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. 2025 May 28;24(6):2906–2915. doi: 10.1021/acs.jproteome.5c00125

Transcriptomics-Informed Proteomics of Venom Glands and Crude Venom from Tityus cf. asthenes from Panama: Enzymes, Proteins, Toxins, and Antimicrobial Peptides

Marcos H Salazar †,‡,*, Octavio Samudio †,‡, Iván Arenas §, Magdalena Hernández-Ortiz ∥, Herlinda Clement §, Lorena Hernandez-Orihuela §, Sergio Encarnación-Guevara ∥, John Cleghorn †, Hildaura Acosta †, Gerardo Corzo §,*
PMCID: PMC12151069  PMID: 40435298

Abstract

The venom gland and the crude venom of Tityus cf. asthenes were analyzed by transcriptomic and proteomic methods. High-throughput RNA sequencing (RNA-seq) and mass spectrometry (MS/MS) allowed for the identification of a wide range of venom proteins. Notably, transcriptomic and proteomic venom approaches identified a hyaluronidase, toxins that affect the voltage-gated Na+ channels, LVPs, cysteine-rich secretory proteins, metalloproteinases, serine proteases, alpha-amylases, toxins that affect the voltage-gated K+ channel, scorpion calcin-like, single insulin-like growth factor-binding domain proteins, nondisulfide-bridged peptides, lectins, chitinases, Kunitz-type serine protease inhibitors, cyclotide trypsin inhibitors, and uncharacterized proteins. These newly identified components enhance the understanding of the venomous nature of Tityus cf. asthenes.

Keywords: Tityus, proteome, transcriptome, scorpion, venom, venom gland

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1. Introduction

Scorpions of the genus Tityus in Panama, including Tityus asthenes, Tityus jaimei, Tityus festae, Tityus championi, and Tityus cerroazul, are known to cause human envenomation, and deaths have been reported. T. asthenes was initially classified under the subgenus Atreus and the family Buthidae. Its distribution includes areas in Costa Rica (Puntarenas), Panama, Colombia, Ecuador, and Peru. Lourenço (1988) placed those scorpion species as a lower synonym of Tityus asthenes. Recent studies have confirmed that T. asthenes does not belong to the subgenus Atreus according to a revised classification within the subgenus Tityus, which encompasses species with very localized distributions that are influenced by environmental factors. For example, some translated coded sequences (TCDS) from the related species T. asthenes from Colombia showed less than a 10% match in the family of Na+ and K+ toxin TCDSs compared to those TCDSs from T. asthenes in Panama analyzed in this study. As a result, T. asthenes from Panama will be named T. cf. asthenes until the species name is confirmed. Furthermore, in a previous work, Salazar et al. (2018) examined the venoms of four species of the genus Tityus from Panama. The authors reported that five protein fractions (identified through HPLC) from the venom of T. asthenes (now T. cf. asthenes) exhibited moderate to lethal activity in mammals. The neurotoxin fractions exhibited the following average peptide molecular masses: fraction 27 (7332.9 Da), 28 (6522.2 and 7101.5 Da), 29 (7399.9 Da), 30 (7400.0 and 7760.0 Da), and 31 (7098.0 Da). The toxins with average molecular masses of 7332.9 and 7101.5 Da were isolated from the venom of T. jaimei (sequence IDs: C0HLZ0 and C0HLZ1, respectively), expressed recombinantly, and used as immunogens to obtain neutralizing antibodies against the venoms of Tityus from Panamá.

To enhance the identification of primary structures of new neurotoxins, enzymes, and other protein components, such as antimicrobials found in the venom of T. cf. asthenes or other toxins that may be considered for their pharmaceutical properties, we performed transcriptomic and proteomic analyses on the venom gland and crude venom, respectively, to characterize the venom components of this species. Also, we have compared the transcriptome and proteome of T. cf. asthenes from Panama with the transcriptome of T. asthenes from Buenaventura, Colombia. Although the transcriptome of T. asthenes species from Buenaventura, Colombia, is not available in current databases, interesting results were obtained.

2. Material and Methods

2.1. Venom and Venom Glands

Tityus cf. asthenes venom and its venom glands were provided by the Centro de Investigación e Información en Medicamentos y Tóxicos de la Universidad de Panamá (CIIMET-UP). Specimens were kept individually in optimal health conditions using plastic cages at a constant temperature of 27 °C. Animals were fed with crickets and tap water ad libitum. A venom pool from 16 scorpions (female) was extracted manually by placing a sterile watch glass covered with parafilm near the scorpion’s stinger to induce venom release, vacuum-dried, and stored at −20 °C until needed. For transcriptomic analysis, venom glands (24 h after venom extraction) were dissected from healthy individuals (six female scorpions), treated with RNAlater (Thermofisher, Asheville, NC, USA), and stored at −70 °C until use. Figure A shows the lateral and frontal images of Tityus cf. asthenes from Palmas Bellas, Colón, Panamá, and Figure B shows the collection site of the scorpion Tityus cf. asthenes in Panamá and the site of T. asthenes collection from Colombia.

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Tityus cf. asthenes. (A) Photos by John Cleghorn of Tityus cf. asthenes and (B) Palmas Bellas, site of specimens’ collection in Panama, and Buenaventura, site of specimens’ collection in Colombia (modified from https://commons.m.wikimedia.org/wiki/File:Buenaventura_in_New_Granada_(1855).svg#mw-jump-to-license).

2.2. Protein Determination

The absorbance at 280 nm was used with one absorbance unit at this wavelength equivalent to 1 mg/mL of protein.

2.3. Biological Activities

2.3.1. Antimicrobial Activity

The antimicrobial activity of the venom peptide fractions was evaluated by measuring the suppression of bacterial growth after applying the chromatographic fractions, which were redissolved in distilled water. A preinoculum of E. coli (Gram-negative) was cultured in LB medium for 18 h at 37 °C with shaking at 180 rpm. After incubation, the optical density (OD) of the culture was measured and subsequently diluted in the same medium to reach an ideal OD value of 0.08. A 1 mL aliquot was then diluted 1:10 in warm (approximately 45 °C) growth medium containing 1.5% (w/v) agar, and it was poured into sterile Petri dishes. Depending on the strain, the bacterial count poured was approximately 106–107 colony-forming units/mL. In parallel, vacuum-dried reverse phase high-performance liquid chromatography (RP-HPLC) fractions were redissolved in 20 μL of distilled water, and 3–5 μL containing 10–20 μg of protein was applied onto the plate surface. Petri dishes were incubated at 37 °C for 12–14 h, and growth inhibition was detected as clear spots on the plate surface. Distilled water was used as a negative control, and 0.5 μL of ampicillin (10 mg/mL) was used as a positive control.

2.4. Mass Spectrometry

For the MS/MS analysis, two venom samples (40 μg each) were solubilized in 50 mM ammonium bicarbonate with 0.5% sodium deoxycholate (SDC). Cysteine reduction and alkylation reactions were performed on both samples. Protein reduction was carried out with 10 mM DTT at 56 °C, followed by alkylation using 40 mM iodoacetamide in the dark at room temperature, with both incubation steps lasting 30 min each. One venom sample was treated with chymotrypsin and the other with trypsin at 1:50 (enzyme/substrate) each. The samples were incubated for 16 h at 37 °C. SDC was removed through ethyl acetate extraction under acidic conditions; specifically, 1 volume of ethyl acetate was added to the sample and acidified with 0.5% trifluoroacetic acid (TFA). After vigorous vortexing and centrifugation, the organic phase was discarded. A second ethyl acetate extraction was performed without TFA. The resulting peptide mixture was desalted using reverse-phase chromatography (ZipTip C18, Millipore, Billerica, MA, USA), then dried and stored at −80 °C until LC–MS/MS analysis. For the analysis, the digested fractions, either from trypsin or chymotrypsin, were redissolved in initial chromatographic conditions and separated on a Dionex Ultimate 3000 RSLCnano UPLC system, in line-coupled with a Q-Exactive Plus high-resolution mass spectrometer (Thermo Fischer Scientific). The mobile phases were: (A) 0.1% formic acid in water and (B) 90:10 (v/v) acetonitrile/water, 0.1% formic acid. Samples (0.5–1 μg) were first trapped on a precolumn (C18 PepMap 100, 5 μm, 100 A, 300 μm inner diameter × 5 mm) and then separated following a 60 min elution gradient using a capillary column at 250 nL/min (EASY Spray Column, PepMap RSLC, C18, 3 μm, 100 Å, 75 μm × 150 mm). The mass spectrometer was operated in positive data-dependent acquisition mode, with the full MS range set from 300 to 2000 m/z. The 10 most intense ions were isolated in the quadrupole and fragmented using high-energy collisional dissociation with a normalized collision energy of 27%. Precursor ions were measured at a resolution of 70,000 (at 200 m/z), and the fragments were estimated at 17,500. Only ions with charge states of 2 and higher were fragmented using an isolation window of 2 m/z. Mass spectra were analyzed using an internal database generated from protein sequences previously obtained from the transcriptomic analyses of T. cf. asthenes from Panama using the MaxQuant program. The mass spectrometry data (both raw files and interpreted files) were deposited in the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org) via the iProX partner repository with the data set identifier PXD058957.

2.5. RNA Extraction and High-Throughput Sequencing from Tityus cf. asthenes

The mRNA of six pairs of venom glands from T. cf. asthenes (female) telsons from Palmas Bellas-Colón-Panama was extracted using an RNAeasy mini kit (Qiagen Inc., Germantown, MD) and the SV Total RNA Isolation System (Promega Co., Madison, WI). RNA quantification was performed using a Nanodrop 1000 (Thermo Scientific) instrument, and its integrity was confirmed using a Bioanalyzer 2100 (Agilent Technologies). A complementary DNA library was constructed from 1 μg of total RNA using the TruSeq Stranded mRNA Sample Preparation Kit, following the supplier’s protocol. Automated DNA sequencing was performed on a Genome Analyzer IIx instrument (Illumina). The resulting library included two FASTQ files (forward reads R1 and reverse reads R2), yielding paired-end reads, each 75 bp in length.

2.6. Bioinformatic Analysis

The T. cf. asthenes transcriptome raw reads were deposited in the Sequence Read Archive (SRAPRJNA1202649. BioSample accession SAMN45962855) under the file names tasthenesR1 (fastq) and tasthenesR2 (fastq). The FastQC program was used to verify the quality of the data. To remove adapter sequences from the sequencing process and filter out low-quality sequences, the cutadapt and Trimmomatic programs were employed. TCDS reconstruction was performed using the Trinity program, with a minimum contig length of 80 bp and all other default settings. The TransDecoder program was then used to identify and extract open reading frame sequences. The command -m 50 was specified to report only the ORFs that met the minimum protein length requirement. Translated TCDSs were annotated using the Blast2GO program. Unless specified otherwise, all programs were run with the default parameters.

3. Results and Discussion

3.1. Transcriptome

An average of 9,703,835 raw paired-end reads were gained from the venom glands of T. cf. asthenes from Panamá. The quality of these raw reads was assessed using the FastQC program. After filtering the raw reads to remove the adapters and the low-quality sequences, a total of 8, 927,621 clean reads remained. The clean reads were assembled into 71,583 contigs using the Trinity program. TransDecoder v3.0.1 extracted all predicted TCDSs of the venom gland transcriptome from T. cf. asthenes, 26,089 TCDSs were obtained and annotated using Blast2GO program. To identify the possible components within the venom gland, BLASTp analyses with an e value of e–10 were performed using the VenomZone database (https://venomzone.expasy.org/), which led to the identification of 490 TCDSs. These reported TCDSs were categorized into 16 protein groups. Figure shows the percentages of each protein group, and the TCDSs are listed in Table S1. This study provides the first comprehensive analysis of the mRNA expression profiles in adult scorpion venom glands from T. cf. asthenes.

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Distribution of TCDSs found in the venom gland of T. cf. asthenes.

3.2. Proteome

For the proteomic analysis of the T. cf. asthenes venom, the Maxquant 2.4.14.0 program was used. As mentioned, venom samples were digested with chymotrypsin and trypsin after protein reduction and alkylation. For each identification, a minimum of two unique peptides were considered and selected under the statistical parameters of the MaxQuant program. Table S2 shows the MS/MS sequences of the digested peptides that match 68 annotated proteins using different protein databases, including the complete genome of Centruroides sculpturatus, the VenomZone, and our own annotated T. cf. asthenes transcriptome (this work).

The proteomic analysis utilizing our annotated T. cf. asthenes transcriptome improved significantly after removing the signal peptide of the TCDSs by using SignalP 5.0. Consequently, the percentage of coverage of the peptides and proteins identified by the MaxQuant program increased. Table S2 shows the digested peptides of sixty-eight venom proteins that match sixty-four TCDSs (Table S3) based on the scoring parameters of the MaxQuant program (Figure ).

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Distribution of proteins found in the venom proteome of T. cf. asthenes.

3.3. Transcriptome–Proteome Correlation

The venom gland transcriptome revealed a total of 490 TCDS (Table S1), of which 64 correspond to the amino acid sequences of the tryptic peptides found in the venom. The transcriptome–proteome correlation revealed the presence of several proteins, including a hyaluronidase (1), toxins that affect the voltage-gated Na+ channel (17), LVP (2), cysteine-rich secretory protein (CRISP) (3), metalloproteinase (9), serine protease (1), alpha-amylase (1), toxins that affect the voltage-gated K+ channel (10), scorpion calcin-like proteins (1), single insulin-like growth factor-binding domain protein (1), nondisulfide peptides (1), lectin (1), Chitinase (1), Kunitz-type serine protease inhibitor (1), cyclotide trypsin inhibitor (1), and uncharacterized proteins (11). Such proteins have a strong identity to venom proteins from Tityus cisandinus, Tityus discrepans, T. festae, T. jaimei, T. perijanensis, and previously reported toxins from T. asthenes. Yet, amylase, lectin, chitinases, and some uncharacterized proteins were more identical to proteins from other scorpion venom genera, such as Centruroides, Mesobuthus eupeus, and Odontobuthus doriae.

3.3.1. Hyaluronidase

Only one TCDS of Hyaluronidase (TaPBP-Hyal-01) was found, and its presence was confirmed through proteomic analysis. This sequence shows 95.8% identity with the hyaluronidase Tcis_Hyal1-like sequence from T. cisandinus (ID: WDU65909.1) (Table S3).

The venom of T. cf. asthenes showed hyaluronidase-positive bands in a zymogram assay. The theoretical molecular mass of the hyaluronidase TCDS is 46,626.8 Da, which matches the apparent experimental molecular mass of the observed hyaluronidase activity (Figure S1A).

3.3.2. Toxins That Affect the Voltage-Gated Na+ Channel

Forty-one TCDSs and 17 toxins that affect voltage-gated Na+ channels were found. They share identities with toxins from T. cisandinus (7), T. discrepans (4), T. festae (1), T. pachyurus (1), T. perijanensis (1), and previously reported toxins from T. asthenes­(3) (Table S3).

The theoretical molecular masses of peptides in fractions 27, 28, and 31 matched the molecular masses. Table compares the experimental average molecular masses of peptide fractions from this venom, as reported by Salazar et al. (2018), with the primary structures found in the transcriptome or proteome.

1. Neurotoxin TCDSs That Matched Venom Proteins Previously Found.

3.3.2.

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a

Protein fractions from Salazar et al. (2018).

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Letters in italics represent amino acids that are enzymatically cleaved to release the mature peptide. Letters in bold or underlined represent amino acid sequences of tryptic peptides found by MS/MS spectrometry (Table S2).

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Theoretical and experimental average molecular masses.

3.3.3. LVP

Only two LVP TCDSs, TaPBP_LVP01 and TaPBP_LVP02, were identified in the proteome analysis, matching an identity of 92.7 and 95.3%, respectively, to an LVP from the venom of T. cisandinus (WDU65901.1) (Table S3). These LVPs were exclusively found in the transcriptome of the venom from T. cisandinus, and only one corresponds to an experimental average molecular mass. LVPs have been detected in the transcriptome of several scorpions, including T. serrulatus (QPD99022), Centruroides hentzi (MBW20167), C. sculpturatus (XP_023227915.1), Hottentotta zagrosensis, and Hottentotta saulcyi, and recently identified in the transcriptome and proteome of Centruroides bicolor (Samudio et al., 2025). Zhu and Gao reported that in the venom gland of Buthus martensii, LVP α- and β-subunits contain seven cysteines forming three intramolecular disulfides and one intermolecular disulfide. Therefore, LVP could form either a homodimer or a heterodimer. Although the target of LVPs in scorpion prey remains unidentified, it may be related to stimulation of lipolysis in an insect hemolymph, potentially facilitating prey digestion.

3.3.4. CRISP

Twelve TCDSs of CRISPs were found, and only three they were corroborated by proteomic analysis (Table S3): two of share amino acid identities with CRISPs from C. sculpturatus and one with a CRISP from T. serrulatus. These CRISPs are distinct from the original ones identified in mammals and snakes, which typically have molecular weights of 20–30 kDa and contain 16 Cys residues. The three TCDSs identified here have molecular weights of 42–44 kDa and from 7 to 9 pairs of cystines. One of them contains 14 Cys residues and is classified as PR1-like, while the two with 18 Cys residues could be classified as Allergens.

3.3.5. Metalloproteinases

A total of 137 TCDSs coding metalloproteinases were identified, and eight were correlated to their tryptic peptides (Table S3). All eight metalloproteinases had primary structure identities from 65 to 88% to metalloproteases from TCDSs from venom glands of T. serrulatus and T. cisandinus. Metalloproteinases have been found in several animal venoms from arachnids, reptiles, and insects. A conserved HEXXH motif is common in metalloproteinases to harbor a metallic ion to exert its catalytic function and generate, depending on the amount injected by a venomous animal, hemorrhages, edema, inflammation, hypotension, and/or necrosis. All eight identified TCDSs possess this HEXXH motif, although some are shorter and contain different numbers of pairs of disulfide bonds.

3.3.6. Serine Protease

Six TCDS that coded for serine protease were identified, and only one (TaPBP_SP01) correlated to the proteome (Table S3). This serine protease TCDS shows an identity (79%) to a serine protease from C. sculpturatus. Similar, serine protease TCDSs have also been reported in the transcriptome of Centruroides limpidus, C. possani, and C. hentzi. , Further investigation of the role of this protein family in venom composition and function would be worthwhile.

3.3.7. Alpha-Amylase

Only one alpha-amylase TCDS (TaPBP_AA01) was found in the transcriptomic analysis, and it correlated with the tryptic peptides from the venom (Table S3). This shares 88.7% identity with a similar TCDS from C. vittatus. Additionally, alpha-amylase TCDSs have been reported in the venom glands of C. limpidus, T. serrulatus, and Tityus obscurus. , Alpha-amylase may play a role in degrading components of the prey’s tissue, such as extracellular polysaccharides, thus facilitating the venom’s diffusion through the tissues. However, further research is needed to clarify the contribution of this protein family to the venom composition and its functional properties.

3.3.8. Toxins That Affect the Voltage-Gated K+ Channel

Sixty-five TCDSs related to the voltage-gated K+ channels were found in the transcriptome (Table S1); however, only ten were identified in the venom proteome (Table S3). Some of these TCDSs have distinct nucleotide sequences but encode the same amino acid sequences, suggesting the presence of transcriptional isoforms that converge into a single functional protein. Among the ten toxins affecting voltage-gated K+ channels, nine share identities with peptides from the venom of T. cisandinus (9) while one is related to that of C. limpidus.

Among the TCDSs found in the venom, eight have molecular masses of around 4 kDa, which are typical for potassium channel toxins. One TCDS, with the sequence KTx-04 (Table S1), has a molecular mass of 7370 Da and shares high identity with the potassium channel toxin TdiKIK (Q0GY43.1) from the scorpion T. discrepans, classified as belonging to the long-chain scorpion toxin family, which exhibits antibacterial and cytotoxic activities. Additionally, the sequence KTx_01 (Table S1), with a molecular mass of 7644 Da, shows 77.9% identity with the scorpine-like peptide precursor from Tityus costatus (AAW72464.1) and 75% identity with the Potassium channel toxin beta-KTx 1 from T. serrulatus (P69940.2). The latter has been reported to induce nociception and can decrease the mechanical nociceptive threshold (hyperalgesia). Further bioassays are required to identify the true pharmacological actions of these two sequences beyond sequence similarity. This will facilitate the evaluation of their potential for new antimicrobial, anticancer, or analgesic drugs to treat chronic or acute pain conditions.

3.3.9. Calcin-Like Family

Three TCDs were identified in the transcriptome (Table S1). But only one member of a scorpion calcin-like family was detected in the proteome (TaPBC_SC_01). This sequence is comparable to the putative CaTx Tcis32 found in the venom gland transcriptome of T. cisandinus (Table S3). Calcins have been identified in scorpion venoms of scorpion species from various families, including Vaejovidae, Euscorpiidae, Hemiscorpidae, Scorpionidae, and Buthidae. The proteins typically consist of 33–36 amino acids linked by three disulfide bridges; however, some are significantly longer and resemble sodium channel toxins (NaTxs). Moreover, most calcins are amphiphilic and highly alkaline, allowing them to penetrate cells. Notable examples include opicalcin, vejocalcin, hemicalcin, imperacalcin, hadrucalcin, maurocalcin, and urocalcin among others. Calcins bind ryanodine receptors (RyRs), which regulate intracellular Ca2+ and act as a secondary messenger in cells, being interesting structures for regulatory functions.

3.3.10. Insulin-Like Growth Factor-Binding Domain Protein

Five TCDSs were found in the transcriptome (Table S1) but only two single insulin-like growth factor-binding domain protein TCDSs in the proteomic analysis (Table S3). Insulin-like growth factors play a role in regulating insulin, which is a commonly coopted peptide for predatory venoms. It is perhaps used to deplete blood sugar during envenomation. Two single insulin-like growth factor-binding domain protein TCDSs found here share identities with those from T. discrepans (70.2%) and C. sculpturatus (66.6%).

3.3.11. Nondisulfide-Bridged Peptide Superfamily

Eleven TCDSs of nondisulfide-bridged peptide (NDPB) peptides were found in the transcriptome (Table S1), but only one (TaPBC_NBDP_01) was found in the proteome. It shares 88.3% identity with a putative hypotensin peptide (Tcis1, WDU65846) from T. cisandinus. Hypotensins lower blood pressure, induce vasodilation and bradycardia, or inhibit angiotensin-converting enzyme like antihypertensive drugs. Nonetheless, further studies are needed to demonstrate the biological activities of these peptides and their potential use as pharmaceuticals.

3.3.12. Lectin

Information regarding venom lectins is quite limited in the literature. In our study, we identified thirty-one TCDSs (Table S1) and only two TCDSs with correspondence to the proteome (TaPBC_Lectin_01 and TaPBC_Lectin_02) that code for lectins similar (78.4 and 74.3% of identity, respectively) to that lectin from the transcriptome of C. vittatus (Table S3). Most known venomous lectins originated from snakes (85.9%) and arachnids (82.8%). In scorpions, hypothetical lectin and lectin-like sequences have been registered for T. obscurus, Hottentotta judaicus (ADY9551, putative hemolectin), and Ixodes scapularis (B7P6 × 9). Within the genus Tityus, two lectin sequences were reported in Tityus stigmurus (Batista et al., 2007). Additionally, lectins have been described in the hemolymph and venom of Heterometrus granulomanus and Buthus occittanus, respectively. , Consequently, there is limited structural information regarding the primary and tertiary structure. Likewise, the nucleotide or amino acid sequences are generally unknown, hindering their recombinant production. Considering that no studies are focusing on the detection, isolation, and biochemical characterization of lectins from scorpion venom, our research focused on an endemic species from the department of Cundinamarca, Tityus macrochirus, to discover and isolate new lectins. These new lectins exhibited specificity toward lactose residues (β-D-Gal (1–4)-β-D-GlcNAc-O-R) and acetylated carbohydrates. It is worth noting that most lectins and lectin-like proteins have been found in transcriptomics studies, likely stemming from TCDSs expressed in hemolymph cells of the telson.

3.3.13. Chitinases

A Chitinase TCDS (TaPBC_Chitinase_01) domain was identified with 84 and 58% identity to a Chitinase domain from the venom glands of C. sculpturatus (XP 023243664) and C. vittatus (XP_067128006), respectively. Chitinases are hydrolytic enzymes that may be involved in the digestion of prey. However, there is limited evidence regarding their presence in scorpion venoms.

3.3.14. Kunitz-Type Serine Protease Inhibitors

Six TCDS Kunitz serine protease inhibitors were found in the transcriptome analysis, but only one (TaPBC_Kunitz_01) was detected in the proteomic analysis. This inhibitor demonstrated 53 and 52% identity to similar venom proteins from the scorpion O. doriae (ALX72370.1) and Lychas mucronatus (P0DJ46). An interesting structural feature in these proteins is the triplet of Tyr, which seems to be important for enzyme inhibition. It is well-known that the Tyr, Trp, and Phe amino acids are key structural residues for receptor recognition and transduction processes. Their noncovalent interactions stabilize specific three-dimensional structures that align with these hydrophobic receptors residues.

3.3.15. Cyclotide Trypsin Inhibitor

Recently, a trypsin inhibitor (A0A6P3CW73) from T. obscurus venom, which shares structural similarities with plant cyclotides, has been found to inhibit trypsin. Similarly, four TCDS of a cyclotide trypsin inhibitor were found in the venom gland of T. cf. asthenes; however, they all encode the same protein sequence (TaPBC_CTI_01). The trypsin inhibitors from T. obscurus and T. cf. asthenes share 97.1% identity.

3.3.16. Uncharacterized Proteins

The peptides and proteins present must play an important biological role in the venom of the scorpion T. cf. asthenes. A total of 12 uncharacterized protein sequences were identified. Seven of them show a high identity to a hypothetical protein reported in the venom gland of scorpion T. discrepans (CAY61861.1). These sequences contain six conserved cysteines and ten identical residues (Figure ). However, further studies are required to determine the biological activity and significance of these peptides in scorpion venom.

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Alignment of toxins found in the venom gland of T. cf asthenes. These sequences have high homology to the CAY61861 (CAY), an uncharacterized sequence of the scorpion T. discrepans.

An unknown sequence, designated TaPBC_Unk_09, shows 64.5% identity with the sequence of the uncharacterized protein LOC111613006 from the venom gland of scorpion C. sculpturatus (XP_023210067.1) and 63.8% identity with uncharacterized protein XP_067128461.1 from the venom gland of scorpion C. vittatus. Additionally, it shares 33.2% identity with adhesion G-protein XP_059082753.1 from the arthropod Tigriopus californicus. Using the web resource InterPro Classification of Protein Families (https://www.ebi.ac.uk/interpro/), it was identified that part of the TaPBC_Unk_09 sequence matches the latrophilin receptor-like protein, a member of the G-protein coupled receptor LN-TM7 family. Interestingly, no similar protein sequences have been reported in the Tityus genus. If this protein facilitates cell adhesion or interaction with cellular membrane components, it might play a role in the internalization of venom into cells; however, further studies are needed to confirm the protein family to which this sequence belongs and its role in the venom. Another sequence, TaPBC_Unk_08, matches the sequence ABR20114.1, identified as a venom toxin-like peptide from M. eupeus. This peptide has been reported in the venom of this scorpion and shares homology with three peptide sequences, HtUy1 (AOF40222.1), HtUy2 (AOF40223.1), and HtUy3 (AOF40224.1), from the transcriptome of the scorpion Hadogenes troglodytes. This is the first report of this type of toxin in scorpions of the genus Tityus. Another uncharacterized protein is the venom protein 164, which was identified from the venom gland of the scorpion L. mucronatus. The sequence TaPBC_Unk_06 shows high identity (81.9%) with the venom protein 164-like sequence obtained from the venom gland of C. vittatus (XP_067139794.1) and 80% identity with the venom protein 164-like isoform X2 sequence obtained from the venom gland of C. sculpturatus (XP_023232232.1). To a lesser extent, it showed 41% identity with the prokineticin-domain-containing protein of the spider Caerostris darwini (GIY27678.1). Using the InterPro web, it was determined that the sequence TaPBC_Unk_06 contains a prokineticin domain. Prokineticins are known to play an important role in regulating the circadian rhythm, modulating pain and inflammation (they are mediators of inflammation and play a role in nociception, the biological process responsible for pain perception), promoting the release of inflammatory cytokines, and modulating pain in inflammatory contexts. They promote smooth muscle contraction in the digestive tract.

All of the factors mentioned above could contribute to the paralyzing effects or pain-induced by the venom. This is the first report identifying this specific type of toxin in the venom of the genus Tityus. Further studies are needed to determine its precise biological activity and potential applications in treatments for pain, inflammation, and circadian rhythm disorders. The sequence TaPBC_Unk_05 showed exclusivity with 14 hypothetical sequences in the NCBI database. These reported sequences are solely from venom gland data of the scorpions C. sculpturatus, C. vittatus, M. eupeus, Isometrus maculatus, and H. judaicus. Figure displays an alignment of some of these sequences, highlighting the conserved nature of 35 residues in the sequence TaPBC_Unk_05 and 26 residues in the sequence TaPBC_Unk_03, among which 8 conserved residues in both sequences are cysteines.

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Alignment of TaPBC-Unk-05 and TaPBC-Unk-03 found in the venom gland of T. cf asthenes.

No families associated with these sequences have been reported. This is the first documented occurrence of this sequence in the venom of the Tityus genus, and further studies are needed to understand the significance and biological activity of these sequences.

3.4. Antimicrobial Peptides and Hyaluronidase Fractions in T. cf. Asthenes Venom

RP-HPLC fractionation was conducted to identify the enzymes and antimicrobial compounds (Figure ). After venom fractionation of T. cf. asthenes, six fractions exhibited antimicrobial activity against E. coli and two for hyaluronidase activity (Figure S2). Of the fractions with antimicrobial activity, AF1 and AF2 showed mild activity compared to fractions AF3 through AF6.

6.

6

Open in a new tab

RP-HPLC profile of the T. cf. asthenes venom. The arrows in the chromatogram indicate the positive fractions for antimicrobial activity for E. coli.

Fractions AF1, AF2, and AF3 exhibited average molecular masses of 7328, 7399.9, and 2147.5 Da, respectively. However, no molecular masses were reported for fractions AF5 and AF6. The TCDS DN1188_c0_g1_i4 matches with a molecular mass of 7325.4 Da and with the tryptic peptides of the MS/MS proteomic data (Tables and S2). No TCDS matching was found for the molecular masses of 7399.9 and 2147.5 Da that correspond to the fractions AF2 and AF3, respectively.

2. TCDS of Peptides with Antimicrobial Activity for E. coli .

3.4.

Open in a new tab
a

Letters in italics represent amino acids that are enzymatically cleaved to release the mature peptide. The bold or underlined letters represent amino acid sequences of tryptic peptides found by MS/MS spectrometry (Table S2).

b

Theoretical and experimental average molecular masses.

Other potential antimicrobial peptides (ID: A0A6P3CW73) include cyclotide trypsin inhibitors. A cyclopeptide (DN88_c0_g2) was found in the venom proteome of T. cf. asthenes; however, it was not found during the RP-HPLC fractionation of the venom (Table ). This type of cyclopeptide in T. cf. asthenes is like the ones isolated from T. obscurus. These peptides are structured with α/β motifs and share structural similarities with plant cyclotides, which have been shown to bind to and disrupt phospholipid membranes, exhibiting lytic activity on cells. , Moreover, chitinases and lectins found in the TCDS venom gland of T. cf. asthenes could also act as antibacterial molecules.

Concerning the hyaluronidase-positive bands, the theoretical molecular mass of the TCDS hyaluronidase is 46 kDa, which aligns with the apparent molecular mass observed in the zymogram (Figure S1).

3.5. Comparative TCDS from Venom Glands of T. cf. Asthenes from Panama and Colombia

The distance between the collection sites of T. cf. asthenes from Palmas Bellas, Colón (Panamá) on the Atlantic side, and T. asthenes from Buenaventura (Colombia) on the Pacific side exceeds 1000 km. To address the entomological differences observed between T. cf. asthenes from Panamá and T. asthenes from Colombia, we focused on identifying the sodium and potassium channel toxins in these two scorpion species. The transcriptome of T. asthenes from Colombia yielded 218 TCDSs with homology to amino acid sequences from various species of the genus Tityus. These TCDSs were categorized into nine categories, with the largest category consisting of metalloproteinases (37%). The ion channel-modulating toxins were represented by sodium (15%) and potassium toxins (10%). Compared with the venom gland transcriptome from T. cf. asthenes, it returned 490 TCDSs with metalloproteinases as the largest group (28%). The ion channel-modulating toxins were represented by sodium (8.4%) and potassium toxins (13.3%). Comparing the available information from T. asthenes from Colombia, six sodium (14.6%) and seven potassium (10.8%) channel toxin TCDSs were identical to the TCDSs found in T. cf. asthenes. The low percentage of identical sodium and potassium toxins between these two scorpion populations suggests biochemical diversification that may reflect underlying genetic or ecological divergence. Further studies integrating genetics, morphology, population differentiation, and environmental data are required to evaluate whether these differences align with species-level boundaries in the Panamanian Tityus scorpions. Other studies on the venom of T. cf asthenes from Caucheras, Mutatá, Municipality, Colombia have reported proteolytic peptides with homology to various sequences documented in databases. Here, only one fragment was found and annotated as a putative protease inhibitor, which exhibits 100% identity and 66% coverage with the full-length sequence TaPBC_CTI_01 or DN88_c0_g2_i8_g (Tables S1/S2/S3), a cyclotide trypsin inhibitor.

4. Conclusions

The combined use of transcriptomics and proteomics has proven to be a powerful tool for identifying proteins present in scorpion venoms. However, it is essential to adjust the parameters of bioinformatic programs to enhance the identification accuracy. In this study, mass spectrometry (MS/MS) using two cleavage enzymes (chymotrypsin and trypsin) allowed for the identification of a significant number of components; however, the prefractionation of the venom could further improve the identification of low-abundance peptides such as antimicrobial peptides.

This work uncovered novel protein sequences in the Tityus genus, offering a deeper understanding of scorpion venom complexities in Panama. These sequences could be of utility in the bioprospecting of new leads with potential antimicrobial, anticancer, or analgesic properties, opening new avenues for pharmacological research.

Supplementary Material

pr5c00125_si_001.pdf (453.5KB, pdf)
pr5c00125_si_002.pdf (171.3KB, pdf)
pr5c00125_si_003.pdf (179.3KB, pdf)
pr5c00125_si_004.pdf (160.2KB, pdf)

Acknowledgments

The authors acknowledge Carolina Guevara, Heymi Sánchez, Eduardo Leiva, William Gonzalez, Maribel Barria, Carlos Moran, and Indalecio Valoy for the in-field collection of scorpions, keeping them in captivity, and venom extraction. They also acknowledge Eileen Rodríguez from Universidad de Panamá-AIP for the excellent management of the funds. This work was supported by grants from Panamanian funds from SENACYT: INF10-045, Proyecto de Movilidad MOV-2023-17, the “Sistema Nacional de Investigación (SNI) Panamá” including undergraduate funds, Ministerio de Ambiente (UNDP-GEF ABS Global Project) and Vicerrectoría de Investigación y Postgrado, Universidad de Panamá (CUFI-2022-CNET-P-012); and Mexicans funds from CONAHCyT-PRONAII 303045, and the “Dirección General de Asuntos del Personal Académico” (DGAPA-UNAM) grant number IT200724.

All data generated during this study will be available upon request. The T. cf. asthenes transcriptome raw reads were deposited in the Sequence Read Archive (SRAPRJNA1202649. BioSample accession SAMN45962855) under the file names tasthenesR1 (fastq) and tasthenesR2 (fastq). The mass spectrometry data (both raw files and interpreted files) were deposited in the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org) via the iProX partner repository with the data set identifier PXD058957.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jproteome.5c00125.

  • Hyaluronidase test zymogram; and antimicrobial activity on E. coli of HPLC fractions from T. cf. asthenes venom (PDF)

  • Translated coding sequences from the venom gland (PDF)

  • Proteomic data from the T. cf. asthenes venom analyzed by MaxQuant (PDF)

  • Translated coding sequences for proteins found in the venom of T. asthenes (PDF)

Conceptualization: MHS, HA, and GC; methodology and analysis: MHS, OS, IA, MHO, SEG; and review and editing: HA, MHS, GC; funding acquisition: MHS, HA, and GC. All researchers agree on publication.

The authors declare no competing financial interest.

Ethical statement: No experiments with humans were performed. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed, and procedures performed in the present study involving animals were done so by the bioethical standards at the “Instituto de BiotecnologíaUNAM” and at the “Universidad de Panamá”. Also, a fair and equitable sharing of genetic resources was accomplished with the Nagoya Protocol.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

pr5c00125_si_001.pdf (453.5KB, pdf)
pr5c00125_si_002.pdf (171.3KB, pdf)
pr5c00125_si_003.pdf (179.3KB, pdf)
pr5c00125_si_004.pdf (160.2KB, pdf)

Data Availability Statement

All data generated during this study will be available upon request. The T. cf. asthenes transcriptome raw reads were deposited in the Sequence Read Archive (SRAPRJNA1202649. BioSample accession SAMN45962855) under the file names tasthenesR1 (fastq) and tasthenesR2 (fastq). The mass spectrometry data (both raw files and interpreted files) were deposited in the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org) via the iProX partner repository with the data set identifier PXD058957.

Articles from Journal of Proteome Research are provided here courtesy of American Chemical Society

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