Profiling the resting venom gland of the scorpion Tityus stigmurus through a transcriptomic survey

Abstract

Background: The scorpion Tityus stigmurus is widely distributed in Northeastern Brazil and known to cause severe human envenoming, inducing pain, hyposthesia, edema, erythema, paresthesia, headaches and vomiting. The present study uses a transcriptomic approach to characterize the gene expression profile from the non-stimulated venom gland of Tityus stigmurus scorpion.

Results: A cDNA library was constructed and 540 clones were sequenced and grouped into 153 clusters, with one or more ESTs (expressed sequence tags). Forty-one percent of ESTs belong to recognized toxin-coding sequences, with transcripts encoding antimicrobial toxins (AMP-like) being the most abundant, followed by alfa KTx- like, beta KTx-like, beta NaTx-like and alfa NaTx-like. Our analysis indicated that 34% of the transcripts encode "other possible venom molecules", which correspond to anionic peptides, hypothetical secreted peptides, metalloproteinases, cystein-rich peptides and lectins. Fifteen percent of ESTs are similar to cellular transcripts. Sequences without good matches corresponded to 11%.

Conclusions: This investigation provides the first global view of gene expression of the venom gland from Tityus stigmurus under resting conditions. This approach enables characterization of a large number of venom gland component molecules, which belong either to known or non yet described types of venom peptides and proteins from the Buthidae family.

Figure 1

Length distribution ofT. stigmurusvenom gland ESTs. A total of 540 clones were analyzed. Abscissa is the length of sequences in 50 bp intervals, whereas the total number of ESTs for each interval is shown in the Y-coordinate. The average length of ESTs was 441 pb.

Figure 2

Functional classification of transcripts fromTityus stigmurusvenom glands. Graph showing the relative proportion of different types of transcripts: ‘Known Toxins’, ‘Other possible venom molecules’, ‘Cellular Proteins’, and no-match sequences (‘No Hit’) [A].

Figure 3

Functional classification of the “Known toxins” and “Other possible venom molecules” categories by relative abundance, considering only these categories. Acronyms are designated as follows: AK, alfa KTX; BK, beta KTX; AN, alfa NaTX; BN, beta NaTX; AM, Antimicrobial peptides; HT, Hypotensins; LT, Lectins; MP, Metalloproteases; AN, Anionic peptides; HS, Hipothetical secreted peptides; CR, Cystein-rich peptides. The blue and gray bars show the classification groups according to EST and cluster abundance, respectively.

Figure 4

Alignment of the amino acid sequence for TSTI0109S, fromT. stigmurusvenom glands, with known potassium channel toxins (α-KTX). Residues are numbered according to the aligned potassium channel toxins (α-KTX) sequences and dots represent gaps introduced to improve alignment. The putative signal peptide is omitted. Conserved cystein residues are indicated by asterisks. The key lysine for blocking activity is detached by a cross. The CXXXC and CXC motifs conserved among scorpion neurotoxins are underlined. Green, red and gray indicate amino acids that are identical, conserved or similar, respectively. The abbreviation and GenBank accession number for the aligned potassium channel toxins sequences are: TsPep2, Tityus serrulatus potassium channel toxin (P0C175); Tst-17, Tityus stigmurus potassium channel toxin (P0C8X6).

Figure 5

Alignment of the amino acid sequence for TSTI0003C, fromT. stigmurusvenom glands, with known potassium channel toxins (β-KTX). Residues are numbered according to the aligned potassium channel toxins (β-KTX) sequences and dots represent gaps introduced to improve alignment. The putative signal peptide is omitted. Conserved cystein residues are indicated by asterisks. The CXXXC and CXC motifs conserved among scorpion neurotoxins are underlined. Green, red and gray indicate amino acids that are identical, conserved or similar, respectively. The abbreviation and GenBank accession number for the aligned potassium channel toxins sequences are: TtrKIK, Tityus trivitattus potassium channel toxin (Q0GY45); TcoKIK, Tityus costatus potassium channel toxin (Q0GY42); TdiKIK, Tityus discrepans potassium channel toxin (Q0GY43) and BmTXKβ, Mesobuthus marteensi potassium channel toxin (Q9NJC6) [A]. Dendogram of β-KTX peptides sequences from scorpion venoms [B].

Figure 6

Alignment of the amino acid partial sequence for TSTI0056S, fromT. stigmurusvenom glands, with known sodium channel toxins (β-NaTX). Residues are numbered according to the aligned sodium channel toxins (β-NaTX) sequences and dots represent gaps introduced to improve alignment. The putative signal peptide is omitted. Conserved cystein residues are indicated by asterisks. Green, red and gray indicate amino acids that are identical, conserved or similar, respectively. The abbreviation and GenBank accession number for the aligned sodium channel toxins sequences are: Tpa8, Tityus pachyurus, sodium channel toxin (CCD31437); IsomTx2, Isometrus vittatus, potassium channel toxin (P0C5H2).

Figure 7

Alignment of the amino acid sequence for TSTI0006C, fromT. stigmurusvenom glands, with known hypotensins. Residues are numbered according to the aligned hypotensin sequences and dots represent gaps introduced to improve alignment. The underlined amino acid sequence indicates the putative signal peptide. Crosses indicate the C-terminal region probably involved in the hypotension effect. The similar BPP amino acid signature represented by a proline doublet is indicated by arrows. Pyr is the N-terminal pyroglutamic acid residue typical of snake BPPs. Green, red and gray indicate amino acids that are identical, conserved or similar, respectively. The abbreviation and GenBank accession number for the aligned hypotensin sequences are: Hypotensin-I, Tityus serrulatus (P84189), Hypotensin-II, Tityus serrulatus (P84190), BPP, Lachesis muta (ABD52884) and BPP, Bothrops jararaca (AAD51326).

Figure 8

Alignment of the amino acid sequence for TSTI0001C, fromT. stigmurusvenom glands, with known sequences of antimicrobial peptides. Residues are numbered according to the aligned antimicrobial peptide sequences and dots represent gaps introduced to improve alignment. The underlined amino acids indicate the putative signal peptide. A possibly promature peptide is shown in yellow font and the putative post-translational signal GRR is in italics. Green, red and gray indicate amino acids that are identical, conserved or similar, respectively. The abbreviation and GenBank accession number for aligned antimicrobial peptide sequences are: Tityus costatus antimicrobial peptide (Q5G8B3), Mucroporin, Lychas mucronatus (B9UIY3), Bmkb1, Mesobuthus martensii (Q718F4) and Caerin-2 Mesobuthus eupeus (ABL68083).

Figure 9

Alignment of the amino acid sequence for TSTI0012C, fromT. stigmurusvenom glands, with known sequences of anionic peptides. Residues are numbered according to aligned anionic peptides sequences and dots represent gaps introduced to improve alignment. The underlined amino acids indicate the putative signal peptide. Conserved acidic residues (D and E) are indicated by asterisks. Green, red and gray indicate amino acids that are identical, conserved or similar, respectively. The abbreviation and GenBank accession number for aligned anionic peptide sequences are: Tityus costatus anionic peptide (Q5G8B1), Tityus discrepans anionic peptide (C9X4J1), Tityus martensii anionic peptide (Q8N0N8).

Figure 10

Alignment of the amino acid sequence for TSTI0052S, fromT. stigmurusvenom glands, with known sequences of IGFBP (insulin-like growth factor-binding protein). Residues are numbered according to aligned IGFBP sequences and dots represent gaps introduced to improve alignment. The underlined amino acids indicate the putative signal peptide. Conserved cystein residues are indicated by asterisks. The IGFBP domain is highlighted by a box. Green, red and gray indicate amino acids that are identical, conserved or similar, respectively. The GenBank accession number for aligned IGFBP sequences are: Lychas mucronatus (P0CJ14), Mesobuthus eupeus (CAY61895), Tityus discrepans (ABR21044), Acromyrmex echinatior (EGI69650).

Figure 11

Relative abundance of ESTs coding for cellular proteins. Acronyms are designated as follows: SP, Structural proteins; CM, Cell Metabolism; PS, Processing and Sorting; CR, Cell Regulation; TT, Transcription and Translation; OF, Other Functions; UF, Unknown Functions. The blue and gray bars show the classification groups relative to EST and Cluster abundance, respectively.