Venom components from Citharischius crawshayi spider (Family Theraphosidae): exploring transcriptome, venomics, and function

Abstract

Despite strong efforts, knowledge about the composition of the venom of many spider species remains very limited. This work is the first report of transcriptome and venom analysis of the African spider Citharischius crawshayi. We used combined protocols of transcriptomics, venomics, and biological assays to characterize the venom and genes expressed in venom glands. A cDNA library of the venom glands was constructed and used to generate expressed sequence tags (ESTs). Sequence comparisons from 236 ESTs revealed interesting and unique sequences, corresponding to toxin-like and other components. Mass spectrometrical analysis of venom fractions showed more than 600 molecular masses, some of which showed toxic activity on crickets and modulated sodium currents in DmNa(v)1 and Na(v)1.6 channels as expressed in Xenopus oocytes. Taken together, our results may contribute to a better understanding of the cellular processes involved in the transcriptome and help us to discover new components from spider venom glands with therapeutic potential.

Fig. 1

Relative proportion of the unique sequences (14 contigs and 30 singlets) of the transcripts from cDNA venom gland library from C. crawshayi. a Frequency distribution of the different EST categories and the corresponding gene clusters. A total of 282 ESTs were analyzed in the current study, of which 236 ESTs according to their specific category are shown. “CellPro” includes transcripts coding for proteins involved in cellular processes (41%). “Toxin-like peptides” includes sequences with high identity to toxin families (25%). “Putative toxins” includes sequences rich in cysteine with identity to toxins or to the ICK-motif (7%). “Unknown function” includes ESTs that presented identity with already described sequences with no functional assessment or hypothetical genes (9%). “No ORF” includes sequences with non-identified open reading frame (16%). “No match” includes ESTs that did not match with currently known sequences (2%). b Proportion of categories and their corresponding number of genes, clones and EST values. “CellPro” category has been divided in different subcategories of functional genes

Fig. 2

Multiple alignments of predicted mature sequences of toxin-like peptides from cDNA library from venom gland of Ci tharischius c rawshayi (Cic). Accession number and name of the putative ICK peptides motif with three disulfide bridges or toxin-like with four disulfide bridges are included on the left with targets from experimental data reported (i.e., ion channels –ex vivo work–, or animal studies –in vivo work–); amino acid number and the percentage of identity with related toxins are shown on the right of the alignment. CaCh effect on calcium channel, KCh effect on potassium channel, TRPV effect on vanilloid receptors, NaCh effect on sodium channel, nAChR effect on nicotinic acetyl choline receptors, toxcric, peptide toxic to crickets; toxmice, peptide toxic to mice; toxfly, peptide toxic to house fly. Abbreviation pre, precursor sequence or amino acid sequence deduced from cDNA or gene. Sequence in bold corresponding to toxins or putative mature sequences. Lower cases in the amino acid sequences show sequence of pro-peptide that did not correspond to the mature peptide; sequences of mature peptides predicted are in bold; filled circles indicate post-translational modification of precursor for amidation of the last residue. Mature peptides were used to obtain theoretical mass data and compared with our mass results of Table 2. ICK and DDH-motif toxins group are indicated in red, conforming of cysteine residues are designed by roman numerals [32]

Fig. 3

Chromatogram of venom fractionation of C. crawshayi by HPLC. a Soluble venom (1 mg protein) was separated in a C18 analytic reverse-phase column, eluted with a linear gradient from 2% solution A (0.12% TFA in water) to 60% solution B (0.10% TFA in acetonitrile) run at a flow rate of 0.5 ml/min, for 70 min. Components labeled with an asterisk were selected for a second separation of fractionation. Panels b, c, and d show examples of some fractions selected and their second fractionation. e Relative abundance of venom mass peptides (frequency/mass)

Fig. 4

Electrophysiological effects of total venom on different voltage-gated ion channels expressed in Xenopus oocytes. Panels show the traces of the effect of total venom in currents of oocytes expressing the Na_v1.4, Na_v1.5 Na_v1.6, DmNa_v1 (Drosophila melanogaster voltage-gated sodium channel), Shaker IR and K_v1.5. The dotted line indicates the zero-current level. The asterisks represent the effect of venom at 20 μg