An ancient, conserved gene regulatory network led to the rise of oral venom systems.

Oral venom systems evolved multiple times in numerous vertebrates enabling the exploitation of unique predatory niches. Yet how and when they evolved remains poorly understood. Up to now, most research on venom evolution has focused strictly on the toxins. However, using toxins present in modern day animals to trace the origin of the venom system is difficult, since they tend to evolve rapidly, show complex patterns of expression, and were incorporated into the venom arsenal relatively recently. Here we focus on gene regulatory networks associated with the production of toxins in snakes, rather than the toxins themselves. We found that overall venom gland gene expression was surprisingly well conserved when compared to salivary glands of other amniotes. We characterized the "metavenom network," a network of ∼3,000 nonsecreted housekeeping genes that are strongly coexpressed with the toxins, and are primarily involved in protein folding and modification. Conserved across amniotes, this network was coopted for venom evolution by exaptation of existing members and the recruitment of new toxin genes. For instance, starting from this common molecular foundation, Heloderma lizards, shrews, and solenodon, evolved venoms in parallel by overexpression of kallikreins, which were common in ancestral saliva and induce vasodilation when injected, causing circulatory shock. Derived venoms, such as those of snakes, incorporated novel toxins, though still rely on hypotension for prey immobilization. These similarities suggest repeated cooption of shared molecular machinery for the evolution of oral venom in mammals and reptiles, blurring the line between truly venomous animals and their ancestors.

The defensive system of tree frog skin identified by peptidomics and RNA sequencing analysis.

The diversity of defensive peptides from skin of amphibians has been demonstrated. These peptides may have resulted from the diversity of microorganisms encountered by amphibians. In this study, peptidomics and RNA sequencing analyses were used to study deeply the defensive peptides of the skin secretions from Polypedates megacephalus. A total of 99 defensive peptides have been identified from the skin secretions. Among these peptides, 3 peptides were myotropical peptides and 34 peptides classified as protease inhibitor peptides. 5 lectins, 8 antimicrobial peptides, 26 immunomodulatory peptides, 10 wound-healing peptides and 13 other bioactive peptides were identified as belonging to the innate immune system. One antimicrobial peptide Pm-amp1 showed high similarity to antimicrobial peptide marcin-18. This peptide was successfully expressed and showed moderate activity against four tested strains. These identified peptides highlight the extensive diversity of defensive peptides and provide powerful tools to understand the defense weapon of frog.

Convergent Substitutions in a Sodium Channel Suggest Multiple Origins of Toxin Resistance in Poison Frogs.

Complex phenotypes typically have a correspondingly multifaceted genetic component. However, the genotype-phenotype association between chemical defense and resistance is often simple: genetic changes in the binding site of a toxin alter how it affects its target. Some toxic organisms, such as poison frogs (Anura: Dendrobatidae), have defensive alkaloids that disrupt the function of ion channels, proteins that are crucial for nerve and muscle activity. Using protein-docking models, we predict that three major classes of poison frog alkaloids (histrionicotoxins, pumiliotoxins, and batrachotoxins) bind to similar sites in the highly conserved inner pore of the muscle voltage-gated sodium channel, Nav1.4. We predict that poison frogs are somewhat resistant to these compounds because they have six types of amino acid replacements in the Nav1.4 inner pore that are absent in all other frogs except for a distantly related alkaloid-defended frog from Madagascar, Mantella aurantiaca. Protein-docking models and comparative phylogenetics support the role of these replacements in alkaloid resistance. Taking into account the four independent origins of chemical defense in Dendrobatidae, phylogenetic patterns of the amino acid replacements suggest that 1) alkaloid resistance in Nav1.4 evolved independently at least seven times in these frogs, 2) variation in resistance-conferring replacements is likely a result of differences in alkaloid exposure across species, and 3) functional constraint shapes the evolution of the Nav1.4 inner pore. Our study is the first to demonstrate the genetic basis of autoresistance in frogs with alkaloid defenses.

Snake venom-like waprin from the frog of Ceratophrys calcarata contains antimicrobial function.

A 255-bp cDNA encoding an 84-amino acid residue (aa) precursor protein containing 8 half-cysteines was cloned from the skin of the frog, Ceratophrys calcarata. By sequence comparison and signal peptide prediction, the precursor was predicted to release a 63-aa mature peptide with amino acid sequence, NVTPATKPTPSKPGYCRVMDELILCPDPPLSKDLCKNDSDCPGAQKCCYRTCIMQCLPPIFRE. The mature was named ceratoxin. Ceratoxin shares significant sequence similarity with the toxin family of waprins containing the whey acidic protein-type (WAP) four-disulfide core domain found in snake venoms. Antimicrobial and trypsin-inhibitory abilities of recombinant ceratoxin were tested. Recombinant ceratoxin showed strong antimicrobial activities against wide spectrum of microorganisms including Gram-negative and Gram-positive bacteria and fungi. It had no serine protease-inhibitory activity. The current results suggested that the snake venom-like waprin with antimicrobial activities in the frog skin plays a role in innate immunity.

Molecular cloning of mRNA from toad granular gland secretion and lyophilized skin: identification of Bo8--a novel prokineticin from Bombina orientalis.

Prokineticins are small (approximately 8 kDa), biologically active secretory proteins whose primary structures have been highly conserved throughout the Animal Kingdom. Representatives have been identified in the defensive skin secretions of several amphibians reflecting the immense structural/functional diversity of polypeptides in such. Here we describe the identification of a prokineticin homolog (designated Bo8) from the skin secretion of the Oriental fire-bellied toad (Bombina orientalis). Full primary structural characterization was achieved using a combination of direct Edman microsequencing, mass spectrometry and cloning of encoding skin cDNA. The latter approach employed a recently described technique that we developed for the cloning of secretory peptide cDNAs from lyophilized skin secretion, and this was further extended to employ lyophilized skin as the starting material for cDNA library construction. The Bo8 precursor was found to consist of an open-reading frame of 96 amino acid residues consisting of a putative 19-residue signal peptide followed by a single 77-residue prokineticin (Mr=7990 Da). Amino acid substitutions in skin prokineticins from the skin secretions of bombinid toads are confined to discrete sites affording the necessary information for structure/activity studies and analog design.

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.

Granular gland transcriptomes in stimulated amphibian skin secretions.

Amphibian defensive skin secretions are complex, species-specific cocktails of biologically active molecules, including many uncharacterized peptides. The study of such secretions for novel peptide discovery is time-limited, as amphibians are in rapid global decline. While secretion proteome analysis is non-lethal, transcriptome analysis has until now required killing of specimens prior to skin dissection for cDNA library construction. Here we present the discovery that polyadenylated mRNAs encoding dermal granular gland peptides are present in defensive skin secretions, stabilized by endogenous nucleic acid-binding amphipathic peptides. Thus parallel secretory proteome and transcriptome analyses can be performed without killing the specimen in this model amphibian system--a finding that has important implications in conservation of biodiversity within this threatened vertebrate taxon and whose mechanistics may have broader implications in biomolecular science.

Xenoxins, a family of peptides from dorsal gland secretion of Xenopus laevis related to snake venom cytotoxins and neurotoxins.

Three new, highly similar peptides from the skin secretion of Xenopus laevis have been purified and analyzed by mass spectrometry and Edman degradation. The 66-amino-acid peptides, termed xenoxin-1, -2, and -3, contain 8 cysteines and show similarity to snake venom cytotoxins and short neurotoxins. Assignment of two out of four disulfide bonds suggests a tertiary structure similar to that of cytotoxins and short neurotoxins. A cDNA encoding pre-xenoxin-1 was isolated from a X. laevis skin cDNA library. The nucleotide sequence predicts the synthesis of a precursor with a signal peptide followed by the sequence of the mature peptide. Xenoxin-1 and -2 lack alpha-neurotoxic activity, have apparently no antibacterial activity, are low in general toxicity as tested in mice, and have no effect on blood coagulation as measured in a Factor VIII procoagulant activity test. Potential functions of xenoxins as well as evolutionary aspects are discussed.