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 characterisation of an unexpected class of insulinotropic peptides in the skin of the frog Agalychnis litodryas

Skin secretions of the frog Agalychnis litodryas were evaluated for the isolation and characterisation of novel insulinotropic peptides. Crude secretions obtained from young adult frogs by mild electrical stimulation of the dorsal skin surface were purified by reverse-phase high-performance liquid chromatography (HPLC) yielding 70 fractions. In acute 20-min incubations with glucose responsive BRIN-BD11 cells, fractions 39-42 (band 1) and fractions 44-46 (band 2) significantly stimulated insulin release by 2-3.5-fold compared with 5.6 mM glucose alone. Pooled fractions in band 1 and band 2 were rechromatographed to reveal 20 homogenous peptide peaks, which elicited significant 1.5-4-fold increases in insulin release. Mass spectrometry analyses indicated molecular masses of between 1649.2 and 4988.9 Da. The two peptides with the greatest insulin-releasing activity were directly subjected to N-terminal amino acid sequence analysis. The sequence of the 3020 Da peptide, called frog skin insulinotropic peptide or FSIP, was determined as AVWKDFLKNIGKAAGKAVLNSVTDMVNE, which has 79% homology with the C-terminal of the 75 amino acid dermaseptin BIV precursor. A partial N-terminal sequence was determined for the 2546.2 Da peptide as MLADVFEKIMGD... These data indicate that the skin secretions of A. litodryas frogs contain biologically active peptides which merit further evaluation as a new class of insulin secretagogues.     

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.     

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.     

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。     

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.     

The Chinese bamboo leaf odorous frog (Rana (Odorrana) versabilis) and North American Rana frogs share the same families of skin antimicrobial peptides

The Chinese bamboo leaf odorous frog (Rana (Odorrana) versabilis) and the North American pickerel frog (Rana palustris) occupy different ecological niches on two different continents with no overlap in geographical distribution. R. palustris skin secretions contain a formidable array of antimicrobial peptides including homologs of brevinin-1, esculentin-1, esculentin-2, ranatuerin-2, a temporin and a family of peptides considered of unique structural attributes when isolated, palustrins 1-3. Here we describe the structures of mature peptides and precursors of eight putative antimicrobial peptides from the skin secretion of the Chinese bamboo leaf odorous frog (Rana (Odorrana) versabilis). Each peptide represents a structural homolog of respective peptide families isolated from R. palustris, including two peptides identical in primary structure to palustrin 1c and palustrin 3b. Additionally, two peptides were found to be structural homologs of ranatuerin 2B and ranatuerin 2P from the closely-related North American species, Rana berlandieri (the Rio Grande leopard frog) and Rana pipiens (the Northern leopard frog), respectively. Both palustrins and ranatuerins have hitherto been considered unique to North American ranid frogs. The use of primary structures of amphibian skin antimicrobial peptides is thus questionable as a taxonomic device or alternatively, the micro-evolution and/or ancestry of ranid frogs is more highly complex than previously thought.     

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.     

Structure-activity relationships of antimicrobial peptides from the skin of Rana esculenta inhabiting in Korea

The anuran (frogs and toads) skin is a rich source of antimicrobial peptides that can be developed therapeutically. We searched the skin secretions of Korean Rana esculenta for antimicrobial peptides, and isolated two cationic peptides with antimicrobial activity and little hemolytic activity: a 46-residue peptide of the esculentin-1 family and a 24-residue peptide of the brevinin-1 family. Their sequences showed some differences from the esculentins-1 and brevinins-1 of European Rana esculenta, indicating that sequence diversification of anuran skin antimicrobial peptides can arise from differences in habitat as well as from species differences. The 46-residue peptide named esculentin-1c had broad antimicrobial activity, while the 24-residue peptide named brevinin-1Ed exhibited limited activity. The solution structure of brevinin-1Ed was in good agreement with that of other brevinin-1-like peptides, with an amphipathic alpha-helix spanning residues 3-20, stabilized in membrane-mimetic environments. The weak bioactivity of brevinin-1Ed was attributable to the unusual presence of an anionic amino acid in the middle of the helical hydrophilic face. This report contributes to world-wide investigations of the structure-activity relationships and evolutional diversification of anuran-skin antimicrobial peptides.     

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.     

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.     

A family of acyclic brevinin-1 peptides from the skin of the Ryukyu brown frog Rana okinavana

The 24 amino-acid residue antimicrobial peptide, brevinin-1 is synthesized in the skins of a wide range of species of Eurasian and North American frogs belonging to the genus Rana. All previously characterized brevinin-1 peptides contain the cyclic heptapeptide domain Cys18-(Xaa)4-Lys-Cys24 at the COOH-terminus of the molecule. Four structurally related peptides were isolated from an extract of the skin of the Ryukyu brown frog Rana okinavana. The amino acid sequences of the peptides [Phe-(Xaa)4-Ile-(Xaa)2-Leu-Ala-Lys-Gly-Leu-Pro-Ser-Leu-Ile-Xaa-Leu-Xaa-Lys-Lys.NH2] identified them as members of the brevinin-1 family that lacked the COOH-terminal cyclic domain but contained a C-terminally alpha-amidated residue. It is suggested, as one possibility, that the Cys18 in the brevinin-1 consensus sequence has been deleted and the Cys24 residue has mutated to a glycine that acts as substrate for peptidyl-glycine alpha-amidating monooxygenase. The peptides potently inhibited the growth of Escherichia coli and Staphylococcus aureus confirming that a cyclic domain is not necessary for antimicrobial activity. A fifth peptide (SFLNFFKGAA10KNLLAAGLDK20LKCKISGTQC30), that also displayed broad-spectrum antimicrobial activity, was isolated from the skin extract and showed structural similarity with members of the ranatuerin-2 family previously isolated from the skin of North American ranid frogs.     

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.     

Antimicrobial peptides and protease inhibitors in the skin secretions of the crawfish frog, Rana areolata

The dorsal skin of the crawfish frog, Rana areolata, is associated with numerous prominent granular glands. Proteomic analysis of electrically stimulated skin secretions from these glands enabled the identification and characterization of eight peptides with antimicrobial and hemolytic activity belonging to the previously identified brevinin-1, temporin-1, palustrin-2, palustrin-3, esculentin-1 (two peptides), and ranatuerin-2 (two peptides) families. The primary structures of the peptides were consistent with a close phylogenetic relationship between R. areolata and the pickerel frog, Rana palustris. Three structurally related cationic, cysteine-containing peptides were identified that show sequence similarity to peptide Leucine–Arginine, a peptide with immunomodulatory and histamine-releasing properties from the skin of the northern leopard frog, Rana pipiens. The skin secretions contained a 61-amino-acid-residue peptide that inhibited porcine trypsin and possessed a 10-cysteine-residue motif that is characteristic of a protease inhibitor previously isolated from the parasitic nematode, Ascaris suum. A 48-amino-acid-residue protein containing eight cysteine residues in the whey acidic protein (WAP) motif, characteristic of elafin (skin-derived antileukoproteinase) and secretory leukocyte protease inhibitor, was also isolated. The data suggest that protease inhibitors in skin secretions may play a role complementary to cationic, amphipathic α-helical peptides in protecting anurans from invasions by microorganisms.     

Peptides with antimicrobial activity from four different families isolated from the skins of the North American frogs Rana luteiventris, Rana berlandieri and Rana pipiens.

The skins of frogs of the genus Rana synthesize a complex array of antimicrobial peptides that may be grouped into eight families on the basis of structural similarity. A total of 24 peptides with differential growth-inhibitory activity towards the Gram-positive bacterium Staphylococcus aureus, the Gram-negative bacterium Escherichia coli and the yeast Candida albicans were isolated from extracts of the skins of three closely related North American frogs, Rana luteiventris (spotted frog), Rana berlandieri (Rio Grande leopard frog) and Rana pipiens (Northern leopard frog). Structural characterization of the antimicrobial peptides demonstrated that they belonged to four of the known families: the brevinin-1 family, first identified in skin of the Asian frog Rana porosa brevipoda; the esculentin-2 family, first identified in the European frog Rana esculenta; the ranatuerin-2 family, first identified in the North American bullfrog Rana catesbeiana; and the temporin family, first identified in the European frog Rana temporaria. Peptides belonging to the brevinin-2, ranalexin, esculentin-1 and ranatuerin-1 families were not identified in the extracts. Despite the close phylogenetic relationship between the various species of Ranid frogs, the distribution and amino-acid sequences of the antimicrobial peptides produced by each species are highly variable and species-specific, suggesting that they may be valuable in taxonomic classification and molecular phylogenetic analysis.     

Pituitary adenylate cyclase-activating peptide (PACAP): assessment of dipeptidyl peptidase IV degradation, insulin-releasing activity and antidiabetic potential

Pituitary adenylate cyclase-activating peptide (PACAP) is a member of the glucagon family of peptides. Like other members, most notably glucagon-like peptide-1 (GLP-1), PACAP is rapidly degraded by dipeptidylpeptidase IV (DPP IV). This study investigated how degradation by DPP IV affected the insulinotropic activity of PACAP, and whether PACAP exerted acute antihyperglycemic properties in normal or ob/ob mice. DPP IV degradation of PACAP(1-27) over 18 h led to the formation of PACAP(3-27), PACAP(5-27) and ultimately PACAP(6-27). In contrast to 1.4-1.8-fold concentration-dependent stimulation of insulin secretion by PACAP(1-27), these peptide fragments lacked insulinotropic activity. While PACAP(1-27) and PACAP(1-38) generated significant insulin responses when given alone or together with glucose in ob/ob and normal mice, they also elevated plasma glucose. These actions were eliminated following degradation of the peptide by incubation with DPP IV. The hyperglycemic effects may be explained at least partly by a potent glucagon-releasing action in ob/ob and normal mice. In conclusion, PACAP is inactivated by DPP IV and despite insulin-releasing effects, its actions on glucagon secretion and glucose homeostasis do not make it a good therapeutic tool for the treatment of type 2 diabetes.