Isolation and cloning of exendin precursor cDNAs from single samples of venom from the Mexican beaded lizard (Heloderma horridum) and the Gila Monster (Heloderma suspectum)

Reptile venoms are complex cocktails of bioactive molecules, including peptides. While the drug discovery potential of most species remains unrealized, many are endangered and afforded protection under international treaties. In this study, we describe how potential clinically important bioactive peptides and their corresponding mRNAs can be structurally characterized from single, small samples of reptile venom. The potential type-2 diabetes therapeutics, exendin-3 and exendin-4, from the Mexican beaded lizard (Heloderma horridum) and the Gila monster (Heloderma suspectum), respectively, have been characterized at both protein and nucleic acid levels to illustrate the efficacy of the technique and its contribution to biodiversity conservation.

Biotransformation of maximakinin, a bradykinin-related nonadecapeptide from toad venom, by mammalian kallikrein and salivary proteases

Maximakinin is an N-terminally extended bradykinin (DLPKINRKGPRPPGFSPFR) from the venom of a Chinese toad (Bombina maxima) that displays highly selective activity at mammalian arterial smooth muscle receptors. In this study, we report that incubation of maximakinin with either kallikrein or human saliva generates catabolites with enhanced bioactivity that retain the tissue selective effects of the parent molecule. In addition, we have observed that kallikrein rapidly cleaves the C-terminal arginyl residue of both maximakinin and bradykinin – a cleavage hitherto considered to be performed by a carboxypeptidase that facilitates selective bradykinin receptor targeting. Maximakinin has thus evolved as a `smart' defensive weapon in the toad with inherent resistance to the signal-terminating protease hardware in the potential predator. Thus, natural selection of amphibian skin peptides for antipredator defence, through interspecies delivery by an exogenous secretory mode, produces subtle structural stabilization modifications that can potentially provide new insights for the design of orally active and selectively targeted peptide therapeutics.

Molecular cloning of a novel putative potassium channel-blocking neurotoxin from the venom of the North African scorpion, Androctonus amoreuxi

Scorpion venoms are a particularly rich source of neurotoxic proteins/peptides that interact in a highly specific fashion with discrete subtypes of ion channels in excitable and non-excitable cells. Here we have employed a recently developed technique to effect molecular cloning and structural characterization of a novel putative potassium channel-blocking toxin from the same sample of venom from the North African scorpion, Androctonus amoreuxi. The deduced precursor open-reading frame is composed of 59 amino acid residues that consists of a signal peptide of approximately 22 amino acid residues followed by a mature toxin of 37 amino acid residues. The mature toxin contains two functionally important residues (Lys27 and Tyr36), constituting a functional dyad motif that may be critical for potassium channel-blocking activity that can be affirmed from structural homologs as occurring in the venoms from other species of Androctonus scorpions. Parallel proteomic/transcriptomic studies can thus be performed on the same scorpion venom sample without sacrifice of the donor animal.

The smooth muscle pharmacology of maximakinin, a receptor-selective, bradykinin-related nonadecapeptide from the venom of the Chinese toad, Bombina maxima.

Structural homologues of vertebrate regulatory peptides found in defensive skin secretions of anuran amphibians often display enhanced bioactivity and receptor binding when compared with endogenous mammalian peptide ligands. Maximakinin, a novel N-terminally extended bradykinin (DLPKINRKGPRPPGFSPFR) from the skin venom of a Chinese toad (Bombina maxima), displays such activity enhancement when compared with bradykinin but is additionally highly selective for mammalian arterial smooth muscle bradykinin receptors displaying a 50-fold increase in molar potency in this smooth muscle type. In contrast, a 100-fold decrease in molar potency was observed at bradykinin receptors in intestinal and uterine smooth muscle preparations. Maximakinin has thus evolved as a “smart” defensive weapon in the toad with receptor/tissue selective targeting. Natural selection of amphibian skin venom peptides for antipredator defence, through inter-species delivery by an exogenous secretory mode, produces subtle structural stabilisation modifications that can potentially provide new insights for the design of selectively targeted peptide therapeutics.

Isolation of scorpion (Androctonus amoreuxi) alpha-neurotoxins and parallel cloning of their respective cDNAs from a single sample of venom

The venoms of buthid scorpions are known to contain basic, single-chain protein toxins (alpha toxins) consisting of 60–70 amino acid residues that are tightly folded by four disulfide bridges. Here we describe isolation and sequencing of three novel putative alpha toxins (AamH1-3) from the venom of the North African scorpion, Androctonus amoreuxi, and subsequent cloning of their precursor cDNAs from the same sample of venom. This experimental approach can expedite functional genomic analyses of the protein toxins from this group of venomous animals and does not require specimen sacrifice for cloning of protein toxin precursor cDNAs.

Monocyte-dependent oncostatin M and TNFa synergize to stimulate unopposed matrix metalloproteinase-1/3 secretion from human lung fibroblasts in tuberculosis

Leukocyte-derived matrix metalloproteinases (MMP) are implicated in the tissue destruction characteristic of tuberculosis (TB). The contribution of lung stromal cells to MMP activity in TB is unknown. Oncostatin M (OSM) is an important stimulus to extrapulmonary stromal MMP induction, but its role in regulation of pulmonary MMP secretion or pathophysiology of TB is unknown. We investigated OSM secretion from Mycobacterium tuberculosis (Mtb)-infected human monocytes/macrophages and the networking effects of such OSM on lung fibroblast MMP secretion. Mtb increased monocyte OSM secretion dose dependently in vitro. In vivo tuberculous granulomas immunostained positively for OSM. Further, conditioned media from Mtb-infected monocytes (CoMTb) induced monocyte OSM secretion (670 ± 55 versus 166 ± 14 pg/mL in controls), implicating an autocrine loop. Mtb-induced OSM secretion was prostaglandin (PG) sensitive, and required activation of surface G-protein coupled receptors. OSM induction was ERK MAP kinase dependent, p38-requiring but JNK-independent. OSM synergized with TNF-, a key cytokine in TB granuloma formation, to stimulate pulmonary fibroblast MMP-1/-3 secretion, while suppressing secretion of tissue inhibitors of metalloproteinases-1/-2. In summary, Mtb infection of monocytes results in PG-dependent OSM secretion, which synergizes with TNF- to drive functionally unopposed fibroblast MMP-1/-3 secretion, demonstrating a previously unrecognized role for OSM in TB.

Cloning of maximakinin precursor cDNAs from Chinese toad, Bombina maxima, venom.

Using a novel technique that we have developed for cloning of amphibian skin secretion peptide cDNAs from lyophilized samples, we report here that maximakinin (DLPKINRKGP-bradykinin) is encoded by two different cDNAs, named BMK-1 and BMK-2, containing either four tandem repeat sequences or a single copy. The open reading frames of both precursor cDNAs were found to be 152 and 116 amino acid residues, respectively. These data provide evidence that the structural diversity of peptides in amphibian skin secretions arising from molecular evolutionary events, can be mediated by parallel diversity in encoding mRNAs that in itself may reflect serial gene duplications.