Analysis of the Venoms of Four Subspecies of the Western Rattlesnake (Crotalus oreganus)

Recently, the benefits venom can have in the discovery and development of different medications to assist in treating a variety of diverse human diseases have been areas of research. In order to develop a better understanding of how to evaluate venoms for potential use as therapeutics, one needs to look at the fundamental composition of venom samples. This project aims at discovering and analyzing the basic components from the venom of four different subspecies of the Western Rattlesnake. Venom samples collected will be subjected to size exclusion chromatography to separate proteins of different sizes. After fractionation of venoms, the following assays are conducted on individual fractions to locate specific activities of enzymatic venom proteins: metalloproteinase activity, kallikrein-like and thrombin-like serine proteinase activities, phospholipase A2 activity, phosphodiesterase activity and L-amino acid oxidase activity. These tests will compare the basic biochemistry of the samples of venoms from the different subspecies to help expand knowledge on chemistry differences between closely related subspecies of rattlesnakes. Snakes use their venoms primarily to obtain food, and results will allow for further evaluation of how their differing venom compositions relate to specific aspects of their ecology, such as use of divergent habitats, potential risks of different predators, effects on various prey species utilized, as well as revealing those components that have remained static and are shared among all subspecies analyzed. Because envenomation symptoms will vary due to venom composition, these results will also help inform what medical sequelae should be expected from particular populations of rattlesnakes

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<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

The venom gland transcriptome of the Desert Massasauga Rattlesnake (Sistrurus catenatus edwardsii): towards an understanding of venom composition among advanced snakes (Superfamily Colubroidea)

<p>Abstract</p> <p>Background</p> <p>Snake venoms are complex mixtures of pharmacologically active proteins and peptides which belong to a small number of superfamilies. Global cataloguing of the venom transcriptome facilitates the identification of new families of toxins as well as helps in understanding the evolution of venom proteomes.</p> <p>Results</p> <p>We have constructed a cDNA library of the venom gland of a threatened rattlesnake (a pitviper), <it>Sistrurus catenatus edwardsii </it>(Desert Massasauga), and sequenced 576 ESTs. Our results demonstrate a high abundance of serine proteinase and metalloproteinase transcripts, indicating that the disruption of hemostasis is a principle mechanism of action of the venom. In addition to the transcripts encoding common venom proteins, we detected two varieties of low abundance unique transcripts in the library; these encode for three-finger toxins and a novel toxin possibly generated from the fusion of two genes. We also observed polyadenylated ribosomal RNAs in the venom gland library, an interesting preliminary obsevation of this unusual phenomenon in a reptilian system.</p> <p>Conclusion</p> <p>The three-finger toxins are characteristic of most elapid venoms but are rare in viperid venoms. We detected several ESTs encoding this group of toxins in this study. We also observed the presence of a transcript encoding a fused protein of two well-characterized toxins (Kunitz/BPTI and Waprins), and this is the first report of this kind of fusion in a snake toxin transcriptome. We propose that these new venom proteins may have ancillary functions for envenomation. The presence of a fused toxin indicates that in addition to gene duplication and accelerated evolution, exon shuffling or transcriptional splicing may also contribute to generating the diversity of toxins and toxin isoforms observed among snake venoms. The detection of low abundance toxins, as observed in this and other studies, indicates a greater compositional similarity of venoms (though potency will differ) among advanced snakes than has been previously recognized.</p

Role of accelerated segment switch in exons to alter targeting (ASSET) in the molecular evolution of snake venom proteins

<p>Abstract</p> <p>Background</p> <p>Snake venom toxins evolve more rapidly than other proteins through accelerated changes in the protein coding regions. Previously we have shown that accelerated segment switch in exons to alter targeting (ASSET) might play an important role in its functional evolution of viperid three-finger toxins. In this phenomenon, short sequences in exons are radically changed to unrelated sequences and hence affect the folding and functional properties of the toxins.</p> <p>Results</p> <p>Here we analyzed other snake venom protein families to elucidate the role of ASSET in their functional evolution. ASSET appears to be involved in the functional evolution of three-finger toxins to a greater extent than in several other venom protein families. ASSET leads to replacement of some of the critical amino acid residues that affect the biological function in three-finger toxins as well as change the conformation of the loop that is involved in binding to specific target sites.</p> <p>Conclusion</p> <p>ASSET could lead to novel functions in snake venom proteins. Among snake venom serine proteases, ASSET contributes to changes in three surface segments. One of these segments near the substrate binding region is known to affect substrate specificity, and its exchange may have significant implications for differences in isoform catalytic activity on specific target protein substrates. ASSET therefore plays an important role in functional diversification of snake venom proteins, in addition to accelerated point mutations in the protein coding regions. Accelerated point mutations lead to fine-tuning of target specificity, whereas ASSET leads to large-scale replacement of multiple functionally important residues, resulting in change or gain of functions.</p

Accelerated exchange of exon segments in Viperid three-finger toxin genes (Sistrurus catenatus edwardsii; Desert Massasauga)

<p>Abstract</p> <p>Background</p> <p>Snake venoms consist primarily of proteins and peptides showing a myriad of potent biological activities which have been shaped by both adaptive and neutral selective forces. Venom proteins are encoded by multigene families that have evolved through a process of gene duplication followed by accelerated evolution in the protein coding region.</p> <p>Results</p> <p>Here we report five gene structures of three-finger toxins from a viperid snake, <it>Sistrurus catenatus edwardsii</it>. These toxin genes are structured similarly to elapid and hydrophiid three-finger toxin genes, with two introns and three exons. Both introns and exons show distinct patterns of segmentation, and the insertion/deletion of segments may define their evolutionary history. The segments in introns, when present, are highly similar to their corresponding segments in other members of the gene family. In contrast, some segments in the exons show high similarity, while others are often distinctly different among corresponding regions of the isoforms.</p> <p>Conclusion</p> <p>Ordered, conserved exon structure strongly suggests that segments in corresponding regions in exons have been exchanged with distinctly different ones during the evolution of these genes. Such a "switching" of segments in exons may result in drastically altering the molecular surface topology and charge, and hence the molecular targets of these three-finger toxins. Thus the phenomenon of accelerated segment switch in exons to alter targeting (ASSET) may play an important role in the evolution of three-finger toxins, resulting in a family of toxins with a highly conserved structural fold but widely varying biological activities.</p

The Venom Gland Transcriptome of the Desert Massasauga Rattlesnake (Sistrurus Catenatus Edwardsii): Towards an Understanding of Venom Composition Among Advanced Snakes (Superfamily Colubroidea)

BACKGROUND: Snake venoms are complex mixtures of pharmacologically active proteins and peptides which belong to a small number of superfamilies. Global cataloguing of the venom transcriptome facilitates the identification of new families of toxins as well as helps in understanding the evolution of venom proteomes. RESULTS: We have constructed a cDNA library of the venom gland of a threatened rattlesnake (a pitviper), Sistrurus catenatus edwardsii (Desert Massasauga), and sequenced 576 ESTs. Our results demonstrate a high abundance of serine proteinase and metalloproteinase transcripts, indicating that the disruption of hemostasis is a principle mechanism of action of the venom. In addition to the transcripts encoding common venom proteins, we detected two varieties of low abundance unique transcripts in the library; these encode for three-finger toxins and a novel toxin possibly generated from the fusion of two genes. We also observed polyadenylated ribosomal RNAs in the venom gland library, an interesting preliminary obsevation of this unusual phenomenon in a reptilian system. CONCLUSION: The three-finger toxins are characteristic of most elapid venoms but are rare in viperid venoms. We detected several ESTs encoding this group of toxins in this study. We also observed the presence of a transcript encoding a fused protein of two well-characterized toxins (Kunitz/BPTI and Waprins), and this is the first report of this kind of fusion in a snake toxin transcriptome. We propose that these new venom proteins may have ancillary functions for envenomation. The presence of a fused toxin indicates that in addition to gene duplication and accelerated evolution, exon shuffling or transcriptional splicing may also contribute to generating the diversity of toxins and toxin isoforms observed among snake venoms. The detection of low abundance toxins, as observed in this and other studies, indicates a greater compositional similarity of venoms (though potency will differ) among advanced snakes than has been previously recognized

Integration of transcriptomic and proteomic approaches for venom profiling

Snake venoms contain many protein and peptide isoforms with high levels of sequence variation, even within a single species. When characterizing venoms, peptide mass fingerprinting using databases built predominately from protein sequences originating from model organisms can be disadvantageous, especially when the intention is to document protein diversity. Therefore, the use of species-specific venom gland transcriptomes corrects for the absence of these unique peptide sequences in databases. The integration of transcriptomics and proteomics improves the accuracy of either approach alone for venom profiling. In this review, we highlight several examples, from both published and unpublished work in our lab, demonstrating how a combined venom gland transcriptome and proteome methodology allows for comprehensive characterization of venoms, including those from understudied rear-fanged snake species, and we provide recommendations for using these approaches. Article highlights: • Use of a species-specific venom gland transcriptome allows for more accurate proteomic quantification of venom components • Different databases bias proteomic results, and smaller databases increase detection sensitivity • Species-specific databases better detect unique peptide sequence

Venom Ontogeny in the Mexican Lance-Headed Rattlesnake (Crotalus polystictus)

As trophic adaptations, rattlesnake venoms can vary in composition depending on several intrinsic and extrinsic factors. Ontogenetic changes in venom composition have been documented for numerous species, but little is known of the potential age-related changes in many rattlesnake species found in México. In the current study, venom samples collected from adult and neonate Crotalus polystictus from Estado de México were subjected to enzymatic and electrophoretic analyses, toxicity assays (LD50), and MALDI-TOF mass spectrometry, and a pooled sample of adult venom was analyzed by shotgun proteomics. Electrophoretic profiles of adult males and females were quite similar, and only minor sex-based variation was noted. However, distinct differences were observed between venoms from adult females and their neonate offspring. Several prominent bands, including P-I and P-III snake venom metalloproteinases (SVMPs) and disintegrins (confirmed by MS/MS) were present in adult venoms and absent/greatly reduced in neonate venoms. Age-dependent differences in SVMP, kallikrein-like, phospholipase A2 (PLA2), and L-amino acid oxidase (LAAO) activity levels were confirmed by enzymatic activity assays, and like many other rattlesnake species, venoms from adult snakes have higher SVMP activity than neonate venoms. Conversely, PLA2 activity was approximately 2.5 X greater in venoms from neonates, likely contributing to the increased toxicity (neonate venom LD50 = 4.5 μg/g) towards non-Swiss albino mice when compared to adult venoms (LD50 = 5.5 μg/g). Thrombin-like (TLE) and phosphodiesterase activities did not vary significantly with age. A significant effect of sex (between adult male and adult female venoms) was also observed for SVMP, TLE, and LAAO activities. Analysis of pooled adult venom by LC-MS/MS identified 14 toxin protein families, dominated by bradykinin-inhibitory peptides, SVMPs (P-I, P-II and P-III), disintegrins, PLA2s, C-type-lectins, CRiSPs, serine proteinases, and LAAOs (96% of total venom proteins). Neonate and adult C. polystictus in this population consume almost exclusively mammals, suggesting that age-based differences in composition are related to physical differences in prey (e.g., surface-to-volume ratio differences) rather than taxonomic differences between prey. Venoms from adult C. polystictus fit a Type I pattern (high SVMP activity, lower toxicity), which is characteristic of many larger-bodied rattlesnakes of North America.Funding for this study was provided in part by the Colorado Office for Economic Development and International Trade (to SPM). Additional funds were provided by the UNC Office of Research

Interrogating the Venom of the Viperid Snake Sistrurus catenatus edwardsii by a Combined Approach of Electrospray and MALDI Mass Spectrometry

The complete sequence characterization of snake venom proteins by mass spectrometry is rather challenging due to the presence of multiple isoforms from different protein families. In the present study, we investigated the tryptic digest of the venom of the viperid snake Sistrurus catenatus edwardsii by a combined approach of liquid chromatography coupled to either electrospray (online) or MALDI (offline) mass spectrometry. These different ionization techniques proved to be complementary allowing the identification a great variety of isoforms of diverse snake venom protein families, as evidenced by the detection of the corresponding unique peptides. For example, ten out of eleven predicted isoforms of serine proteinases of the venom of S. c. edwardsii were distinguished using this approach. Moreover, snake venom protein families not encountered in a previous transcriptome study of the venom gland of this snake were identified. In essence, our results support the notion that complementary ionization techniques of mass spectrometry allow for the detection of even subtle sequence differences of snake venom proteins, which is fundamental for future structure-function relationship and possible drug design studies