Effects of the amphibian skin-derived bradykinin B2 receptor antagonist peptide, kinestatin, on the proliferation of human prostate cancer cell lines

Bradykinin and related peptides are found in the defensive skin secretions of many frogs and toads. While the physiological roles of bradykinin-related peptides in sub-mammalian vertebrates remains obscure, in mammals, including humans, canonical bradykinin mediates a multitude of biological effects including the proliferation of many types of cancer cell. Here we have examined the effect of the bradykinin B2 receptor antagonist peptide, kinestatin, originally isolated by our group from the skin secretion of the giant fire-bellied toad, Bombina maxima, on the proliferation of the human prostate cancer cell lines, PC3, DU175 and LnCAP. The bradykinin receptor status of all cell lines investigated was established through PCR amplification of transcripts encoding both B1 and B2 receptor subtypes. Following this demonstration, all cell lines were grown in the presence or absence of kinestatin and several additional bradykinin receptor antagonists of amphibian skin origin and the effects on proliferation of the cell lines was investigated using the MTT assay and by counting of the cells in individual wells of 96-well plates. All of the amphibian skin secretion-derived bradykinin receptor antagonists inhibited proliferation of all of the prostate cancer lines investigated in a dose-dependent manner. In addition, following incubation of peptides with each cell line and analysis of catabolites by mass spectrometry, it was found that bradykinin was highly labile and each antagonist was highly stable under the conditions employed. Bradykinin signalling pathways are thus worthy of further investigation in human prostate cancer cell lines and the evidence presented here would suggest the testing of efficacy in animal models of prostate cancer as a positive outcome could lead to a drug development programme for the treatment of this disease.

Kassorins: Novel innate immune system peptides from skin secretions of the African hyperoliid frogs, Kassina maculata and Kassina senegalensis

From defensive skin secretions acquired from two species of African hyperoliid frogs, Kassina maculata and Kassina senegalensis, we have isolated two structurally related, C-terminally amidated tridecapeptides of novel primary structure that exhibit a broad spectrum of biological activity. In reflection of their structural novelty and species of origin, we named the peptides kassorin M (FLEGLLNTVTGLLamide; 1387.8 Da) and kassorin S (FLGGILNTITGLLamide; 1329.8 Da), respectively. The primary structure and organisation of the biosynthetic precursors of kassorins M and S were deduced from cloned skin secretion-derived cDNA. Both open-reading frames encoded a single copy of kassorin M and S, respectively, located at the C-terminus. Kassorins display limited structural similarities to vespid chemotactic peptides (7/13 residues), temporin A (5/13 residues), the N-terminus of Lv-ranaspumin, a foam nest surfactant protein of the frog, Leptodactylus vastus, and an N-terminal domain of the equine sweat surfactant protein, latherin. Both peptides elicit histamine release from rat peritoneal mast cells. However, while kassorin S was found to possess antibacterial activity against Staphylococcus aureus, kassorin M was devoid of such activity. In contrast, kassorin M was found to contract the smooth muscle of guinea pig urinary bladder (EC50 = 4.66 nM) and kassorin S was devoid of this activity. Kassorins thus represent the prototypes of a novel family of peptides from the amphibian innate immune system as occurring in defensive skin secretions.

Peptide IC-20, encoded by skin kininogen-1 of the European yellow-bellied toad, Bombina variegata, antagonizes bradykinin-induced arterial smooth muscle relaxation

The objectives were to determine if the skin secretion of the European yellow-bellied toad (Bombina variegata), in common with other related species, contains a bradykinin inhibitor peptide and to isolate and structurally characterize this peptide. Materials and Methods: Lyophilized skin secretion obtained from this toad was subjected to reverse phase HPLC fractionation with subsequent bioassay of fractions for antagonism of the bradykinin activity using an isolated rat tail artery smooth muscle preparation. Subsequently, the primary structure of the peptide was established by a combination of microsequencing, mass spectroscopy, and molecular cloning, following which a synthetic replicate was chemically synthesised for bioassay. Results: A single peptide of molecular mass 2300.92 Da was resolved in HPLC fractions of skin secretion and its primary structure determined as IYNAIWP-KH-NK-KPGLL-. Database interrogation with this sequence indicated that this peptide was encoded by skin kininogen-1 previously cloned from B. variegata. The blank cycles were occupied by cysteinyl (C) residues and the peptide was located toward the C-terminus of the skin kininogen, and flanked N-terminally by a classical -KR- propeptide convertase processing site. The peptide was named IC-20 in accordance (I = N-terminal isoleucine, C = C-terminal cysteine, 20 = number of residues). Like the natural peptide, its synthetic replicate displayed an antagonism of bradykinin-induced arterial smooth muscle relaxation. Conclusion: IC-20 represents a novel bradykinin antagonizing peptide from amphibian skin secretions and is the third such peptide found to be co-encoded with bradykinins within skin kininogens.

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.

Antimicrobial peptides and alytesin are co-secreted from the venom of the Midwife toad, Alytes maurus (Alytidae, Anura): Implications for the evolution of frog skin defensive secretions

The skin secretions of frogs and toads (Anura) have long been a known source of a vast abundance of bioactive substances. In the past decade, transcriptome data of the granular glands of anuran skin has given new impetus to investigations of the putative constituent peptides. Alytes obstetricans was recently investigated and novel peptides with antimicrobial activity were isolated and functionally characterised. However, genetic data for the evolutionarily ancient lineage to which Alytes belongs (midwife toads; Alytidae) remains unavailable.

Here we present the first such genetic data for Alytidae, derived via the granular gland transcriptome of a closely-related species of midwife toad, Alytes maurus. First, we present nucleotide sequences of the entire peptide precursors for four novel antimicrobial peptides (AMPs). The two precursors resemble those from Bombinatoridae in both their structural architecture and amino acid sequence. Each precursor comprises two AMPs as tandem repeats, with a member of the alyteserin-1 family (alyteserin-1Ma: GFKEVLKADLGSLVKGIAAHVAN-NH2 or alyteserin-1Mb: GFKEVLKAGLGSLVKGIPAHVAN-NH2) followed by its corresponding member from the alyteserin-2 family (alyteserin-2Ma: FIGKLISAASGLLSHL-NH2 or alyteserin-2Mb: ILGAIIPLVSGLLSHL-NH2). Synthetic replicates of the four AMPs possessed minimal inhibitory concentrations (MICs) ranging from 9.5 to 300 µM, with the most potent being alyteserin-2Ma. Second, we also cloned the cDNA encoding an alytesin precursor, with the active alytesin exhibiting high sequence identity to bombesin-related peptides from other frogs. All putative mature peptide sequences were confirmed to be present in the skin secretion via LC/MS.

The close structural resemblance of the alyteserin genes that we isolated for A. maurus with those of Bombina provide independent molecular evidence for a close evolutionary relationship between these genera as well as more support for the convergent evolution of the AMP system within anurans. In contrast to the more evolutionarily conserved nature of neuropeptides (including alytesin, which we also isolated), the more variable nature of the AMP system together with the sporadic distribution of AMPs among anuran amphibians fuels in part our hypothesis that the latter system was co-opted secondarily to fulfil a function in the innate immune system, having originally evolved for defence against potential macropredators.

The chains of the heterodimeric amphibian skin antimicrobial peptide, distinctin, are encoded by separate messenger RNAs

Using a primer to a conserved nucleotide sequence of previously-cloned skin peptides of Phyllomedusa species, two distinct cDNAs were “shotgun” cloned from a skin secretion-derived cDNA library of the frog, Phyllomedusa burmeisteri. The two ORFs separately encode chains A and B of an analog of the previously-reported heterodimeric peptide, distinctin. LC-MS/MS analysis of native versus dithiotreitol reduced crude venom, confirmed the predicted primary sequences as well as the cystine link between the two monomers. Distinctin predominantly exists in the venom as a heterodimer (A-B), neither of the constituent peptides were detected as monomer, whereas of the two possible homodimers (A-A or B-B), only B-B was detected in comparatively low quantity. In vitro dimerization of synthetic replicates of the monomers demonstrated that besides heterodimer, both homodimers are also formed in considerable amounts. Distinctin is the first example of an amphibian skin dimeric peptide that is formed by covalent linkage of two chains that are the products of different mRNAs. How this phenomenon occurs in vivo, to exclude significant homodimer formation, is unclear at present but a “favored steric state” type of interaction between chains is most likely.

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).

Identification and Functional Analysis of a Novel Tryptophyllin Peptide from the Skin of the Red-eye Leaf Frog, Agalychnis callidryas

Amphibian skin has proved repeatedly to be a largely untapped source of bioactive peptides and this is especially true of members of the Phyllomedusinae subfamily of frogs native to South and Central America. Tryptophyllins are a group of peptides mainly found in the skin of members of this genus. In this study, a novel tryptophyllin (TPH) type 3 peptide, named AcT-3, has been isolated and structurally-characterised from the skin secretion and lyophilised skin extract of the red-eye leaf frog, Agalychnis callidryas. The peptide was identified in and purified from the skin secretion by reverse-phase HPLC. MALDI-TOF mass spectrometry and MS/MS fragmentation sequencing established its primary structure as: pGlu-Gly-Lys-Pro-Tyr-Trp-Pro-Pro-Pro-Phe-Leu-Pro-Glu, with a non-protonated molecular mass of 1538.19Da. The mature peptide possessed the canonical N-terminal pGlu residue that arises from post-translational modification of a Gln residue. The deduced open-reading frame consisted of 63 amino acid residues encoding a highly-conserved signal peptide of approximately 22 amino acid residues, an intervening acidic spacer peptide domain, a single AcT-3 encoding domain and a C terminal processing site. A synthetic replicate of AcT-3 was found to antagonise the effect of BK on rat tail artery smooth muscle and to contract the intestinal smooth muscle preparations. It was also found that AcT-3 could dose-dependently inhibit the proliferation of human prostate cancer cell lines after 72h incubation.

The inguinal macroglands of the frog Physalaemus nattereri (Leptodactylidae): structure, toxic secretion and relationship with deimatic behaviour

Amphibian skin is characterized by the presence of mucous glands, related to cutaneous breathing, reproduction and water balance, and granular glands, related to the production of toxins used in defence. In some species the granular glands can form accumulations in certain regions of the body. This is the case for inguinal macroglands of the leptodactylid frog Physalaemus nattereri, where these structures form a pair of black discs associated with deimatic behaviour. The morphology of the inguinal macroglands and their secretion were studied in this species and correlated to deimatic behaviour. The inguinal macroglands are formed from elongated granular glands that, in contrast with the granular glands of the rest of the skin, have small spherical granules with a proteinic content. In the dermis of the whole body, except for the inguinal macroglands and the inguinal region, a well-developed calcified dermal layer is observed. During deimatic behaviour these macroglands discourage a potential predator from attacking, but if visual cues are insufficient and the predator persists in the attack, atoxic secretion is eliminated in its mouth. This elimination is favoured by the absence of a calcified dermal layer in the macroglands, which makes the dermal region softer than the rest of the dorsal skin.

Skin glands, poison and mimicry in dendrobatid and leptodactylid amphibians

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Anuran skin and basking behavior: the case of the treefrog Bokermannohyla alvarengai (Bokermann, 1956)

We investigated the morphology of the skin and the biochemistry of the lipids in the skin secretion of Bokermannohyla alvarengai, a montane treefrog that is known to bask regularly, motionless in full sunlight for extended periods of time. Our primary goal was to identify structural and biochemical modifications that might assist this frog species to accommodate the conflicting demands for heat exchange and water balance while basking. The modulation of heat exchange in basking B. alvarengai involves changes in skin coloration. We found that this response was supported by a prominent monolayer of large iridophores, whose light reflectance property is adjusted by the response of intervening melanophores. Mucosubstances and lipid compounds, mainly consisted of saturated fatty acids and presumably secreted from granular glands, were detected on the skin of B. alvarengai. These compounds formed an extra-epidermal layer over the animal's dorsal surface that might assist in the prevention of excessive water loss through evaporation. Additionally, we found well-developed skin folds at the ventral region of the frogs that lead to an increment of surface area. This feature combined with the extensive hypervascularization, also noticed for the skin of B. alvarengai, may play an important role in water reabsorption. The suite of structural and biochemical modifications identified for the integument of B. alvarengai seems to conjugate aspects relevant to both, heat exchange and water balance, allowing for this species to explore basking as an efficient thermoregulatory strategy. J. Morphol. 276:1172-1182, 2015. © 2015 Wiley Periodicals, Inc.

Anti-proliferative and cytotoxic activity of pentadactylin isolated from Leptodactylus labyrinthicus on melanoma cells

Nowadays, the emergence of resistance to the current available chemotherapeutic drugs by cancer cells makes the development of new agents imperative. The skin secretion of amphibians is a natural rich source of antimicrobial peptides (AMP), and researchers have shown that some of these wide spectrum molecules are also toxic to cancer cells. The aim of this study was to verify a putative anticancer activity of the AMP pentadactylin isolated for the first time from the skin secretion of the frog Leptodactylus labyrinthicus and also to study its cytotoxic mechanism to the murine melanoma cell line B16F10. The results have shown that pentadactylin reduces the cell viability of B16F10 cells in a dose-dependent manner. It was also cytotoxic to normal human fibroblast cells; nevertheless, pentadactylin was more potent in the first case. The studies of action mechanism revealed that pentadactylin causes cell morphology alterations (e.g., round shape and shrinkage morphology), membrane disruption, DNA fragmentation, cell cycle arrest at the S phase, and alteration of mitochondrial membrane potential, suggesting that B16F10 cells die by apoptosis. The exact mechanism that causes reduction of cell viability and cytotoxicity after treatment with pentadactylin is still unknown. In conclusion, as cancer cells become resilient to death, it is worthwhile the discovery of new drugs such as pentadactylin that induces apoptosis.

两栖类动物皮肤分泌物及其生物学适应意义--大蹼铃蟾皮肤分泌物蛋白质多肽组的启示

以大蹼铃蟾(Bombina maxima)为代表性物种,揭示了两栖类皮肤分泌物蛋白质多肽组丰富的分子和功能多样性.目前在大蹼铃蟾已发现的分子包括3类不同的抗菌肽、缓激肽、缓激肽增强肽和缓激肽拮抗肽、缓激肽基因相关肽、富含脯氨酸铃蟾肽及其基因相关肽、神经调节素U、Bv8肽、三叶型蛋白和蛋白酶抑制剂等.抗菌肽分子多样性及其形成机制、缓激肽及其基因相关肽功能和表达模式的多样性都较好地揭示了在多样的生态条件下,两栖类皮肤活性肽环境适应的分子基础及生物合成的调控机制.富含血红素辅基的白蛋白广泛分布在大蹼铃蟾皮肤外皮层细胞膜上和真皮海绵层内,表明它在皮肤的生理功能,如在呼吸、物质交换和渗透压调节中有重要作用.两栖类皮肤分泌物蛋白质多肽组,由于其分子结构的多样性、新颖性和哺乳类同系物的存在,在生物医学研究和天然药物开发中具有独特和不可替代的价值;同时,两栖类皮肤功能基因组具有多样性丰富、快速重组突变的特征,是探讨生物适应的基因基础、基因形成机制和进化特性等生物学基本问题的优秀模型.

A small trypsin inhibitor from the frog of Odorrana grahami

A novel peptide inhibitor (OGTI) of serine protease with a molecular weight of 1949.8, was purified from the skin secretion of the frog, Odorrana grahami. Of the tested serine proteases, OGTI only inhibited the hydrolysis activity of trypsin on synthetic chromogenic substrate. This precursor deduced from the cDNA sequence is composed of 70 amino acid residues. The mature OGTI contains 17 amino acid residues including a six-residue loop disulfided by two half-cysteines (AVNIPFKVHFRCKAAFC). In addition to its unique six-residue loop, the overall structure and precursor of OGTI are different from those of other serine protease inhibitors. It is also one of the smallest serine protease inhibitors ever found. (C) 2008 Elsevier Masson SAS. All rights reserved.

大蹼铃蟾（Bombina maxima）皮肤分泌物非晶状体-晶状体蛋白与三叶因子复合物细胞生物学活性和分子作用机制

非晶状体-晶状体蛋白质(non-lens -crystallins)在脊椎动物中以一簇的基 因家族的形式存在，在各种上皮细胞中广泛表达，但对其在体内承担的功能，人 们几乎一无所知。三叶因子蛋白(trefoil factors, TFFs)主要分布在胃肠道上皮和两 栖动物皮肤表面，许多研究表明该家族的蛋白质，在粘膜保护，损伤修复和肿瘤 抑制中具有重要的作用；但是自发现以来，TFFs 一直作为孤儿配基存在，对于 其作用的机制了解得很少。人们从来没有把两个家族的蛋白质联系在一起来考虑 过。 本实验室从中国特有的两栖动物大蹼铃蟾(Bombina maxima)皮肤分泌物中， 分离到这两个家族的蛋白质的天然结合在一起的复合物，并命名为：非晶状体- 晶状体蛋白和三叶因子蛋白复合物（non-lens -crystallin and treifol factor complex, -CAT）。尽管在低剂量下，-CAT 已对小鼠，大鼠和兔有致死活性， 但其结构与人源的non-lens -crystallins 和TFFs 的同源性，提示了它可能在正 常的生理功能中起到重要作用，也为揭示两类重要的蛋白质的功能和作用机制提 供了可能性。本研究工作，在多种细胞株中，对-CAT 的生物学活性作了详细 的研究，并对其作用机制进行了进一步深入的探讨。 我们原代培养了人脐静脉血管内皮细胞(HUVEC) ，兔主动脉内皮细胞 (RAEC)，兔心内皮细胞(REEC)；培养了多种肿瘤细胞株。-CAT 能够引起多种 贴壁细胞的脱落。-CAT 在高剂量下能够引起这些细胞的凋亡；但是在不同的 细胞株中，发生凋亡的通路可能是不同的。在低剂量下，-CAT 能够促进细胞 的迁移，对HUVEC 具有诱导伤口修复的活性。 以HUVEC 细胞为模型，我们探讨了-CAT 的作用机制。在激光共聚焦显 微镜下，观察到-CAT 诱导HUVEC 发生囊泡化，这个效应是剂量依赖的；囊 泡化的发生不依赖于NH4Cl 的存在，但是NH4Cl 能够增大囊泡化的效应。在荧 光染料Cy3 直接标记-CAT 时，观察到-CAT 被定向运输到细胞核上。荧光染 料FITC 分别标记-CAT 的轻链和重链的多克隆抗体，免疫荧光染色发现，在较 短的时间(5 min)内, -CAT 已经进入细胞，并有部分分子被运输到细胞核上。随 着时间增加到30 min，轻链和重链在核上的比例也渐增加；到2 小时，-CAT的重链全部集中在细胞核上，而-CAT 的轻链则退出核外，主要聚集在细胞核 周围。核定位显示-CAT 分子进入HUVEC 细胞核，可能在基因的转录调节中 发挥作用。我们运用基因芯片检测了加药处理前后细胞蛋白质表达水平的变化。 在四组重复实验中均显示，加入-CAT 处理后，有121 个基因发生上调，这些 基因在调节细胞的生存和死亡中具有复杂的功能。其中核受体蛋白质家族 （NR4A1 等）的变化最为明显。而其他的基因包括调节细胞早期生长，凋亡， 炎症反应的相关基因和金属蛋白酶。同时，加入-CAT 处理后，仅有2 个基因 发生下调，包括胶原I，这为解释细胞发生脱落和调亡提供了分子基础。 本研究工作揭示了非晶状体-晶状体蛋白和三叶因子蛋白的相互作用，共 同定位到细胞核上，并调节基因的转录，首次提示非晶状体-晶状体蛋白可能 参与一条全新的细胞信号调节途径，在组织平衡，肿瘤发生和胚胎发育中起到重 要的作用；同时也为三叶因子蛋白的分子作用机制的解析提供了一种新的可能 性。