Mackessy, Stephen P.
Keenan, Susan M.
University of Northern Colorado
Type of Resources
Place of Publication
University of Northern Colorado
Snake venoms are complex mixtures of many enzymatic and non-enzymatic proteins, as well as small peptides. Several major venom protein superfamilies, including three-finger toxins, phospholipases A2, serine proteinases, metalloproteinases, proteinase inhibitors and lectins, are found in almost all snake venoms, from front-fanged viperids (vipers and pit vipers) and elapids (cobras, mambas, sea snakes, etc.) to rear-fanged colubrids. However, these proteins vary in abundance and functionality between species. Variation in snake venom composition is attributed to both differences in the expression levels of toxin encoding genes and occurrence of amino acid sequence polymorphisms. Documenting intraspecific venom variation has both clinical (antiserum development) and biological (predator and prey coevolution) implications. Venom is primarily a trophic adaptation and as such, the evolution and abundance of venom proteins relates directly to prey capture success and organism natural history. Without this biologically relevant perspective, proteomic and transcriptomic approaches could produce simply a list of proteins, peptides, and transcripts. It is therefore important to consider the presence and evolution of venom proteins in terms of their biological significance to the organism. Three-finger toxins (3FTx) comprise a particularly common venom protein superfamily that contributes significantly to differences in envenomation symptomology, toxicity, and overall venom composition. Three-finger toxins are non-enzymatic proteins that maintain a common molecular scaffold, and bind to different receptors/acceptors and exhibit a wide variety of biological effects. These toxins are the main lethal neurotoxins in some snake venoms and are currently the only known venom proteins associated with prey-specific toxicity. This dissertation has four major objectives: (i) to examine 3FTxs in front-fanged Elapidae and rear-fanged snake venoms for prey-specific toxicity, (ii) to examine differences in 3FTx expression within rear-fanged snake venom glands, (iii) to determine if mRNA transcripts obtained from crude venoms can be utilized for molecular evolutionary studies and venom proteomic studies, and (iv) to determine if a transcriptomic and proteomic integrated approach can more thoroughly characterize differences in rear-fanged snake venom composition. Three-finger toxins were isolated from the venom of the front-fanged Naja kaouthia (Family Elapidae; Monocled Cobra) and rear-fanged Spilotes (Pseustes) sulphureus (Family Colubridae; Amazon Puffing Snake) using chromatographic techniques, and toxicity assays were performed to evaluate prey specificity. Despite various 3FTxs being present in abundance within N. kaouthia venom, only one 3FTx (alpha-cobratoxin) demonstrated lethal toxicity (<5 >µg/g) toward both NSA mice (Mus musculus) and House Geckos (Hemidactylus frenatus). For P. sulphureus, the most abundant 3FTx (sulmotoxin A), a heterodimeric complex, displayed prey-specific toxicity towards House Geckos, and the second most abundant 3FTx (sulmotoxin B) displayed prey-specific toxicity towards mice. This demonstrates how a relatively simple venom with toxins dominated by one venom protein superfamily (3FTXs) can still allow for the targeting of a diversity of prey. Venom gland toxin transcriptomes and crude venom transcriptomes were obtained via individual transcripts with 3’RACE (Rapid Amplification of cDNA Ends) and next- generation sequencing to evaluate the abundance, diversity, and molecular evolution of 3FTxs. Venom protein gene expression within rear-fanged snake venom glands revealed trends towards either viper-like expression, dominated by snake venom metalloproteinases, or elapid-like expression, dominated by 3FTxs. For non-conventional 3FTxs transcripts within these glands and within crude venom, approximately 32% of 3FTx amino acid sites were under positive selection, and approximately 20% of sites were functionally critical and conserved. RNA isolated from crude venom demonstrated to be a successful approach to obtain venom protein transcripts for molecular evolutionary analyses, resulting in a novel approach without the need to sacrifice snakes for tissue. The use of a combined venom gland transcriptome with proteomic approaches aided in characterizing venom composition from previously unstudied rear-fanged snake venoms. This dissertation represents an important step in the incorporation of multiple high-throughput characterization methods and the addition of multiple assays to explore the biological roles of toxins, in particular 3FTxs, within these venoms.
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