Novel Venom Proteins Produced by Differential Domain-Expression Strategies in Beaded Lizards and Gila Monsters (genus Heloderma)

The origin and evolution of venom proteins in helodermatid lizards were investigated by multidisciplinary techniques. Our analyses elucidated novel toxin types resultant from three unique domain-expression processes: 1) The first full-length sequences of lethal toxin isoforms (helofensins) revealed this toxin type to be constructed by an ancestral monodomain, monoproduct gene (beta-defensin) that underwent three tandem domain duplications to encode a tetradomain, monoproduct with a possible novel protein fold; 2) an ancestral monodomain gene (encoding a natriuretic peptide) was medially extended to become a pentadomain, pentaproduct through the additional encoding of four tandemly repeated proline-rich peptides (helokinestatins), with the five discrete peptides liberated from each other by posttranslational proteolysis; and 3) an ancestral multidomain, multiproduct gene belonging to the vasoactive intestinal peptide (VIP)/glucagon family being mutated to encode for a monodomain, monoproduct (exendins) followed by duplication and diversification into two variant classes (exendins 1 and 2 and exendins 3 and 4). Bioactivity characterization of exendin and helokinestatin elucidated variable cardioactivity between isoforms within each class. These results highlight the importance of utilizing evolutionary-based search strategies for biodiscovery and the virtually unexplored potential of lizard venoms in drug design and discovery.

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.