Abstract
The venoms of toxic animals are chemical pools composed of various proteins, peptides, and small organic molecules used for predation and defense, in which the peptidic toxins have been intensively pursued mining modulators targeting disease-related ion channels and receptors as valuable drug pioneers. In the present study, we uncovered the molecular diversity of peptide toxins in the venom of the spider Heteropoda pingtungensis (H. pingtungensis) by using a combinatory strategy of venom gland cDNA library and transcriptome sequencing (RNA-seq). An amount of 991 high-quality expressed sequence tags (ESTs) were identified from 1138 generated sequences, which fall into three categories, such as the toxin-like ESTs (531, 53.58%), the cellular component ESTs (255, 25.73%), and the no-match ESTs (205, 20.69%), as determined by gene function annotations. Of them, 190 non-redundant toxin-like peptides were identified and can be artificially grouped into 13 families based on their sequence homology and cysteine frameworks (families A–M). The predicted mature toxins contain 2–10 cysteines, which are predicted to form intramolecular disulfide bonds to stabilize their three-dimensional structures. Bioinformatics analysis showed that toxins from H. pingtungensis venom have high sequences variability and the biological targets for most toxins are unpredictable due to lack of homology to toxins with known functions in the database. Furthermore, RP-HPLC and MALDI-TOF analyses have identified a total of 110 different peptides physically existing in the H. pingtungensis venom, and many RP-HPLC fractions showed potent inhibitory activity on the heterologously expressed NaV1.7 channel. Most importantly, two novel NaV1.7 peptide antagonists, µ-Sparatoxin-Hp1 and µ-Sparatoxin-Hp2, were characterized. In conclusion, the present study has added many new members to the spider toxin superfamily and built the foundation for identifying novel modulators targeting ion channels in the H. pingtungensis venom.
Highlights
Rather than strength, speed, and intelligence which make most predator species greatly adapted to the cruel “struggle for existence” on earth, toxic animals have evolved to utilize another smart and high-efficiency strategy, envenomation, for predation and defense [1]
We revealed the great diversity of H. pingtungensis venom peptides using a combinatory strategy of cDNA library and transcriptome sequencing, from which 190 novel toxin-like peptides belonging
Clone sequencing of the H. pingtungensis (Figure 1A) venom gland in the cDNA library resulted in 953 high-quality expressed sequence tags (ESTs), which are grouped into three distinct functional clusters by blasting against the non-redundant protein sequence database: 493 ESTs encode putative toxins, with the cDNA length ranging from 0.3–1.0 Kb; 255 ESTs are other cellular protein-encoding sequences, with the cDNA length ranging from 0.45–1.0 Kb; and 205 ESTs do not match any sequences in the database, with the cDNA length ranging from 0.2–1.05 Kb
Summary
Rather than strength, speed, and intelligence which make most predator species greatly adapted to the cruel “struggle for existence” on earth, toxic animals have evolved to utilize another smart and high-efficiency strategy, envenomation, for predation and defense [1]. The secretory epithelial cells in it produce the toxin as a precursor, usually comprising the signal peptide, the propeptide, and the mature peptide, which will be cleaved to release the functional mature peptide during the exocytosis process [9] These peptide toxins are small cysteinerich proteins with a molecular weight of less than 10 kDa. the cysteines in toxin form stable intramolecular disulfide bonds which assist it in refolding into a highly compacted globin-like structure, with the hydrophobic residues interacting to make a hydrophobic core and hydrophilic residues exposed to the aqueous phase. More than 1000 spider species are identified in the Heteropodidae family and the venom peptides from one member, the Heteropoda venatoria (H. venatoria), are intensively explored by both venom fractionation-based active peptides screening and venom gland cDNA library sequencing [11,16,17,18,19]. The present study has expanded the capacity of the existing spider toxins’ superfamily and set the foundation for identifying novel modulators targeting ion channels and receptors from the H. pingtungensis venom
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