Transcriptome analysis of the venom gland of the Mexican scorpion Hadrurus gertschi (Arachnida: Scorpiones)

Abstract

Background: Scorpions like other venomous animals possess a highly specialized organ that produces, secretes and disposes the venom components. In these animals, the last postabdominal segment, named telson, contains a pair of venomous glands connected to the stinger. The isolation of numerous scorpion toxins, along with cDNA-based gene cloning and, more recently, proteomic analyses have provided us with a large collection of venom components sequences. However, all of them are secreted, or at least are predicted to be secretable gene products. Therefore very little is known about the cellular processes that normally take place inside the glands for production of the venom mixture. To gain insights into the scorpion venom gland biology, we have decided to perform a transcriptomic analysis by constructing a cDNA library and conducting a random sequencing screening of the transcripts.

Results: From the cDNA library prepared from a single venom gland of the scorpion Hadrurus gertschi, 160 expressed sequence tags (ESTs) were analyzed. These transcripts were further clustered into 68 unique sequences (20 contigs and 48 singlets), with an average length of 919 bp. Half of the ESTs can be confidentially assigned as homologues of annotated gene products. Annotation of these ESTs, with the aid of Gene Ontology terms and homology to eukaryotic orthologous groups, reveals some cellular processes important for venom gland function; including high protein synthesis, tuned posttranslational processing and trafficking. Nonetheless, the main group of the identified gene products includes ESTs similar to known scorpion toxins or other previously characterized scorpion venom components, which account for nearly 60% of the identified proteins.

Conclusion: To the best of our knowledge this report contains the first transcriptome analysis of genes transcribed by the venomous gland of a scorpion. The data were obtained for the species Hadrurus gertschi, belonging to the family Caraboctonidae. One hundred and sixty ESTs were analyzed, showing enrichment in genes that encode for products similar to known venom components, but also provides the first sketch of cellular components, molecular functions, biological processes and some unique sequences of the scorpion venom gland.

Figure 1

Reads length distribution of H. gertschi venom gland ESTs. A total of 147 ESTs were analyzed in the current study. Abscissa is the length of sequences in 50 bp intervals, whereas the total number of ESTs for each cluster is shown in the Y-coordinate.

Figure 2

Relative proportion of each category of the transcripts from H. gertschi venom gland library. A) Relative proportion of each category of the 147 total transcripts from H. gertschi venom gland. B) Relative proportion of the unique sequences (20 contigs and 48 singlets). "Unknown function" includes ESTs that presented identity with already described sequences with no functional assessment. "NoORF" includes sequences with non identified open reading frame. "No match" includes ESTs that did not match with currently known sequences. "GO-sorted" includes transcripts coding for proteins involved in cellular processes. "α and β-KTx" transcripts encode for putative K⁺ toxins from α and β-families, respectively. "NDBP" comprises non-disulfide-bridged peptides. "Other venom components" includes both H. gertschi PLA2 and the Kunitz-type serine proteinase inhibitor.

Figure 3

Gene Ontology-sorted sequence annotation. Functional classification of all nr-matched transcripts from the H. gertschi venom gland. The vertical axis shows the relative proportion of ESTs. The abscissa shows the categories within each of three ontologies: cellular component, molecular function and biological processes. For comparison, the relative proportion of toxin-like ESTs is also shown. All toxin-like sequences were assigned to the special set of the "biological process" ontology called "multi-organism process" (GO:0051704).

Figure 4

Scorpion toxin-like precursors in H. gertschi venom gland library. A) Predicted amino acid sequences of the potential α-KTx. HGE024|Contig2 predicted sequence is aligned with all members of the α-KTx 6 subfamily. HGE025|Contig5 is aligned with anuroctoxin (α-KTx 6.12). PSI-BLAST e-values for the third iteration are shown. B) Predicted amino acid sequence of Hg scorpine like 2 and its alignment with others members of the scorpine-like group. The percentage of identity with scorpine is shown. See Supplementary Figure 1 for the complete nucleotide sequences of HGE024|Contig2, HGE025|Contig5 and Hg scorpine like 2. Each sequence starts with its SwissProt accession number followed by common names and Protein Data Bank codes between parentheses (where available). Systematic numbering (sensu [47,49]) for α-KTx is included between accession numbers and common names. Identical amino acids are in red colour and conserved ones in green.

Figure 5

Predicted amino acid sequences of the novel non-disulfide-bridged peptides (NDBP). A) NDBP-5.5 and NDBP-5.6 are aligned with others scorpion cytolytic peptides; the percentage of identity with IsCT is shown. Putative signal peptides are in italics, whereas identified C-terminal prosequences and mature forms are underlined or in bold characters, respectively. B) Alignment of NDBP-3.7 with members of the NDBP 3 subfamily. See Supplementary Figure 2 for the complete nucleotide sequences encoding for NDBP-5.5, NDBP-5.6 and NDBP-3.7. Each sequence starts with its SwissProt accession number followed by common names. Identical amino acids are in red colour and conserved ones in green.

Figure 6

Putative mature sequence of phospholipase A2 precursor. Predicted amino acid sequence of H. gertschi PLA2 (HGE031|PLA2) aligned with other scorpion venom PLA2. BLAST e-values are shown. See Supplementary Figure 3 for the nucleotide sequence of HGE031|PLA2. Each sequence starts with its SwissProt accession number followed by common names. Identical amino acids are in red colour and conserved ones in green.

Figure 7

Multiple sequence alignment of the KU-type proteins of venomous organisms. Predicted amino acid sequence of HGE030|Hg1 aligned with other venom-derived members of the Kunitz-type serine proteinase inhibitors. BLAST e-values with P68425 (spider Ornithoctonus huwena), P31713 (sea anemone Stichodactyla helianthus), P00981 (snake Dendroaspis polylepis polylepis) and P0C1X2 (cone snail Conus striatus) are shown. See Supplementary Figure 4 for the complete nucleotide sequence of HGE030|Hg1. Each sequence starts with its SwissProt accession number followed by common names. Identical amino acids are in red colour and conserved ones in green.