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

Eggshell Matrix Protein Mimetics: Elucidation of Molecular Mechanism of Goose Eggshell Calcification using Designed Peptides

Model peptides were designed, synthesized and conducted a detailed structure-property study to unravel the molecular mechanism of goose eggshell calcification. The peptides were designed based on the primary structural features of the eggshell matrix proteins ansocalcin and OC-17. In vitro CaCO₃ crystal growth experiments in presence of these peptides showed calcite crystal aggregation as observed in the case of the parent protein ansocalcin. The structure of these peptides in solution was established using intrinsic tryptophan fluorescence studies and quasi-elastic light scattering experiments. The structural features are correlated with observed results of the in vitro crystallization studies.Singapore-MIT Alliance (SMA

Designer peptides to understand the mineralization of calcium salts

Recently, we reported the extraction, purification and amino acid sequence of ansocalcin, the major goose eggshell matrix protein. In vitro studies showed that ansocalcin induces spherical calcite crystal aggregates. We designed two peptides using the unique features of the sequence of ansocalcin and the role of these peptides in CaCO&#x2083; crystallization was investigated. The peptides showed similar activities as compared to ansocalcin, but at a higher concentration. The full characterization of the peptides and a rational for the observed morphology for the calcite crystals are discussed in detail.Singapore-MIT Alliance (SMA

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

Transcriptomic analysis of the venom gland of the red-headed krait (Bungarus flaviceps) using expressed sequence tags

<p>Abstract</p> <p>Background</p> <p>The Red-headed krait (<it>Bungarus flaviceps</it>, Squamata: Serpentes: Elapidae) is a medically important venomous snake that inhabits South-East Asia. Although the venoms of most species of the snake genus <it>Bungarus </it>have been well characterized, a detailed compositional analysis of <it>B. flaviceps </it>is currently lacking.</p> <p>Results</p> <p>Here, we have sequenced 845 expressed sequence tags (ESTs) from the venom gland of a <it>B. flaviceps</it>. Of the transcripts, 74.8% were putative toxins; 20.6% were cellular; and 4.6% were unknown. The main venom protein families identified were three-finger toxins (3FTxs), Kunitz-type serine protease inhibitors (including chain B of β-bungarotoxin), phospholipase A₂(including chain A of β-bungarotoxin), natriuretic peptide (NP), CRISPs, and C-type lectin.</p> <p>Conclusion</p> <p>The 3FTxs were found to be the major component of the venom (39%). We found eight groups of unique 3FTxs and most of them were different from the well-characterized 3FTxs. We found three groups of Kunitz-type serine protease inhibitors (SPIs); one group was comparable to the classical SPIs and the other two groups to chain B of β-bungarotoxins (with or without the extra cysteine) based on sequence identity. The latter group may be functional equivalents of dendrotoxins in <it>Bungarus </it>venoms. The natriuretic peptide (NP) found is the first NP for any Asian elapid, and distantly related to Australian elapid NPs. Our study identifies several unique toxins in <it>B. flaviceps </it>venom, which may help in understanding the evolution of venom toxins and the pathophysiological symptoms induced after envenomation.</p

Unusual accelerated rate of deletions and insertions in toxin genes in the venom glands of the pygmy copperhead (Austrelaps labialis) from kangaroo island

<p>Abstract</p> <p>Background</p> <p>Toxin profiling helps in cataloguing the toxin present in the venom as well as in searching for novel toxins. The former helps in understanding potential pharmacological profile of the venom and evolution of toxins, while the latter contributes to understanding of novel mechanisms of toxicity and provide new research tools or prototypes of therapeutic agents.</p> <p>Results</p> <p>The pygmy copperhead (<it>Austrelaps labialis</it>) is one of the less studied species. In this present study, an attempt has been made to describe the toxin profile of <it>A. labialis </it>from Kangaroo Island using the cDNA library of its venom glands. We sequenced 658 clones which represent the common families of toxin genes present in snake venom. They include (a) putative long-chain and short-chain neurotoxins, (b) phospholipase A₂, (c) Kunitz-type protease inhibitor, (d) CRISPs, (e) C-type lectins and (f) Metalloproteases. In addition, we have also identified a novel protein with two Kunitz-type domains in tandem similar to bikunin.</p> <p>Conclusion</p> <p>Interestingly, the cDNA library reveals that most of the toxin families (17 out of 43 toxin genes; ~40%) have truncated transcripts due to insertion or deletion of nucleotides. These truncated products might not be functionally active proteins. However, cellular trancripts from the same venom glands are not affected. This unusual higher rate of deletion and insertion of nucleotide in toxin genes may be responsible for the lower toxicity of <it>A. labialis </it>venom of Kangroo Island and have significant effect on evolution of toxin genes.</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

Niemann–Pick Type C2 Proteins in Aedes aegypti: Molecular Modelling and Prediction of Their Structure–Function Relationships

Aedes aegypti is a major vector that transmits arboviruses through the saliva injected into the host. Salivary proteins help in uninterrupted blood intake and enhance the transmission of pathogens. We studied Niemann–Pick Type C2 (NPC2) proteins, a superfamily of saliva proteins that play an important role in arbovirus infections. In vertebrates, a single conserved gene encodes for the NPC2 protein that functions in cholesterol trafficking. Arthropods, in contrast, have several genes that encode divergent NPC2 proteins. We compared the sequences of 20 A. aegypti NPC2 proteins to the cholesterol-binding residues of human and bovine, and fatty-acid-binding residues of ant NPC2 protein. We identified four mosquito NPC2 proteins as potential sterol-binding proteins. Two of these proteins (AAEL006854 and/or AAEL020314) may play a key role in ecdysteroid biosynthesis and moulting. We also identified one mosquito NPC2 protein as a potential fatty-acid-binding protein. Through molecular modelling, we predicted the structures of the potential sterol- and fatty-acid-binding proteins and compared them to the reference proteins