three peptides), and a novel peptide, RV-23 (RIGVLLARLPKLFSLFKLMGKKV) that has limited structural similarity to the bee venom peptide, melittin. This distribution of peptides contrasts with that found previously in skin secretions from R. aurora aurora collected under the same conditions and at the same time of year (one ranatuerin-2 peptide, two brevinin-1 peptides, and one temporin peptide). The variation in amino acid sequences between corresponding R. aurora draytonii and R. aurora aurora peptides is comparable with the variation in sequences of orthologs from other members of the Amerana group of New World ranid frogs (Rana boylii, Rana muscosa, and Rana luteiventris). It is proposed, therefore, that the red-legged frogs should be regarded as separate species (R. aurora and R. draytonii) within the Amerana group rather than conspecific subspecies. The data emphasize that amino acid sequences of antimicrobial peptides in skin secretions may be used to infer taxonomic and phylogenetic relationships between species of ranid frogs.     

The ascaphins: a family of antimicrobial peptides from the skin secretions of the most primitive extant frog, Ascaphus truei

The tailed frog Ascaphus truei occupies a unique position in phylogeny as the most primitive extant anuran and is regarded as the sister taxon to the clade of all other living frogs. Eight structurally related peptides, termed ascaphins 1-8, were isolated from norepinephrine-stimulated skin secretions of A. truei and were shown to possess differential growth inhibitory activity against Escherichia coli and Staphylococcus aureus. Ascaphins 2-7 may be represented by the consensus amino acid sequence GX2DX2KGAAKX3KTVAX2IANX.COOH whereas ascaphin-1 (GFRDVLKGAAKAFVKTVAGHIAN.NH2) and ascaphin-8 (GFKDLLKGAAKALVKTVLF.NH2) contain a C-terminally alpha-amidated residue. The ascaphins show no appreciable structural similarity with other families of antimicrobial peptides from frog skin but display limited sequence identity with the cationic, amphipathic alpha-helical peptides pandinin 1 and opistoporin 1, isolated from the venoms of African scorpions. Ascaphin-8 shows the highest potency against a range of pathogenic microorganisms but has the greatest haemolytic activity. The data indicate that the host defence strategy of using antimicrobial peptides in skin secretions arose early in the evolution of anurans.     

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.     

An antimicrobial peptide from the skin secretions of the mountain chicken frog Leptodactylus fallax (Anura:Leptodactylidae)

A 25 amino-acid-residue, C-terminally alpha-amidated peptide with antimicrobial activity, which has been termed fallaxin, was isolated in high yield from the norepinephrine-stimulated skin secretions of the mountain chicken frog Leptodactylus fallax (Anura:Leptodactylidae). The amino acid sequence of the peptide (Gly-Val-Val-Asp-Ile-Leu-Lys-Gly-Ala-Ala-Lys-Asp-Ile-Ala-Gly-His-Leu-Ala-Ser-Lys-Val-Met-Asn-Lys-Leu.NH2) shows structural similarity with members of the ranatuerin-2 family previously isolated from the skins of frogs of the genus Rana that are only distantly related to the Leptodactylidae. This observation is consistent with the hypothesis that many frog skin antimicrobial peptides are related evolutionarily, having arisen from multiple duplications of an ancestral gene that existed before the radiation of the different families. Fallaxin inhibited the growth of reference strains of Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella pneumoniae) but with relatively low potency (MIC> or =20 microM) and was inactive against the Gram-positive bacterium (Staphylococcus aureus) and the yeast Candida albicans. The hemolytic activity of fallaxin was very low (HC50>200 microM). A second peptide, comprising residues (1-22) of fallaxin, was also isolated from the skin secretions but this component was inactive against the microorganisms tested.     

A novel bradykinin-like peptide from skin secretions of rufous-spotted torrent frog, Amolops loloensis

A bradykinin-like peptide has been isolated from skin secretions of rufous-spotted torrent frog, Amolops loloensis. This bradykinin-like peptide was named amolopkinin. Its primary structure, RAPVPPGFTPFR, was determined by Edman degradation and mass spectrometry. It is structurally related to bradykinin-like peptides identified from skin secretions of other amphibians. Amolopkinin is composed of 12 amino acid residues and is related to bradykinin composed of nine amino acid residues, identified from the skin secretions of Odorrana schmackeri. Amolopkinin was found to elicit concentration-dependent contractile effects on isolated guinea pig ileum. cDNA clones encoding the precursor of amolopkinin were isolated by screening a skin cDNA library of A. loloensis and then sequenced. 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 a deficiency of an18-nucleotide fragment (TCAAGAATGATCAGACGC in the cDNA encoding bradykinin from O. schmackeri) in the peptide-coding region resulted in absence of a di-basic site for trypsin-like proteinases and an unusual - APV - insertion in the N-terminal part of amolopkinin. This is the first report of a bradykinin-like peptide comprised of bradykinin with an insertion in its N-terminal part. Our results demonstrate the hypervariability of amphibian bradykinin-like peptides, as well as the diversity of antimicrobial peptides in amphibians.     

Peptides with antimicrobial activity of the brevinin-1 family isolated from skin secretions of the southern leopard frog, Rana sphenocephala.

Three peptides with growth-inhibitory activity towards the gram-negative bacterium Eschericia coli were isolated from electrically stimulated secretions from the skin of the southern leopard frog, Rana sphenocephala. Structural characterization demonstrated that the peptides [brevinin-1Sa, minimum inhibitory concentration (MIC) = 55 microM; brevinin-1Sb, MIC = 17 microM; brevinin-1Sc, MIC = 14 microM] represent new members of the brevinin-1 family of antimicrobial peptides, previously isolated from several other species of frogs of the genus Rana. Their high concentration in skin secretions and extreme variability in amino acid sequence suggest that the brevinin family of peptides may be of value as molecular markers for the identification and taxonomic classification of Ranid frogs.     

Antimicrobial peptides from the skin of the Japanese mountain brown frog Rana ornativentris: evidence for polymorphism among preprotemporin mRNAs

A previous study led to the isolation of antimicrobial peptides belonging to the temporin and brevinin-2 families from a pooled extract of the skin of adult specimens of the Japanese mountain brown frog Rana ornativentris Werner 1903. In order to ascertain whether individual frogs expressed the full complement of temporin genes, we individually cloned cDNAs encoding the temporin precursors from total RNA extracted from the skins of 12 frogs by RT-PCR using a set of preprotemporin-specific primers. All the specimens examined contained mRNAs directing the synthesis of the novel, but inactive, temporin-1Oe (ILPLLGNLLNGLL x NH2). Nucleotide sequence analysis revealed marked polymorphism among individual frogs. Twenty-seven distinct preprotemporin-1Oe mRNAs were identified that contained synonymous substitutions in the antimicrobial peptide region and both synonymous and non-synonymous substitutions in the signal peptide and intervening sequence regions. Up to eight preprotemporin-1Oe mRNA variants were found within a single frog. In addition, several cDNAs encoding preprotemporin-1Oa and -1Ob and a single cDNA encoding preprotemporin-1Oc were characterized. Peptidomic analysis of norepinephrine-stimulated skin secretions revealed the presence of temporin-1Oe, temporin-1Of (SLILKGLASIAKLF x NH2), temporin-1Og (FLSSLLSKVVSLFT x NH2), four members of the ranatuerin-2 family and one member of the palustrin-2 family in addition to previously characterized temporin and brevinin-2 peptides.     

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.     

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.     

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.     

Purification and characterization of antimicrobial peptides from the skin secretions of the mink frog (Rana septentrionalis)

Skin secretions were obtained from male, female, and juvenile specimens of the mink frog (Rana septentrionalis) by electric stimulation and shown to contain 10 peptides that differentially inhibited the growth of microorganisms. The elution profiles of secretions from the three groups following reverse-phase HPLC were almost identical indicating that there were no major sexual or developmental differences in chemical composition. Four peptides of the brevinin-1 family, with potent antimicrobial activity and strong hemolytic activity, two members of ranatuerin-2 family and three members of the temporin family, were purified and characterized structurally. A 21-amino-acid C-terminally alpha-amidated peptide (GIWDTIKSMGKVFAGKILQNL.NH(2)) with broad-spectrum antimicrobial activity was also isolated from the skin secretions. This peptide shows limited structural similarity with the N-terminal region of brevinin-2 peptides previously isolated from R. temporaria skin but lacks the C-terminal cyclic heptapeptide domain associated with this family. Molecular and morphological data support the placement of R. septentrionalis in the R. catesbeiana species group, but analysis based upon the distribution of the molecular forms of the antimicrobial peptides is indicative of a closer phylogenetic relationship between R. septentrionalis and the frogs of the R. pipiens and R. boylii groups.     

Characterization of an insulinotropic peptide from skin secretions of Odorrana andersonii

Insulinotropic peptide agents are regarded as potential candidates for anti‐diabetic treatment. In the present study, a novel insulinotropic peptide, termed OA‐A1, was purified from frog skin secretions of Odorrana andersonii. Mature OA‐A1 was determined to be a 1965.049 Da peptide with an amino acid sequence of LVGKLLKGAVGDVCGLLPIC, in which an intramolecular disulfide bridge was formed by two cysteine residues. At the cellular level, OA‐A1 exhibited potent proliferation promoting effects on mouse‐derived pancreatic β‐TC‐6 cells and significantly stimulated insulin release in β‐TC‐6 cells at a minimum concentration of 1 nM. In the animal model, OA‐A1 also showed a dose‐dependent insulin‐releasing role in mice. At concentrations ranging from 1 nmol/kg to 1 μmol/kg, OA‐A1 had a significant acute hypoglycemic effect on streptozotocin (STZ)‐induced diabetic mice. The pancreatic islet areas of diabetic mice increased dose‐dependently after 21 days of OA‐A1 treatment (1–100 nmol/kg) compared with those of the saline control group. Moreover, OA‐A1 significantly improved the oral glucose tolerance of STZ‐induced diabetic mice. Taken together, these results suggest that OA‐A1 provides an excellent template for the development of novel anti‐diabetic therapeutic agents. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.