DNA in Amphibian and Reptile Venom Permits Access to Genomes Without Specimen Sacrifice

Amphibian defensive skin secretions and reptile venoms are rich sources of bioactive peptides with potential pharmacological/pharmaceutical applications. As amphibian and reptile populations are in rapid global decline, our research
group has been developing analytical methods that permit generation of robust molecular data from non-invasive skin secretion samples and venom samples. While previously we have demonstrated that parallel proteome and venom gland
transcriptome analyses can be performed on such samples, here we report the presence of DNA that facilitates the more widely-used applications of gene sequencing, such as molecular phylogenetics, in a non-invasive manner that circumvents specimen sacrifice. From this “surrogate” tissue, we acquired partial 12S and 16S rRNA gene sequences that are presented for illustration purposes. Thus from a single sample of amphibian skin secretion and reptile venom, robust and complementary proteome, transcriptome and genome data can be generated for applications in diverse scientific disciplines.

Structural and functional analogs of the novel mammalian neuropeptide, neuromedin S (NmS), in the dermal venoms of Eurasian bombinid toads.

We report the isolation and structural characterization of two neuromedin S (NmS) analogs, (NmS-17 and NmS-33), from the dermal venoms of Eurasian bombinid toads. NmS is a novel neuromedin U (NmU)-related peptide with potent anorexigenic and circadian rhythm-modulating properties recently discovered in mammals. Cloning of NmS precursor-encoding cDNAs from skin venom-derived libraries revealed the presence of a high degree of transcript splice variation comparable to that found previously for NmU in both amphibian skin and mammalian brain. Synthetic replicates of both amphibian NmS peptides evoked robust and dose-dependent transient increases in intracellular calcium ion concentrations in CHO cells that had been stably transfected with either FM-3/GPR66 or FM-4/TGR-1 human NmU receptors. The potency and efficacy of these amphibian skin peptides at such receptors were comparable to those observed with human NmS and rat NmS. These data show that NmS and NmU genes had already diverged at the level of the Amphibia and that differential splicing of their transcribed mRNAs has been highly conserved throughout tetrapod vertebrate evolution indicative of fundamental biological function. NmS is additionally a novel neuropeptide homolog that can be added to the biologically active peptide arsenal of amphibian venom/defensive skin secretions.

Helokinestatins: A novel family of bioactive peptides with drug-lead potential fromthe venomof the Gila Monster (Heloderma suspectum)

Venom of the Gila Monster (Heloderma suspectum) has proven to be an unlikely source of lead compounds (exendins) for the development of new injectable peptide therapeutics for the treatment of Type 2 diabetes. However, no systematic searches for new classes of bioactive peptides in lizard venom have appeared until recently. Here we describe the discovery of a new class of peptides – the helokinestatins – from H. suspectum venom, their structural characterisation and that of their biosynthetic precursors from cloned cDNA. In addition, we have subjected members of the family to preliminary pharmacological characterisation. Helokinestatins 1–6 are a family of proline-rich peptides containing 10–15 amino acid residues terminating in a common -Pro-Arg.OH motif. They are encoded in tandem within two virtually identical biosynthetic precursors of 177 and 178 amino acid residues, differing by only a single Pro residue. Each precursor also encodes a single copy of a C-type natriuretic peptide located at the C-terminus. Synthetic replicates of all helokinestatins were shown to be devoid of any direct action on the smooth muscle of rat tail artery but were found to be potent inhibitors of bradykinin-induced relaxation in this preparation in a manner that is suggestive of a non-competitive mechanism. Helokinestatin-3 (VPPPPLQMPLIPR) and helokinestatin-5 (VPPPLQMPLIPR) were found to be most potent in this respect causing almost complete inhibition of bradykinin-induced relaxation. Helokinestatins and BPPs may have a shared evolutionary history but the former do not inhibit ACE. The bradykinin inhibitory potential of helokinestatins may be exploited in the local control of chronic inflammation.

Helokinestatin-7 peptides from the venoms of Heloderma lizards

Helokinestatins 1–6 constitute a family of bradykinin antagonist peptides originally isolated from the venoms of the Gila Monster, Heloderma suspectum and the Mexican beaded lizard, Heloderma horridum. Here we report the identification, isolation and preliminary pharmacological characterization of two novel tridecapeptides, named helokinestatin-7S (FDDDSTELILEPR – 1550 Da) and helokinestatin-7H (FDDDSRKLILEPR – 1604 Da), whose primary structures were predicted from cDNAs cloned from venom libraries of respective Heloderma lizards. Computed molecular masses of putative helokinestatin-7 peptides were used as tools to locate these peptides in archived LC/MS fractions from respective venoms and sequences were confirmed by MS/MS fragmentation. A synthetic replicate of helokinestatin-7H was found to antagonize the relaxation effect of bradykinin on rat arterial smooth muscle but to have no measurable effects alone. In contrast, synthetic helokinestatin-7S was found to directly contract this preparation. Studies on related natural peptides with subtle differences in primary structure can provide the tools for structure/activity studies in pharmacological investigations directed toward unraveling the molecular basis of venom toxicity and for the evaluation of potential therapeutic leads.

Cloning of cDNAs and molecular characterisation of C-type lectin-like proteins from snake venoms

C-type lectin-like proteins (CTLPs) isolated from snake venoms are the largest and most complex non-mammalian vertebrate C-type lectin-like domain family. In the present study, we simultaneously amplified four cDNAs encoding different types of CTLP subunits from the venoms of two different species of snakes by RT-PCR with a single sense primer and a nested universal primer - two CTLP subunit-encoding cDNAs were cloned from Deinagkistrodon acutus venom and two from Agkistrodon halys Pallas venom. All four cloned CTLP subunits exhibited typical motifs in their corresponding domain regions but with relatively-low sequence similarities to each other. Compared with previously-published CTLPs, the four cloned CTLPs subunits showed slight variations in the calcium-binding sites and the disulphide bonding patterns. To our knowledge, these data constitute the first example of co-expression of CTLP platelet glycoprotein Ib-binding subunits and coagulation factors in Agkistrodon halys Pallas venom.

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.

Cloning and characterisation of three novel disintegrin precursors from the venoms of three Atheris species: Atheris chlorechis, Atheris nitschei and Atheris squamigera

Snake venom constitutes one of the most complex mixtures of naturally-occurring toxic proteins/polypeptides and a large number of these possess very profound biological activities. Disintegrins, that are commonly found in viper venoms, are low molecular weight proteins that usually contain an -Arg-Gly-Asp- (-RGD-) motif that is known to be involved in cell adhesion ligand recognition, binding specifically to cell surface integrin receptors and also exhibiting platelet anti-aggregation activity.

Here, we report for the first time, the successful cloning of three cDNAs encoding disintegrin precursors from lyophilised venom-derived libraries of Atheris chlorechis, Atheris nitschei and Atheris squamigera, respectively. All of these disintegrins belong to the short-coding class and all exhibit high degrees of structural identity, both in their amino acid sequences and in the arrangement of their functional domains. Mass spectrometric analyses of the HPLC-separated/in-gel digested venom proteins was performed to characterise the mature disintegrins as expressed in the venom proteome. Studies on both the structures and conserved sites within these disintegrins are of considerable theoretical interest in the field of biological evolution and in the development of new research tools or novel templates for drug design.