Evolution and Biological Roles of Three-Finger Toxins in Snake Venoms

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 (\u3c5 \u3eµg/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

Snakebite Therapeutics Based on Endogenous Inhibitors from Vipers

Venomous snakebite is a major human health issue in many countries and has been categorized as a neglected tropical disease by the World Health Organization. Venomous snakes have evolved to produce venom, which is a complex mixture of toxic proteins and peptides, both enzymatic and nonenzymatic in nature. In this current era of high-throughput technologies, venomics projects, which include genome, transcriptome, and proteome analyses of various venomous species, have been conducted to characterize divergent venom phenotypes and the evolution of venom-related genes. Additionally, venomics can also inform about mechanisms of toxin production, storage, and delivery. Venomics can guide antivenom and therapeutic strategies against envenomations and identify new toxin-derived drugs/tools. One potentially promising drug development direction is the use of endogenous inhibitors present in snake venom glands and serum that could be useful for snakebite therapeutics. These inhibitors suppress the activity of venom proteases, enzymatic proteins responsible for the irreversible damage from snakebite. This book chapter will focus on insights from venomous snake adaptations, such as the evolution of venom proteases to generate diverse activities and snake natural resistance to inhibit activity, and how this information can inform and have applications in the treatment of venomous snakebite

総会抄録

<p><b>Aligned Middle American Rattlesnake (<i>Crotalus simus tzabcan</i>) C-type lectins (A) and serine proteases (B).</b> A) Four unique venom-based C-type lectin transcripts (asterisks) were identified for <i>C</i>. <i>s</i>. <i>tzabcan</i> and aligned to other crotaline species. Identical nucleotide sequences are shaded and corresponding GenBank accession numbers are as follows: Crotalus_adamanteus (AEJ31974.1), Deinagkistrodon_acutus (AAM22790.1), Crotalus_d_terrificus (Q719L8.1), and Crotalus_o_helleri (AEU60004.1). B) Venom-based serine proteases cDNA sequences (asterisks) were also obtained from <i>C</i>. <i>s</i>. <i>tzabcan</i> and were aligned with toxins from several other species; identical nucleotide sequences are shaded, and the catalytic triad composed of Ser195, Asp102, and His57 associated with thrombin-like activity in snake venom serine proteases are identified (arrowheads). Isoform 3 from <i>C</i>. <i>s</i>. <i>tzabcan</i> is a partial sequence. GenBank accession numbers are as follows: Agkistrodon_p_leucostoma (HQ270466.1), Bothrops_asper (DQ247724.1), Crotalus_d_terrificus7 (EU360954.1), Crotalus_d_terrificus4 (EU360952.1), Crotalus_d_terrificus3 (EU360951.1), Crotalus_d_durissus (DQ164401.1), Sistrurus_c_edwardsi (DQ464239.1), Trimeresurus_mucrosquamatus (X83225.1), Crotalus_adamanteus (HQ414118.1), Calloselasma_rhodostoma (L07308.1), Deinagkistrodon_acutus (AY861382.1), Trimeresurus_stejnegeri (AF545575.1), and Crotalus_atrox (AF227153.1).</p

Experimental and computational aspects of bioactive proteins from animal venoms: an insight into pharmacological properties and drug discovery

Editoria

Highly Evolvable: Investigating Interspecific and Intraspecific Venom Variation in Taipans (Oxyuranus spp.) and Brown Snakes (Pseudonaja spp.)

Snake venoms are complex mixtures of toxins that differ on interspecific (between species) and intraspecific (within species) levels. Whether venom variation within a group of closely related species is explained by the presence, absence and/or relative abundances of venom toxins remains largely unknown. Taipans (Oxyuranus spp.) and brown snakes (Pseudonaja spp.) represent medically relevant species of snakes across the Australasian region and provide an excellent model clade for studying interspecific and intraspecific venom variation. Using liquid chromatography with ultraviolet and mass spectrometry detection, we analyzed a total of 31 venoms covering all species of this monophyletic clade, including widespread localities. Our results reveal major interspecific and intraspecific venom variation in Oxyuranus and Pseudonaja species, partially corresponding with their geographical regions and phylogenetic relationships. This extensive venom variability is generated by a combination of the absence/presence and differential abundance of venom toxins. Our study highlights that venom systems can be highly dynamical on the interspecific and intraspecific levels and underscores that the rapid toxin evolvability potentially causes major impacts on neglected tropical snakebites

Relationship between the number of colony picks and the number of observed unique phospholipase A₂ isoforms.

<p>Relationship between the number of colony picks and the number of observed unique phospholipase A₂ isoforms.</p

RNA and mRNA isolation protocols used to obtain extracellular RNA within venom, and the resulting yields and cDNA amplification success.

<p>RNA and mRNA isolation protocols used to obtain extracellular RNA within venom, and the resulting yields and cDNA amplification success.</p

<i>Alsophis portoricensis</i> venom PIII metalloproteinase sequence aligned with amino acid sequences from rear-fanged and elapid snake species.

<p>Identical residues are shaded, demonstrating PIII metalloproteinase sequence conservation in these diverse species. Only a partial sequence of the complete transcript from <i>A</i>. <i>portoricensis</i> (asterisks) was used for the alignment. Genbank accession numbers are as follows: Philodryas_chamissonis (AJB84503.1), Philodryas_olfersii (ACS74987.1), Cerberus_rynchops (ADJ51055.1), Pseudechis_porphyriacus (ABQ01133.1), and Demansia_vestigiata (ABK63559.1).</p

Aligned sequences of non-conventional three-finger toxins (3FTxs) from venoms of rear-fanged and elapid snakes.

<p>Venom-derived 3FTx sequences (asterisks) obtained from <i>Boiga irregularis</i>, <i>B</i>. <i>dendrophila</i>, <i>B</i>. <i>nigriceps</i>, <i>B</i>. <i>cynodon</i>, <i>Oxybelis fulgidus</i>, <i>Ahaetulla prasina</i>, and <i>Trimorphodon biscutatus lambda</i> were aligned with various other rear-fanged and Elapidae species; identical nucleotide sequences are shaded. GenBank accession numbers are as follows: Trimorphodon_biscutatus_Tri3 (EU029678.1), Trimorphodon_biscutatus_Tri2 (EU029677.1), Telescopus_dhara_Tel4 (EU029686.1), Boiga_dendrophila_denmo (DQ366293.1), Boiga_irregularis_irditoxinB (DQ304539.1), Boiga_irregularis_irditoxinA (DQ304538.1), Boiga_irregularis_1f (GBSH01000015.1), Thrasops_jacksoni_Thr3 (EU029685.1), Dispholidus_typus_Dis1 (EU029674.1), Telescopus_dhara_Tel1 (EU029675.1), Thrasops_jacksoni_Thr5 (EU036635.1), Trimorphodon_biscutatus_Tri1 (EU029675.1), Naja_atra (AF031472.1), Bungarus_multicinctus (AF056400.1), Ophiophagus_hannah (FJ952515.1), Psammophis_mossambicus_Psa1 (EU029669.1), Leioheterodon_madagascariensis (EU029676.1), Bungarus_candidus (AY057878.1), and Dendroaspis_angusticeps (AF241871.1).</p

Full-Length Venom Protein cDNA Sequences from Venom-Derived mRNA: Exploring Compositional Variation and Adaptive Multigene Evolution - Fig 3

<p><b>Aligned Group IIA phospholipase A₂ acidic (A) and basic (B) subunit isoforms.</b> The neurotoxic, heterodimeric PLA₂ complex of crotoxin/Mojave toxin homologs consists of both an acidic A subunit and basic B subunit. A) Sequence alignments of the acidic (A) subunit of crotoxin or Mojave toxin homologs with identical residues shaded; the conserved signal peptide region is indicated by the red bar, and the Ca²⁺ binding loop is indicated by the blue bar. Regions of the A subunit which are post-translationally cleaved in the mature protein are indicated by red brackets below sequences. Sequences similar to crotoxin/Mojave toxin acidic subunit A derived from from venom (asterisks) were discovered in <i>Crotalus oreganus concolor</i> and <i>C</i>. <i>simus tzabcan</i> venoms. GenBank accession numbers of known toxins are as follows: Crotalus_s_scutulatus_MojaveTX (U01026.1), Crotalus_d_terrificus_CrotoxinA (X12606.1), Sistrurus_c_tergeminus_SistruxinA (Q6EAN6.1), and Gloydius_intermedius_GintexinA (AID56658.1). B) Sequence alignments of the basic (B) subunit of crotoxin/Mojave toxin homologs with identical residues shaded; the conserved signal peptide region is indicated by the red bar, and the Ca²⁺ binding loop is indicated by the blue bar. The asparagine-6 (N6) associated with neurotoxic PLA₂ functionality is indicated by arrowheads. Sequences similar to the crotoxin or Mojave toxin basic B subunits derived from venom (asterisks) were discovered in <i>Crotalus oreganus concolor</i>, <i>C</i>. <i>simus tzabcan</i>, and <i>C</i>. <i>basiliscus</i> venoms, with N6 sequences also found in <i>C</i>. <i>m</i>. <i>nigrescens</i> and <i>C</i>. <i>o</i>. <i>cerberus</i> venoms. GenBank accession numbers of known toxins are as follows: Crotalus_s_scutulatus_MojaveTX (U01027.1), Crotalus_d_terrificus_CrotoxinB (X12603.1), Crotalus_v_viridis_N6 (AF403138.1), Sistrurus_c_tergeminus_N6 (AY355169.1), Sistrurus_c_tergeminus_SistruxinB (Q6EER2.1), Bothriechis_schlegelii_N6 (AY355168.1), Protobothrops_mucrosquamatus (AF408409.1), and Deinagkistrodon_acutus (X77649.1).</p