Molecular Diversification Of Peptide Toxins From Two Tarantulas

Spider venom is a mixture of various compounds with diversified biological activities, which are used for capturing prey and defending against predators. The peptide components bind to a broad range of cellular targets with high affinity and selectivity and appear to have remarkable structural diversity. Although spider venoms have been intensively investigated in the past few decades, venomic strategies have focused on peptides of high abundance of peptides. In addition of complete spider genomes or representative cDNA libraries frustrate researchers interested in molecular diversity and understanding the genetic mechanisms of toxin evolution.In the present study, second generation sequencing technologies combined with proteomic analysis was applied to rapidly reveal the diversity of peptide toxins in the venom of the Chinese bird spider Ornithoctonus huwena.454 sequencing revealed a total of 123,922 reads(amounting to ?42 Mb) from the venom gland of Ornithoctonus huwena with an average length of ?327 bases per read, ranging from 40 to 836 bp. We observed high identity of 32,267 reads with toxin families, accountingfor 29% raw reads. ‘Cellular Proteins’ includes transcripts coding for proteins involved in cellular processes(44%),including peptidases, cell signaling, cell structure and motility, metabolism and protein processing. The group‘Putative toxins’ includes sequences rich in cysteine that display identity to toxins(0.1%). ‘Unknown function’encompasses ESTs homologous to described sequences with no functional assessment or hypothetical genes(7%). About 20%reads were assigned to the “No Hit” category, indicating no match with currently known sequences. A total of 626 toxin precursors were retrieved from transcriptomic data. All toxin precursors, clustered into 16 gene superfamilies, which included 6 novel superfamilies and 6 novel cysteine patterns.A large number of toxin variants are identified at low levels due to hypermutations and fragment insertions/deletions.Furthermore, the intraspecific venom variability, in combination with various transcripts and the variable peptide processing identified previously, contributes to the hypervariability of toxins in venoms and associated rapid and adaptive evolution of toxins for prey capture and defense.In order to investigate the mechanisms responsible for toxin diversity, transcriptomics based on second-generationhigh-throughput sequencing was combined with peptidomic assays to characterize the venom of the tarantula Haplopelma hainanum.The genes expressed in the venom glands were identified and the bioactivity of their protein products was analyzed using the patch-clamp technique. Sequencing revealed a total of 249,549 reads with an average length of ~328 bases per read(max. 830 bp, min. 40 bp). 52,570 reads displayed similarity to known peptide toxins or ‘Toxin-Like’ sequences; the ‘Putative Toxins’ category includes sequences rich in cysteine residues and sharing sequence identity with toxins or proteins including the ICK motif(5%) that were not identified by a BLAST search;‘the ‘Cellular Proteins’ category includes transcripts coding for proteins involved in cellular processes(44%); The‘Unknown Function’ category includes reads that shared sequence identity with previously described sequences with no functional assessment or hypothetical genes; The ‘No Hit’category indicates no match with currently known sequences. A total of 1,136 potential toxin precursors were identified that clustered into 90 toxin groups, of which 72 were novel. The toxin peptides clustered into 20 cysteine scaffolds that included between 4 to 12 cysteines, and 14 of these groups were newly identified in this spider. In combination with toxindiversity, the diverse bioactivities of spider toxins facilitate adaptation to changing environments, types of prey,and milking regimes in captivity.Cardiac ion channels are membrane-spanning proteins that allow the passive movement of ions across the cell membrane along its electrochemical gradient, which regulates the resting membrane potential as well as the shape and duration of the cardiac action potential. Additionally, they have been recognized as potential targets for the actions of neurotransmitters, hormones and drugs of cardiac diseases.Spider venoms contain high abundant of toxins that target diverse ion channels and have been considered as a potential resource of new constituents with specific pharmacological properties. However, few peptides from spider venoms were detected as cardiac channel antagonists. In order to explore the effects of the venom of Ornithoctonus huwena and O.hainana on the action potential and ionic currents of neonatal rat ventricular myocytes(NRVMs), whole cell patch clamp technique was used to record action potential duration(APD), sodium current(INa), L calcium current(ICaL),rapidly activating and inactivating transient outward currents(Ito1), rapid(IKr) and slow(IKs) components of the delayed rectifier currents and theinward rectifier currents(IK1). Our results showed that both100 μg/mL huwen and hainan venom obviously prolonged APDs.Huwen venom prolongs APD90 in a frequency-dependent manner in isolated neonatal rat ventricular myocytes. 100 μg/mL huwen venom inhibited 72.3 ± 3.6% INacurrent, 58.3 ± 4.2% summit current and 54 ± 6.1% the end current of IKr, and 65 ± 3.3%ICaLcurrent, yet, didn’t have obvious effect on IKs, Ito1 and IK1currents. Similarity, hainan venom could inhibit INaand ICa Leffectively, while no evident inhibitory effects on cardiac K+channels(Ito1, Iks, Ikrand Ik1) were observed.In conclusion, the O.huwena and H.hainana venom represented a multifaceted pharmacological profile. It contains abundant of cardiac channel antagonists and might be valuable tools for investigation of both channels and anti- arrhythmic therapy development.In conclusion, we performed the strategy that transcriptome combined with proteome and patch-clamp approaches and revealed the mechanism of toxin diversity from tarantula Ornithoctonus huwena and Haplopelma hainanum venom.