Molecular Characterization of Three Novel Phospholipase A2 Proteins from the Venom of Atheris chlorechis, Atheris nitschei and Atheris squamigera

Secretory phospholipase A2 (sPLA2) is known as a major component of snake venoms and displays higher-order catalytic hydrolysis functions as well as a wide range of pathological effects. Atheris is not a notoriously dangerous genus of snakes although there are some reports of fatal cases after envenomation due to the effects of coagulation disturbances and hemorrhaging. Molecular characterization of Atheris venom enzymes is incomplete and there are only a few reports in the literature. Here, we report, for the first time, the cloning and characterization of three novel cDNAs encoding phospholipase A2 precursors (one each) from the venoms of the Western bush viper (Atheris chlorechis), the Great Lakes bush viper (Atheris nitschei) and the Variable bush viper (Atheris squamigera), using a “shotgun cloning” strategy. Open-reading frames of respective cloned cDNAs contained putative 16 residue signal peptides and mature proteins composed of 121 to 123 amino acid residues. Alignment of mature protein sequences revealed high degrees of structural conservation and identity with Group II venom PLA2 proteins from other taxa within the Viperidae. Reverse-phase High Performance Liquid Chromatography (HPLC) profiles of these three snake venoms were obtained separately and chromatographic fractions were assessed for phospholipase activity using an egg yolk suspension assay. The molecular masses of mature proteins were all identified as approximately 14 kDa. Mass spectrometric analyses of the fractionated oligopeptides arising from tryptic digestion of intact venom proteins, was performed for further structural characterization.

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.