Unveiling the Protein Components of the Secretory-Venom Gland and Venom of the Scorpion Centruroides possanii (Buthidae) through Omic Technologies

Abstract

Centruroides possanii is a recently discovered species of "striped scorpion" found in Mexico. Certain species of Centruroides are known to be toxic to mammals, leading to numerous cases of human intoxications in the country. Venom components are thought to possess therapeutic potential and/or biotechnological applications. Hence, obtaining and analyzing the secretory gland transcriptome and venom proteome of C. possanii is relevant, and that is what is described in this communication. Since this is a newly described species, first, its LD₅₀ to mice was determined and estimated to be 659 ng/g mouse weight. Using RNA extracted from this species and preparing their corresponding cDNA fragments, a transcriptome analysis was obtained on a Genome Analyzer (Illumina) using the 76-base pair-end sequencing protocol. Via high-throughput sequencing, 19,158,736 reads were obtained and ensembled in 835,204 sequences. Of them, 28,399 transcripts were annotated with Pfam. A total of 244 complete transcripts were identified in the transcriptome of C. possanii. Of these, 109 sequences showed identity to toxins that act on ion channels, 47 enzymes, 17 protease inhibitors (PINs), 11 defense peptides (HDPs), and 60 in other components. In addition, a sample of the soluble venom obtained from this scorpion was analyzed using an Orbitrap Velos apparatus, which allowed for identification by liquid chromatography followed by mass spectrometry (LC-MS/MS) of 70 peptides and proteins: 23 toxins, 27 enzymes, 6 PINs, 3 HDPs, and 11 other components. Until now, this work has the highest number of scorpion venom components identified through omics technologies. The main novel findings described here were analyzed in comparison with the known data from the literature, and this process permitted some new insights in this field.

Keywords: Centruroides possanii; next generation sequencing; omics technologies; proteome; scorpion; transcriptome; venom; venom gland.

Figure 1

Distribution and morphology of the scorpion Centruroides possanii. (A) The orange dot indicates the geographical distribution and collection area of the scorpion C. possanii in Minatitlan, Colima, Mexico. (B) A female specimen of scorpion C. possanii. The scale bar 1:1 represents that 1 cm in the image equals 1 cm in reality.

Figure 2

RNA analysis using Bioanalyzer 2100 (Agilent). The left image shows the representative electropherogram of the total RNA extracted from the venom-producing gland of the scorpion C. possanii. The separation of the 18S rRNA fragment in the gel is shown in the right image. The dotter points indicated in green refer to the internal standard of the equipment.

Figure 3

Alignment of some alpha toxins that act on sodium channels. The signal peptide is shown in gray and italics, and the mature peptide is in bold. Dots indicate identical amino acids. In blue is the identical pattern of the eight cysteines forming the disulfide bridges (positions 31–83, 35–56, 42–66, and 46–68). In yellow are the hydrophobic residues essential for their function on sodium channels; in cyan the variable region of the domain functional amino-carboxyl “NC”; and in pink, the amino acids of the functional “core” domain. Percent identity (% ID) is shown for mature peptides only. The amidation signal is represented in red color (position 85).

Figure 4

Alignment of beta toxins that act on sodium channels. The signal peptide shown is in italics and a gray color, and the mature peptide is in bold. Dots indicate identical amino acids. The identity percentage (% ID) refers only to the region of mature peptides. Cysteines (blue) present a conserved pattern formed by disulfide bridges at positions 31–84, 35–60, 44–65, and 48–67. Glutamic acid of position 35 (E35; green) is essential for binding β-NaTxs with the Nav channel. The crucial residues for the activity are shown in magenta, and the amidation and cut signals are in red.

Figure 5

Alignment of alpha toxins that act on potassium ion channels. The signal peptide is shown in gray and italics. The underlined region represents the propeptide, the mature peptide is shown in bold, and in blue are the cysteines that form the disulfide bridges (positions 31–50, 35–55, and 40–57). Dots indicate identical amino acids. Amidation (G) and cleavage (K) signals are shown in red. The identity percentage (% ID) takes into account only mature peptides. The green shaded regions indicate the amino acids that form the functional dyad (K and Y) and interact with the potassium channel.

Figure 6

Alignment of gamma toxins acting on potassium ion channels. The signal peptide is shown in a gray color and italics. The mature peptide is shown in bold, and the cysteines forming the disulfide bridges are blue. Dots indicate identical amino acids. Highlighted in magenta is the amino acid involved with the binding site of the channel. The identity percentage (% ID) was estimated only for mature peptides.

Figure 7

Alignment of some group 1 delta toxins acting on potassium channels. The signal peptide is shown in gray and italics, the mature peptide in bold, and the cysteines forming the disulfide bridges in blue. Dots indicate identical amino acids. In yellow, the amino acids Lys³⁶ and Ala³⁷ of the BmKTT-2 toxin are essential for function. The identity percentage (% ID) took into consideration only mature peptides.

Figure 8

Alignment of group 2 delta toxins acting on potassium ion channels. The signal peptide is shown in gray and italics, the mature peptide in bold, and the cysteines forming the disulfide bridges in blue. Dots indicate identical amino acids. In yellow is the amino acid Lys³⁵ of the LmKTT-1a toxin, which interacts directly with trypsin. The identity percentage (% ID) considered only the region of mature peptides.

Figure 9

Alignment of Host Defense Peptides of the Defensin type. In italics are the amino acids of the signal peptide, in bold are the mature peptides, and dots indicate identical amino acids. The conserved cysteine pattern is in blue, and the amino acids indicate cleavage (RK) in red. The percentages of identity (% ID) between sequences correspond to the mature peptide.