Abstract
Spider venom is a complex mixture of bioactive peptides to subdue their prey. Early estimates suggested that over 400 venom peptides are produced per species. In order to investigate the mechanisms responsible for this impressive diversity, transcriptomics based on second generation high 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. 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 and 12 cysteines, and 14 of these groups were newly identified in this spider. Highly abundant toxin peptide transcripts were present and resulted from hypermutation and/or fragment insertion/deletion. In combination with variable post-translational modifications, this genetic variability explained how a limited set of genes can generate hundreds of toxin peptides in venom glands. Furthermore, the intraspecies venom variability illustrated the dynamic nature of spider venom and revealed how complex components work together to generate diverse bioactivities that facilitate adaptation to changing environments, types of prey, and milking regimes in captivity.
Highlights
Spider venom is a complex mixture and contains over 400 venom peptides/species
As outlined under “Experimental Procedures,” we searched for toxin peptide sequences directly from the sequencing reads, because the average read length of Ͼ200 bp allowed the identification of full-length toxin precursors
52,570 reads displayed similarity to known peptide toxins or toxin-like sequences; the category of putative toxins includes sequences rich in cysteine residues and sharing sequence identity with toxins or proteins including the inhibitor cysteine knot (ICK) motif (5%) that were not identified by a BLAST search; the category of cellular proteins 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; and the no hit category indicates no match with currently known sequences
Summary
Spider venom is a complex mixture and contains over 400 venom peptides/species. Results: Highly abundant hypermutation, fragment insertion/deletion, and variable post-translational modifications were observed in venom gland, and the highly diverse toxins exhibit diverse functions. 420 peptides were detected by mass spectrometry, but few could be paired with peptide precursors identified from cDNA and genomic DNA sequencing [10] This limited data focused mainly on highly abundant and smaller toxin precursors, whereas less. The relatively new 454 Life Sciences pyrosequencing technology has been successfully implemented in a number of species, including spiders [12,13,14,15,16] This approach provides a more comprehensive landscape of the transcriptomic content of venom glands and has improved technical capabilities that identify longer sequences, a wider range of sensitivity, and greater accuracy than traditional Sanger sequencing [17, 18]. Variable peptide processing and selective expression explain how a limited set of gene transcripts can generate hundreds of toxin peptides in spider venom that have diverse activities and cooperate to subdue potential prey species
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