An overview of <i>Phoneutria nigriventer</i> spider venom using combined transcriptomic and proteomic approaches

<div><p><i>Phoneutria nigriventer</i> is one of the largest existing true spiders and one of the few considered medically relevant. Its venom contains several neurotoxic peptides that act on different ion channels and chemical receptors of vertebrates and invertebrates. Some of these venom toxins have been shown as promising models for pharmaceutical or biotechnological use. However, the large diversity and the predominance of low molecular weight toxins in this venom have hampered the identification and deep investigation of the less abundant toxins and the proteins with high molecular weight. Here, we combined conventional and next-generation cDNA sequencing with Multidimensional Protein Identification Technology (MudPIT), to obtain an in-depth panorama of the composition of <i>P</i>. <i>nigriventer</i> spider venom. The results from these three approaches showed that cysteine-rich peptide toxins are the most abundant components in this venom and most of them contain the Inhibitor Cysteine Knot (ICK) structural motif. Ninety-eight sequences corresponding to cysteine-rich peptide toxins were identified by the three methodologies and many of them were considered as putative novel toxins, due to the low similarity to previously described toxins. Furthermore, using next-generation sequencing we identified families of several other classes of toxins, including CAPs (Cysteine Rich Secretory Protein—CRiSP, antigen 5 and Pathogenesis-Related 1—PR-1), serine proteinases, TCTPs (translationally controlled tumor proteins), proteinase inhibitors, metalloproteinases and hyaluronidases, which have been poorly described for this venom. This study provides an overview of the molecular diversity of <i>P</i>. <i>nigriventer</i> venom, revealing several novel components and providing a better basis to understand its toxicity and pharmacological activities.</p></div

Conventional sequencing annotation.

<p>Annotation distribution of unique sequences (A) and ESTs (B) against UniProt. C) Distribution of EST annotation per Contig size (number of ESTs used to generate contig sequences). Numbers inside the bars are the raw numbers of ESTs per annotation class.</p

Diversity and abundance of putative venom components from <i>P</i>. <i>nigriventer</i> venom gland transcriptome sequenced by NGS.

<p>Unique sequences were searched against UniProt database and classified into known toxin subfamilies. Left graph shows relative proportions expressed as percentages of unique entries. Right graph shows relative proportions expressed as percentages of abundance (FPKM) of transcripts belonging to each subfamily.</p

Relative abundance, expressed as FPKM, of subfamilies of the putative venom components found in the NGS analysis of <i>P</i>. <i>nigriventer</i> venom glands.

<p>A) Cysteine-rich peptide toxins and B) Other families of venom components. Unique sequences were classified into known toxin subfamilies according to UniProt database. Bars represent the sum of FPKM for each transcript belonging to the described groups.</p

Venn diagram representing the total number of unique cysteine-rich peptide toxins found in <i>P</i>. <i>nigriventer</i> venom by each technique used.

<p>Venn diagram representing the total number of unique cysteine-rich peptide toxins found in <i>P</i>. <i>nigriventer</i> venom by each technique used.</p

<i>P</i>. <i>nigriventer</i> venom composition analyzed by MudPIT proteomic technique.

<p>Left graph shows the proportion of components detected by venom analysis. The peptide sequences found were searched against the NGS transcriptomic database and classified according to their UniProt annotation as ‘putative venom components’ or ‘cellular functions’. Eighteen percent of the retrieved proteins did not match any sequence from the database. Right graph shows putative venom components divided into subfamilies of putative toxins. The proportion of each category was calculated by the sum of the emPAI.</p

Classification of the cysteine-rich peptide toxins identified, according to their cysteine frameworks.

<p>Classification of the cysteine-rich peptide toxins identified, according to their cysteine frameworks.</p

General composition of <i>P</i>. <i>nigriventer</i> venom gland transcriptome sequenced by NGS.

<p>Unique sequences were searched against UniProt database and classified as ‘putative venom components’ or ‘cellular function proteins’. Left graph shows relative proportions expressed as percentages of unique sequences. Most of the unique sequences (66%) did not match any sequence from UniProt database (e-value < 1e^-5). Right graph shows relative proportions expressed as percentages of abundance (FPKM) of transcripts belonging to each category.</p

Sequence alignments of cysteine-rich peptide toxin precursors from group II.

<p>Alignment was performed with MUSCLE, Signal peptide is highlighted in yellow, propeptide is highlighted in green and processing quadruplet motif (PQM) is highlighted in cyan. Conserved cysteines are marked in blue. Percentage of identity (ID%) with the reference protein was calculated using the tool EMBOSS Stretcher for pairwise sequence alignment using either the complete or processed mature sequence. CSTX-10 (UniProt: B3EWT0), from <i>C</i>. <i>salei</i> spider; U7-cntx-Pn1a (UniProt: P81791), U10-cntx-Pn1a (UniProt: P0C2S9), U6-cntx-Pn1a (UniProt: P81793), ω-cntx-Pn1a (UniProt: O76201), κ-cntx-Pn1a (UniProt: O76200), U9-cntx-Pn1a (UniProt: P0C2S6), from <i>P</i>. <i>nigriventer</i> spider; and U1-cntx-Pk1a (UniProt: P83895), from <i>P</i>. <i>keyserlingi</i> spider were used as references.</p

List of the main families of molecules for the venom components found in NGS transcriptomic analysis.

<p>List of the main families of molecules for the venom components found in NGS transcriptomic analysis.</p