Identification, classification and characterization of the dermonecrotic toxins in venom glands of Hottentotta saulcyi, Androctonus crassicauda and Hemiscorpius lepturus using transcriptome analysis

doi:10.21203/rs.3.rs-2501970/v1

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Identification, classification and characterization of the dermonecrotic toxins in venom glands of Hottentotta saulcyi, Androctonus crassicauda and Hemiscorpius lepturus using transcriptome analysis

https://doi.org/10.21203/rs.3.rs-2501970/v1

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Venom phospholipase D (PLDs), known as dermonecrotic toxins, are the major molecules in the crude venom of scorpions that are mainly responsible for lethality and dermonecrotic lesions during scorpion envenoming. The purpose of this study was fourfold: First to identify transcripts coding for venom dermonecrotic toxin by transcriptomic analysis of the venom from Androctonus crassicauda, Hottentotta saulcyi, and Hemiscorpius lepturus; second to classify them by sequence similarity to known PLDs and motif extraction method; third to characterize scorpion PLDs; and fourth to investigate phylogenetic relationships of the PLD proteins. This is the first report of the presence of dermonecrotic toxin sequence in A. crassicauda and H. saulcyi venom. We found that the venom gland of scorpions encodes two PLD isoforms; PLD1 ScoTox-beta and PLD2 ScoTox-alpha I. Two highly conserved regions shared by all PLDs beta are GAN and HPCDC (HX2PCDC), and the most important conserved regions shared by all PLDs alpha are two copies of the HKDG (HxKx4Dx6G) motif. We found that PLD1 is a 31–43 kDa acidic protein containing signal sequences and PLD2 is a 128 kDa basic protein without known signal sequences. The gene structures of PLD1 and PLD2 contain 6 and 21 exons respectively.

Biological sciences/Biochemistry

Biological sciences/Biotechnology

Biological sciences/Genetics

Biological sciences/Molecular biology

Phospholipase D

Dermonecrotic toxins

Transcriptomic analysis

Conserved regions

Envenoming caused by scorpion species, including Androctonus crassicauda, Hottentotta saulcyi, Mesobuthus eupeus, Odontobuthus doriae, and Hemiscorpius lepturus, are public health challenges in Iran ¹, so their venom is used for the production of antivenom by the Razi Vaccine and Serum Research Institute, Hesarak, Karaj, Iran. Each year, the variety of symptoms including local pain, sweating, vomiting, delirium, anxiety, necrosis, inflammation, muscle paralysis, even hematuria and extended edema following scorpion sting are announced by the Iranian Ministry of Health ^2,3. Many studies have been conducted to find the major contributing factor that causes these symptoms, most of which are based on the identification of venom compounds.

Venom of scorpions contains complex mixtures of different biologically active compounds, including enzymes (such as hyaluronidases, phospholipases, and proteases), toxic peptides, free amino acids, carbohydrates, lipids and other metabolites ^4,5. Some of the venom components are critical for the life of the scorpion because the scorpion uses them for capturing prey, defending against predators, and, reproduction ⁴. However, many studies investigated that some peptides and proteins of scorpion venoms have cytotoxic, neurotoxic, and immunosuppressive properties ⁶. Among complex secretory mixture of venom, phospholipases D (PLDs), also called dermonecrotic toxins, sphingomyelinases D or SMases, were identified as the most abundant enzymes with many isoforms. These hydrolase enzymes are the main enzymes of venom that are known to be responsible for the symptoms of neurotoxicity and lethality during envenomation and may involve in the adaptation of the organism, and the effectiveness of the venom ⁷. Phospholipase D (PLD) is known to be a ubiquitous enzyme that its activity has been discovered in a range of species, such as plants, mammals and microorganisms⁸. Venom phospholipase D (PLD), can transform sphingomyelin (SM) to ceramide 1-phosphate (C1P) by degradation of cell membrane phospholipids ⁹. Subsequently, the release of ceramide 1-phosphate (C1P) can proceed to a pro-inflammatory mediator which can regulate the TNF-α and recruit neutrophils ^7,10. This enzyme is mainly responsible for dermonecrotic lesions, and these reactions are involved in components of the complement activation, migration of polymorphonuclear leukocytes, platelets aggregation and inflammatory response ¹¹.

Furthermore, PLD can associate with a large number of proteins unites involved in a variety of physiological cellular purposes, such as cytoskeletal dynamics, chemotaxis of leukocytes and cell proliferation ^12,13. Although the PLD is the most studied and well-known toxin derived from arthropod venoms, especially in the venoms of brown spiders, Loxosceles ¹⁴, and scorpion venoms of H. lepturus, Centruroides sculpturatus, Megacormus gertschi, and Paravaejovis schwenkmeyeri ^6,15−18, there are only general and preliminary studies on scorpion venom PLDs. Therefore, molecular and structural characterization of this enzyme in scorpions is required. In the present study, along with identification of dermonecrotic toxin-like sequences from venom glands of A. crassicauda, H. saulcyi, and H. lepturus using de novo transcriptome reconstruction, the classification and characteristics of this enzyme were performed. Furthermore, the current study attempted to predict the exon-intron pattern of PLD gene of scorpion venom gland. In addition, the evolutionary relationships between scorpion species and spiders have been developed. This is the first report of the presence of dermonecrotic toxin sequence in the venom of A. crassicauda and H. saulcyi.

This paper presents a transcriptome analysis of the H. saulcyi venom gland. H. lepturus and A. crassicauda cDNA libraries used in this study were produced in our previous study. Detailed information about the experimental design, samples preparation, RNA extraction and high-throughput RNA-sequencing methods have been described previously ¹⁹. Identification, classification and characterization of the dermonecrotic toxin sequences were done as described for H. saulcyi. A graphical description of the study steps is given in a work flow diagram (Fig. 1).

Sample preparation

The H. saulcyi scorpions were captured from deserts around the Khuzestan province, south-west of Iran in autumn 2021 according to ethical guidelines of the world health organization (WHO) method. The scorpion species were identified in the laboratory of Razi Vaccine and Serum Research Institute and Toxicology Research Center laboratory of Ahvaz Jundishapur University of Medical Sciences. The milking was carried out by electroshock to allow the venom cells to enter the secretory phase. Three days after milking, the telsons of scorpion individuals were removed and preserved in RNAprotect Tissue Reagent® (Cat. No. /ID: 76104), for immediate stabilization of RNA.

RNA isolation and cDNA library construction from venom gland

Total RNA was isolated from freshly isolated venom gland tissues according to the protocol instruction of RNeasy Mini Kit (Cat. 74104). The quality of extracted RNA was evaluated using Nanodrop (co. Thermo, USA). Each ten RNA samples of H. saulcyi venom glands were pooled together in equal concentrations to generate two pooled RNA samples. The samples were evaluated for RNA Integrity Number (RIN) by Agilent Bioanalyzer 2100. The RNA sample with a RIN > 7 were selected for cDNA library construction. The library was synthesized using TruSeq® Stranded mRNA Library Prep (cat. number 20020594) according to the manufacturer’s instructions. Finally, cDNA library (from 2 H. saulcyi individuals) was sequenced by High-throughput RNA-sequencing (Illumina Next-Generation Sequencing). Sample was subjected to a paired-end sequencing of 150 bp on the Illumina HiSeq 2000 platform (Illumina, Macrogen Co Macrogen, Seoul, Korea).

Assembly and annotation of deep sequencing data

Trimmomatic and FastQC java programs (http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/) were used to filter poor quality reads and assess the quality of raw reads, respectively. The clean reads of each species individual were separately de novo assembled into contigs using Trinity software (v2.10.0) ²⁰ with the default parameters. Since no reference sequence is provided for studied scorpion species, the de novo assembled transcriptome sequences were considered as the reference sequences to evaluate the quality of the transcripts. Bowtie2 v2.3.5.1 was used to map the reads back to the transcriptome assemblies.

Open Reading Frames (ORFs) of the non-redundant transcript isoforms and potential coding sequences were predicted by TransDecoder software v3.0.1 with the ‘single best ORF’ option ²¹.

Identification of the dermonecrotic toxins in venom glands transcriptome

To identify the protein and nucleotide sequences with the highest sequence similarities to dermonecrotic toxins, the local dermonecrotic toxin database was generated using scorpion manually reviewed dermonecrotic proteins and some from closely related species (Table 1). Then, The Transdecoder-predicted proteins and nucleotide sequences from each species were searched against local dermonecrotic toxin database using blastx and blastp with an E value threshold le^− 3. Finally, the homologous sequences representing dermonecrotic toxins were preserved from three scorpion species mRNA candidates. The classification of those sequences first was performed based on sequence similarity to already known families/classes. To classify based on sequence similarity, newly extracted sequences were searched against NCBI and Uniprot databases. A sequence alignment was generated with an online server in EBI (https://www.ebi.ac.uk/Tools/msa/clustalo/). For confirmation, we used known motif extraction method by searching the protein sequences against NCBI Conserved Domain Database (CDD) (https://www.genome.jp/tools/motif/), PROSITE collection of motifs online tool (https://prosite.expasy.org/scanprosite/) and RCSB Protein Data Bank (RCSB PDB) (https://www.rcsb.org/search/advanced/sequence). To assist in fast identifying PLD family members from insects, we attempted to find a diagnostic peptide pattern by aligning the motif sequences of PLDs from scorpions, spiders, ticks, termite, ants, flies and wasp species using MUSCLE tool, a multiple sequence alignment program.

To achieve this purpose, two more scorpion transcriptomes from Androctonus australis (SRR1724216.1) and Hottentotta trilineatus (SRR1721800.1) were downloaded from NCBI. We conducted de novo assemblies of these transcriptomes with Trinity v2.10.0 ^20,22 and open reading frames prediction with TransDecoder pipeline (https://transdecoder.github.io/) as described above.

Characterization of gene and protein of dermonecrotic toxins

To enhance the confidence of the predicted dermonecrotic toxins, the newly discovered protein and nucleotide sequences were subjected to further characterization. An intron-exon maps of dermonecrotic toxin genes were predicted based on our previous proposed methodology ¹⁹, the comparison of whole genome shotgun sequences with mRNAs. The mRNA sequence of phospholipase D SpeSicTox-beta (XM_023367013.1) with its genomic DNA (NW_019384571.1) and mRNA sequence of phospholipase D2 (XM_023381888.1) with its genomic DNA (NW_019385241.1) from Centruroides sculpturatus were used as templates to compare with the dermonecrotic toxin mRNA sequences found in this study. A sequence alignment was generated with MAFT (high speed multiple sequence alignments program) online server in EBI (https://mafft.cbrc.jp/alignment/server/index.html).

In the following, physicochemical properties of toxin were determined with ProtParam tool from Expasy (https://web.expasy.org/protparam/) and proteomics tool from INNOVAGEN (http://pepcalc.com). Molecular weight, iso-electric point, instability index and water solubility of PLDs were estimated. Potential signal peptides of toxins were predicted via SignalP-6.0 server (https://services.healthtech.dtu.dk/service.php?SignalP).

3D-structure determination and refinement of dermonecrotic toxins

Homology modeling was used to determine the 3-D structures of newly found dermonecrotic toxins. Four models were generated for each toxin using four different methods as follows: I-TASSER by multiple threading approaches, Phyre2 with Poing² method, and SWISS-MODEL. The best model was selected via quality assessment using SAVESv6.0 (https://saves.mbi.ucla.edu/) as well as by Z-score through the ProSA-web (https://prosa.services.came.sbg.ac.at/prosa.php). For further analysis of the predicted models, Ramachandran plot was also analyzed for each structure.

The best quality protein structures were used for comparison of the structural homology between PLDS from all scorpion species. Visualization and structural alignment of the selected structures of PLDs from A. crassicauda were performed using UCSF Chimera software (https://www.cgl.ucsf.edu/chimera/).

Phylogenetic analysis

The homologous peptides of dermonecrotic toxins were extracted using BlastP by searching of newly found dermonecrotic toxins against NCBI database. The amino acid sequences of dermonecrotic toxins were then aligned with other representative sequences from scorpions, spiders, ticks, termite, ants, flies and wasp species using multiple alignments with MUSCLE tool ²³. The obtained multiple alignment results were used to estimate consensus phylogenetic trees by a Maximum Likelihood Algorithm ²⁴ using PhyML 3.0 program ²⁵ hosted on the Phylogeny.fr web server (http://www.phylogeny.fr/simple_phylogeny.cgi).

Ethical statement

The manuscript and data were not previously or simultaneously submitted elsewhere. All experiments in this paper were carried out under the standard procedures of scientific ethics, including the care of experimental animals. Permission was obtained from Environmental Protection Agency of Iran to collect scorpions of A. crassicauda, H. saulcyi, and H. lepturus. No animals were euthanized as part of this study and all sample collection methods and experimental procedures described herein were rigorously reviewed and approved by the Institutional Animal Care Committee of Razi Vaccine and Serum Research Institute (Permit number IR.RVSRI.REC.1401.017) and AREEO protocols, which are in compliance with Iran guidelines for work with animals. This study also adheres to the ARRIVE Guidelines for reporting animal research. All procedures were performed in accordance with relevant guidelines' in the manuscript. All authors have read the manuscript and agree to its publication in Journal of Scientific Report and agree that it has followed the rules of ethics presented in the guidelines for journal publication.

Assembly and annotation raw sequences from RNA sequencing

In this work, we carried a transcriptome analysis of the H. saulcyi venom gland for extracting and characterizing the sequences of the phospholipase D. In addition, we used the previously reconstructed venom gland transcriptome from H. lepturus and A. crassicauda ¹⁹. After sequencing and filtering out of low-quality reads, the cDNA library was constructed from approximately 48 million paired-end of clean reads of >200 bp by the Trinity program ²⁰. Among the 191150 assembled transcripts, 110126 unigenes were obtained and 98365 potential coding sequences were predicted using TransDecoder software. We found that a high percentage of those protein coding sequences (95167) had a match in non-redundant protein (Nr), Swissprot, and Pfam databases.

In our previous study, following removal of the adaptor, short reads and quality filtering, approximately 51 and 48 million paired-end of clean reads were obtained in A. crassicauda and H. lepturus, respectively. In total, 260663 and 222989 transcript sequences were reconstructed by de novo transcriptome assembler of Trinity software, in which 152704 and 122618 unigenes (>200 bp) were clustered from deep RNA-Seq data of A. crassicauda and H. lepturus, respectively. After comparing the protein coding sequences against NCBI, Swissprot, and Pfam databases, 11415 and 11869 transcripts were identified as known mRNAs in A. crassicauda and H. lepturus transcriptomes, respectively.

Identification of the dermonecrotic toxins

The first step is building a local database of dermonecrotic toxin to target BLAST searches for phospholipase sequences. This database was created by collecting the known sequences of phospholipase D1, D2 or phospholipase alpha and beta from different scorpion species and closely related species such as spiders, ticks, termite, ants, flies and wasp species. Using our local dermonecrotic toxin database, we conducted exhaustive BLAST searches of the H. saulcyi, A. crassicauda and H. lepturus venom glands transcriptome and found multiple transcripts with high sequence similarity to PLDs. Generating a target database of PLD sequences from a few widely used databases facilitated the use of comprehensive search strategies to extract subsets of PLD sequences from our dataset. To increase the sequence identification confidence and to classify the sequences, the obtained sequences were directly searched against NCBI and Uniprot databases. Sequences sharing high sequence identity with previously classified PLD sequences belonging to species mentioned above were considered members of this group. Accordingly, we found that scorpions encode two genes of phospholipase D: PLD1 and PLD2.

Domain and motif identification

For definitive categorization, newly obtained PLD sequences were subjected to protein motif and domain identification. For domain identification, we used BLASTP searches of the local copy of the NCBI CDD. Furthermore, PROSITE collection of motifs online tool and RCSB PDB server were utilized for protein motif exploration. By searching PROSITE database, we found that some PLD sequences are sharing a motif of phosphatidylinositol-specific phospholipase C (PIPLC_X_DOMAIN) and glycerophosphodiester phosphodiesterases domain (GP-PDE), which were classified as PLD1 family. The rest, which have share similar motifs including two copies of HKD motif (PLD) and a phox homology domain (PX), were considered as PLD2 family. By analyzing the results of searches for protein motifs in the NCBI CDD database, two copies of catalytic domains of phospholipases were found for sequences classified into PLD2 family, while a Glycerophosphodiester phosphodiesterase-like domain (GDPD_like_SMaseD_PLD) of spider venom sphingomyelinases D was found for sequences classified into PLD1 family. Using RCSB PDB resource, we found that PLD1 show structure similarity with 6U8Z (Crystal Structure of Catalytic Domain of Human Phospholipase D1), and PLD2 ScoTox alpha shows structure similarity with 3K72 (Structure of integrin alphaX beta2). In general, based on sequence similarity and conserved segments of protein or nucleic acid sequences analyses of PLDs from scorpions, three types of phospholipase D (PLD) discovered in H. saulcyi, A. crassicauda and H. lepturus scorpion species were named PLD alpha, beta I and beta II. Results of the BLAST searches against NCBI-CDD and PROSITE databases are presented in Figure 2.

The discovery of a specific diagnostic peptide pattern from the conserved sequence or known motifs of PLDs family assist to recognize members of this highly diverged but homologous family in insects. To achieve this goal, the alignments of conserved segment of PLD sequences from scorpions, spiders, ticks and mites were performed with Clustal Omega program. Based on sequence analysis of PLDs beta from insects, we found that those family members had highly conserved segments (Figure 3). All members of PLDs beta are characterized by the sequence HMX13GANX4DX13HX2PCDC, for which two domains termed GAN (HMX13GAN) and HPCDC (HX2PCDC) can be defined for them.

All members of beta PLDs are characterized by the sequence HMX13GANX4DX13HX2PCDC, for which two domains can be defined, namely GAN (HMX13GAN) and HPCDC (HX2PCDC).

Therefore, enzymes with the characteristic HMGAN and HPCDC domains are categorized as part of a PLD1 family and include PLD beta enzymes. This large family was divided into two subgroups (beta I and beta II) based on sequence similarity that we named Phospholipase D1 Scorpion Toxin-beta I (PLD1 ScoTox-beta I) or Dermonecrotic Scorpion Toxin-beta I (DScorTox-betaI), and Phospholipase D1 Scorpion Toxin-beta II (PLD1 ScoTox-beta II) or Dermonecrotic Scorpion Toxin-beta II (DScorTox-beta II).

Various numbers of PLD1 betaI were identified from venom glands transcriptome of H. saulcyi and A. crassicauda and H. lepturus, which the highest diversity was observed in H. lepturus scorpion. In general, three PLD1 were discovered from venom gland of A. crassicauda, called PLD1 ScoTox-betaI PLD1, ScoTox-betaII isoform x1, and PLD1 ScoTox-betaII isoform x2; eight from H. lepturus, named PLD1 ScoTox-betaI isoform x1 to PLD1 ScoTox betaI isoform x8; and one from H. saulcyi, called PLD1 ScoTox-betaI isoform x1. The nucleic acid and protein sequence data displayed in this study are available in the NCBI under the accession number OP867052, OP867053, OP867054, OP867055, OP867056, OP867057, OP867058, OP867059, OP867060, OP867061, OP180075.

Furthermore, we aligned the PLD2 or PLD alpha sequences from scorpion species and similar sequences from other insects (spiders, ticks, termite, ants, flies and wasp species) using Clustal Omega program and found that PLD alpha from insects is highly conserved and contains duplicate phosphodiesterase active sites. The profiles of phospholipase D2 phosphodiesterase active sites are LWAHHEKX4DQX2AFXGG and ELXYVHSKX2IXDDX3IXGSANINDRS for first and second catalytic sequences respectively (Figure 4), in which characterized by the sequence HxKx4Dx6G (HKDG domain). Based on these analyses of PLDs, sequences sharing two copies of catalytic HKDG domain were categorized as part of the PLD2 family. We named these PLDs, Phospholipase D2 Scorpion Toxin-alpha I (PLD2 ScoTox-alpha I) or Dermonecrotic Scorpion Toxin-alpha I (DScorTox-alpha I). The nucleic acid and protein sequence data displayed in this study are available in the NCBI under the accession number OP867062 and OP820507.

Exon-intron organization of scorpion PLDs

The exon-intron patterns of PLD beta (Fig. 5, Fig. 6) and alpha (Fig. 5, Fig. 7) genes were predicted by aligning the mRNA sequences of PLD beta or alpha discovered in this study with the genomic sequence and mRNA of dermoncrotic toxins from Centruroides sculpturatus using MAFT program. The total sequence of PLD beta gene from START-codon (ATG) to STOP-codon (TAA) is around 5400bp and full-length cDNAs spans from ~1100 to 1600bp. As show in figure 6, PLD beta gene was found to consist of 6 coding exons and 5 introns. Exons 1–6 vary in size from ~45bp (exon 1) to ~600bp (exon 6), whereas the introns vary in length between ~70bp (intron 4) to ~1630bp (intron 3). Exons 1 to 6 containing ~45, 250, 132, 125, 228, and 601bp, respectively. The introns 1 to 5 consist of ~1012, 77, 1630, 71, and 1217bp, respectively. We found that exon 6 with the size of ~600bp and intron 3 with the size of ~1630bp are the longest regions in the PLD1 genes. All introns of PLD1 gene follow the standard form of GT-AG.

As show in figure 7, PLD alpha gene is quite large and predicted to consist of 21 coding exons ranging from 68bp (exon 5) to 245bp (exon 13). The total sequence of PLD alpha gene and full-length cDNAs from START-codon (ATG) to STOP-codon (TAA) are ~45596bp and ~2800bp respectively, in which exons 1 to 21 containing ~120, 154, 125, 110, 68, 160, 153, 150, 166, 110, 139, 149, 245, 80, 150, 120, 123, 113, 90, 160, 115bp respectively. We found that PLD2 gene contains 20 GT-AG type introns. The introns 1 to 20 consist of 10700, 4102, 109, 3384, 3332, 5000, 80, 8066, 74, 168, 110, 4446, 550, 89, 2242, 70, 60 84, 70, 92bp. Exon 13 with the size of 245bp and intron 1 with the size of 10700bp are the longest regions in the PLD2 genes.

Protein structures of PLDs

Computational models of protein structures for dermonecrotic toxins were determined with I-TASSER, Phyre2, and SWISS-MODEL web servers. The quality of each model was assessed by overall quality factor in SAVES server and Z-score in ProSA-web server (Table 2). All Phyre2 generated models had less quality according to SAVES server assessment. Models predicted by Swiss-model with high overall quality factor and a Z-score in the range of native conformations have the best quality. The Ramachandran map analysis of three-dimensional structures predicted by Swiss-model showed that the percentage distribution of dihedral angles of the PLD protein residues at the allowable zone is over 95% which indicating high stability of structures; therefore, they were utilized for structural homology comparison.

Structural homology analysis of the dermonecrotic toxin sequences

The best computational models of PLDs were used for comparison of the structural homology between A. crassicauda–PLDs, H. saulcyi-PLDs and the structure of dermonecrotic toxins derived from H. lepturus. For structural alignment and visualization, we used molecular graphics program UCSF Chimera (ver. 1.11.2, University of California, San Francisco, CA, USA). The analysis indicated that the predicted PLD structures contain both α-helices and β-pleated sheets which had significant structural homology when compered together (Fig. 8, 9). Comparative modeling analysis of PLD alpha showed that the number of α-helix and β-sheet in A. crassicauda and H. saulcyi were similar. Structures of PLD beta from A. crassicauda, H. saulcyi and H. lepturus were also generally similar and minor differences observed between PLD beta structures are related to the amino acid composition of the proteins.

Characterization of the gene and protein of PLD1 and PLD2

SignalP (version 6.0) was implemented to propose the presence, cleavage site positions and general structure of the signal peptides. Signal peptide score, as the confidence in the signal peptide prediction, was predicted over than 0.97 for all PLD1 members (Table 3). General structure of the signal peptides has suggested and it consists of three main parts, including N-terminal region (N-region), intermediate hydrophobic region (H-region) and C-terminal region (C-region). This study revealed differences in the amino acid composition and length of these three regions between PLD1 ScoTox-betaI proteins of the Buthidae and Hemiscorpiidae families, such that the longer amino acid signal peptides (32-amino acids signal peptide) were predicted for isoforms X3-X6 of H. lepturus.

In addition, such differences were also observed between PLD1-betaI and PLD1-betaII group of proteins. The N-regions of PLD1 ScoTox-betaI and betaII from Buthidae family (A. crassicauda and H. saulcyi) were predicted to compose of 2–3 amino acid residues, and they have a cleavage site at position 21 and 19, respectively. We found that the cleavage site is absent from PLD2 ScoTox-alpha proteins.

The physiochemical properties were analyzed for all mature PLDs using Expasy’s ProtParam software (Tables 4, 5). We found that PLD1-beta gene from scorpions encodes a mature protein with molecular weight ranging from ~ 31 to 44 kDa. Based on IP results, scorpion PLD1-beta is an acidic peptide with isoelectric points ranging from 5.5 to 6.9. The PLD1-betaI sequences in this study indicated high stability, high aliphatic indices and negative values of GRAVY. The aliphatic indices for those protein sequences ranged from 74.74 to 83.59. Mature PLD2-alpha with molecular weight of approximately 128 kDa and polar nature (negative GRAVY value), has a basic isoelectric point (~9) and high aliphatic index (Table 5).

Phylogenetic relationships of the arthropods

Phylogenetic analyses of the PLD proteins were conducted using the PhyML 3.0 program ¹⁰ to evaluate the evolutionary relationship of PLD proteins in insects. To build the phylogenetic tree, we included dermoncrotic toxins sequences from this study, alongside available PLD homologous peptides in GenBank from A. australis and H. trilineatus scorpions and six out-groups including spiders, ticks, termite, ants, flies and wasp species (Table 1). Our phylogenetic analysis classified the PLD family of proteins into two main subfamilies, PLD1-beta and PLD2-alpha (Fig. 10). Clade 1 consist of PLD1-beta and clade 2 includes PLD2-alpha, which clade 1 further divided into the subclades betaI and betaII. The phylogenetic tree represents that PLD alpha members share close evolutionary relationship, as well as PLD beta members of different insect species. However, there are long evolutionary distances between PLD alpha and PLD beta.

The analysis of PLDs showed that A. crassicauda and H. saulcyi PLD members have a close phylogenetic relationship with their other Buthidae family orthologs, but they have a long phylogenetic distance to their H. lepturus orthologs. Furthermore, among all insects, the closest phylogenetic relationship of PLD members was observed between scorpions and spiders.

Lethal potency, neurotoxicity and cytotoxicity of venom of different scorpion species have been reported in human ²⁵, mice ²⁶ and Sesamia nonagrioides ²⁷. In terms of venom lethality, the venoms of A. crassicauda, H. saulcyi, and H. lepturus are the most toxic. For this purpose, transcriptomic analyses on the venom glands of these three scorpion species from two families of Buthidae (A. crassicauda and H. saulcyi) and Hemiscorpiidae (H. lepturus) were conducted in order to identify enzymes which potentially play important roles during scorpion envenoming. In the last two decades, numerous studies have evaluated various components in the venom of poisonous animals including scorpions using genetic engineering strategies, transcriptomics and proteomics approaches^12,28-30, some of which have identified toxin transcript sequences similar to phospholipase D responsible for neurotoxicity and lethality^6,16-18,31. Phospholipase D, a superfamily of transphosphatidylase, generates phosphatidic acid as a lipid second messenger through hydrolysis of the phosphatidylcholine (PC) ³². Previous researches have shown that PLD plays its role in several cellular processes include hypertension, vesicular trafficking, exocytosis, endocytosis, phagocytosis, regulation of cellular metabolism, and tumorigenesis, indirectly through its catalytic products, phosphatidic acid^4,32,33. In Vitro study of recombinant dermonecrotic toxins from brown spider (Loxosceles intermedia) and scorpion of H. lepturus venom showed that those venom components caused a wide range of pathophysiological effects in rabbit and mice including; induce a local inflammatory response, dermonecrosis, lysis of red blood cells, platelet adhesion, increased permeability of blood vessels and acute kidney injury^15,28,34. These findings made us focus on the identification and investigation of scorpion venom PLDs.

Identification and classification of the PLD isoforms

The current study was conducted to report of transcriptome analysis of venom glands of H. saulcyi H. lepturus and A. crassicauda with insight into identification, classification and characterization of the putative phospholipase D, known as dermonecrotic toxin. This report marks the first evidence of PLDs in the venom of H. saulcyi and A. crassicauda scorpions. Multiple transcripts with high sequence similarity to PLDs were identified in the venom glands transcriptome of three studied scorpions by BLAST searching against the local dermonecrotic toxin database. This study showed that two forms of phospholipase D, PLD1 and PLD2, are encoded by the scorpion’s venom gland.

First, recombinant PLD of venom, with molecular weight of 32–35 kDa were purified from the venom of the spider ³⁵, then a 33 kDa protein representing sphingomyelinase D and dermonecrosis activity was purified from the venom of H. lepturus ³⁶. Although after reporting the first evidence of phospholipase D activity in a scorpion venom of H. lepturus ¹⁵, several studies, including the Reference Sequence (RefSeq) Genome Assembly bioproject of Centruroides sculpturatus (PRJNA422877), venom gland transcriptomic analyses of H. lepturus ¹⁶, Megacormus gertschi (JAW07090.1)⁶ and Paravaejovis schwenkmeyeri ⁶ identified toxin transcript sequences similar to phospholipase D as well as, recombinant phospholipase D1 from H. lepturus ^18,31, so far characterization of catalytic motifs of scorpions PLDs and classification of them have not been done. Two PLD isoforms, PLD1 and PLD2, have already been found in the mammals ³⁷, plants and yeast ³⁸. In another classification, the PLD superfamily from plants, microbes and eukaryotes was classified into two groups, HKD PLD and non-HKD PLD, based on the presence of the HKD catalytic motif ³⁹. Given the fact that conserved protein sequence regions are very useful for identifying, grouping and studying of protein families due to their importance in terms of structure or function of the molecule ⁴⁰, we used motif extraction to classify the newly found sequences. Interestingly, it can be inferred from our motifs and domains classification that venom gland of scorpions encodes two PLD isoforms; PLD1, known as Phospholipase D1 Scorpion Toxin-beta (PLD1 ScoTox-beta) or Dermonecrotic Scorpion Toxin-beta (DScorTox-beta), and PLD2 that known as Phospholipase D2 Scorpion Toxin-alpha I (PLD2 ScoTox-alpha I) or Dermonecrotic Scorpion Toxin-alpha I (DScorTox-alpha I). Furthermore, two classes of PLD1 gene: betaI and betaII have been identified from venom glands transcriptome of H. saulcyi, A. crassicauda and H. lepturus. Similar to the results of the conserved regions, in our phylogenetic analysis of PLDs, PLD1 and PLD2 clustered as a unique subfamily, while betaI and betaII were grouped in one cluster, these results are also consistent with the results of sequence alignment. Indeed, we found that scorpion PLD beta and alpha differ in their regulatory motifs.

By multiple sequence alignments of the catalytic motifs of PLD superfamily from insect, it was found that the beta group of insect PLDs lacks the HKD conserved motif, while the alpha group has duplicate HKD motifs and new key residues were identified as common features of PLD alpha (HxKx4Dx6G) and beta (GAN and HPCDC). Interestingly, the results of our motif extraction, sequence and structural homology searches revealed that scorpion PLDs beta share homology with secreted mammalian glycosylphosphatidylinositol phospholipase D (GPI-PLD), phosphatidylcholine-specific PLD (PC-PLD), and Loxosceles PLDs (brown spider). Meanwhile, PLD alpha shares a common feature with PX/PH proteins.

Consistent with our study, the GPI-PLD, PC-PLD, and Loxosceles PLDs, which shares distant homologies, grouped as non-HKD enzymes and plant and mammalian PXPH-PLD enzymes grouped as HKD PLDs ^4,39,41. Citing that Loxosceles PLD with SMase activity had been classified as lacking HKD enzymes ⁴¹, and considering previous research in which by comparing the recombinant phospholipase D toxins from Hemiscorpius and Loxosceles venom, the equal ability of these two PLDs for hydrolyzing lysophosphatidylcholine (LPC) and sphingomyelin was shown ⁴², so the scorpion PLD beta enzymes can be considered structurally and functionally homologous with most of PLDs from Loxosceles (this must be proven experimentally), while scorpion PLD alpha is estimated to have similar functions to several members of mammalian PLDs that bear a PX domain and are represented by a duplicated HKD motif. It has been explained that these members of mammalian PLDs, in addition to lipase activity, hydrolyze phosphodiester bonds through the HKD catalytic motif ⁴¹. Since previous studies have reported different activities for these three PLDs; GPI-PLD hydrolyzes GPIs to produce PA and a free head group, PC-PLD catalyzes the hydrolysis of PC to produce phosphatidic acid and choline, and Loxosceles PLDs exhibit SMase activity ^4,39,41, thus, a more accurate classification of PLDs in scorpions requires studying the physicochemical properties of these isoforms in scorpions.

Characterization of the gene and protein of PLD1 and PLD2

We found that PLD1 with molecular weights ranging from 31 to 43 kDa is an acidic protein containing a signal peptide and PLD2 with molecular weights of 128 kDa is a basic protein without known signal peptide. Intron-exon pattern analysis of PLD1 and PLD2 genes resulted in the prediction of 6 and 21 exons respectively. The different characteristics of PLD1 and PLD2 prove their difference in localization or functions. Basically, phospholipases differ in structure, function, regulation, and mode of action which are divided into two general groups, the acylhydrolases and the phosphodiesterases ⁴³. Functionally, most cells comprise a large number of phospholipases that can be acted as secreted forms, membrane associated, and intracellular located ⁴³. Overall, based on the results of this study and previous studies, the difference between PLD1 and PLD2, in addition to the primary sequence and function, is also in their intracellular location and the types of their activating proteins ^41,44. Initial studies based on overexpression and monoclonal antibody studies suggested that PLD1 is mostly found in intracellular membranes, while PLD2 is located on the plasma membrane ^9,38,45. These studies; based on the localization and activating proteins of PLDs, support the role of both PLDs in facilitating receptor-mediated endocytosis, where PLD1 mediates vesicular traffic, which is part of the main activities of PLDs reported in previous studies and PLD2 facilitates endocytosis of the angiotensin II type 1 receptor. However, the role of PLDs in endocytosis may be complex, and it is likely that both PLD1 and PLD2 work together to stimulate receptor endocytosis, such that activation of one lead to the activation of the other ⁴⁵. Basically, phospholipases differ in structure, function, regulation, and mode of action which are divided into two general groups, the acylhydrolases and the phosphodiesterases ⁴³. Functionally, most cells comprise a large number of phospholipases that can be acted as secreted forms, membrane associated, and intracellular located ⁴³. In any case, the available data about the role of each of these two are few and more studies are needed in this matter, but the difference between these two PLDs in the presence of the signal peptide sequence can be another reason for the difference in their cellular location. Because all proteins from eukaryotic cells are synthesized in cytoplasm and signal peptides direct linked proteins to their specific locations.

Many biotechnological studies have suggested that PLDs derived from scorpion and spider venom have the potential to be used in next-generation immunization strategies due to their important structural and functional diversity. This study provides relevant findings for improving their use as valuable tools for the development of antivenoms and new drugs. In this study, we reported the transcriptome information of H. saulcyi, A. crassicauda and H. lepturus, with an emphasis on the identification and classification of PLDs, which can provide insight into their identification and classification in insects. In scorpions, genes encoding PLD isoforms are classified into two groups: alpha and beta. This classification is highly based on similarities in sequence identity, motifs and domains conservation, exon-intron architecture, protein structures and phylogenetic analysis, and can serve as a reference in grouping of newly discovered PLDs from scorpions.

PLD: Phospholipase D

SM: Sphingomyelin

C1P: Ceramide 1-phosphate

WHO: World health organization

RIN: RNA Integrity Number

ORFs: Open Reading Frames

CDD: Conserved Domain Database

PLD1 ScoTox-beta I: Phospholipase D1 Scorpion Toxin-beta I

DScorTox-betaI: Dermonecrotic Scorpion Toxin-beta I

PLD1 ScoTox-beta II: Phospholipase D1 Scorpion Toxin-beta II

DScorTox-beta II: Dermonecrotic Scorpion Toxin-beta II

PLD2 ScoTox-alpha I: Phospholipase D2 Scorpion Toxin-alpha I

DScorTox-alpha I: Dermonecrotic Scorpion Toxin-alpha I

LPC: lysophosphatidylcholine

LPA: lysophosphatidic acid

PC: Phosphatidylcholine

PI: Phosphatidylinositol

GPI-PLD: Glycosylphosphatidylinositol phospholipase D

PC-PLD: Phosphatidylcholine-specific PLD

SMase: Sphingomyelinase

Ethics declarations

Ethics approval and consent to participate

Permission was obtained from Environmental Protection Agency of Iran to collect scorpions of A. crassicauda, H. saulcyi, and H. lepturus. No animals were euthanized as part of this study and all sample collection methods and experimental procedures described herein were rigorously reviewed and approved by the Institutional Animal Care Committee of Razi Vaccine and Serum Research Institute (Permit number IR.RVSRI.REC.1401.017) and AREEO protocols, which are in compliance with Iran guidelines for work with animals. This study also adheres to the ARRIVE Guidelines for reporting animal research.

Consent for publication

Not applicable.

Availability of data and materials

The mRNA and protein sequences reported in this paper are appearing in the NCBI under the accession numbers OP867052, OP867053, OP867054, OP867055, OP867056, OP867057, OP867058, OP867059, OP867060, OP867061, OP180075, OP867062, and OP820507.

Competing interests

The authors declare that they have no conflict of interest.

Funding

Financial support for the all authors was provided by Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran (960354-020-18-83-2).

Author's contributions

Conceived and designed the experiments: F.S. and M.B. Collect and identify the scorpion specimens: M.B and F.S. Analyzed the data: F.S. wrote the manuscript: F.S. All authors have reads and approved the manuscript.

Acknowledgements

We thank Razi Vaccine and Serum Research Institute and Ahvaz Jundishapur University of Medical Sciences, of Iran for their financial support and our colleagues for many stimulating discussions.

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Table 1

Phospholipase D protein sequences used to generate the multiple sequence alignment for detection of a specific diagnostic peptide pattern from the conserved sequence and phylogenetic analyses.
Taxon	GenBank ID
Hemiscorpius lepturus	A0A1L4BJ98.1
Hemiscorpius lepturus	API81379.1
Centruroides sculpturatus	XP_023221538.1
Centruroides sculpturatus	XP_023222781.1
Centruroides sculpturatus	XP_023222756.1
Centruroides sculpturatus	XP_023222755.1
Centruroides sculpturatus	XP_023222769.1
Centruroides sculpturatus	XP_023237656.1
Megacormus gertschi	JAW07090.1
Araneus ventricosus	GBL90950.1
Nephila pilipes	GFT87468.1
Ixodes scapularis	XP_040074083.1
Tetranychus urticae	XP_015792030.1
Halotydeus destructor	KAI1280660.1
Parasteatoda tepidariorum	XP_015910970.2
Caerostris darwini	GIY48762.1
Nephila pilipes	GFT14276.1
Rhipicephalus microplus	XP_037273479.1
Dermacentor silvarum	XP_049512073.1
Dermacentor andersoni	XP_050050470.1
Zootermopsis nevadensis	XP_021931643.1
Orussus abietinus	XP_012275762.1
Diachasma alloeum	XP_015117785.1
Cotesia glomerata	XP_044577252.1
Atta colombica	XP_018056921.1
Apis florea	XP_003691388.1

Table 2

Quality assessment of predicted models of PLDs beta from different servers.
		PHYRE2		I-TASSER		Swiss-Prot
		o.q.f	Z-score	o.q.f	Z-score	o.q.f	Z-score
H. lepturus	PLD1 ScoTox-betaI isoform x1	75.8364	-7.39	96.4413	-8.16	94.052	-7.7
	PLD1 ScoTox-betaI isoform x3	81.5451	-6.84	95.7854	-7.18	95.6897	-7.39
	PLD1 ScoTox-betaI isoform x4	72.9614	-7.07	85.8238	-7.34	93.9655	-7.38
	PLD1 ScoTox-betaI isoform x5	70.9559	-6.04	90.2778	-7.78	94.4649	-7.65
	PLD1 ScoTox-betaI isoform x6	85.6618	-6.83	89.9306	-7.77	95.9559	-7.92
A.crassicauda	PLD1 ScoTox-betaI	78.4387	-6.25	82.906	-4.76	94.7955	-6.49
	PLD1 ScoTox-betaII isoform x2	66.2879	-7.11	74.6795	-8.26	93.9394	-7.43
	PLD2 ScoTox-alphaI	64.6362	-8.21	67.3052	-5.33	80.3191	-7.94
H. saulcyi	PLD1 ScoTox-betaI isoform x1	70.2602	-5.67	82.8804	-5.99	94.4238	-6.79
H. saulcyi	PLD2 ScoTox-alphaI	66.9477	-8.28	68.1023	-8.68	76.8617	-8.69
The overall quality factors (o.q.f) were estimated by SAVES server and Z-scores were estimated by ProSA-web server.

Table 3

Signal peptides and their cleavage sites of PLD1-beta genes.
		Signal Peptide (Sec/SPI)	Cleavage site	N-region	H-region	C-region
H. lepturus	PLD1 ScoTox-betaI isoform x1	0.9991	20 and 21	2	13	5
	PLD1 ScoTox-betaI isoform x3	0.97	32 and 33	16	13	3
	PLD1 ScoTox-betaI isoform x4	0.9989	32 and 33	16	13	3
	PLD1 ScoTox-betaI isoform x5	0.9951	32 and 33	16	13	3
	PLD1 ScoTox-betaI isoform x6	0.97	32 and 33	16	13	3
A.crassicauda	PLD1 ScoTox-betaI	0.9991	21 and 22	2	15	4
A.crassicauda	PLD1 ScoTox-betaII isoform x2	0.9991	19 and 20	3	13	3
H. saulcyi	PLD1 ScoTox-betaI isoform x1	0.9992	21 and 22	2	15	4
Sec/SPI: "standard" secretory signal peptides transported by the Sec translocon and cleaved by Signal Peptidase I (Lep)

Table 4

Physiochemical properties of PLD1-beta genes.
		Mature protein amino acids No	Molecular weight	Theoretical pI	Instability index	Aliphatic index	GRAVY
H. lepturus	PLD1 ScoTox-betaI isoform x1	289	33138.65	6.91	36.46	83.59	-0.497
	PLD1 ScoTox-betaI isoform x3	270	31106.86	5.82	37.61	75.81	-0.569
	PLD1 ScoTox-betaI isoform x4	270	31183.90	5.63	37.61	74.74	-0.612
	PLD1 ScoTox-betaI isoform x5	296	34224.60	5.81	28.78	81.01	-0.537
	PLD1 ScoTox-betaI isoform x6	296	34111.44	5.81	28.10	81.01	-0.550
A.crassicauda	PLD1 ScoTox-betaI	378	43823.41	6.29	38.19	82.30	-0.494
A.crassicauda	PLD1 ScoTox-betaII isoform x2	320	37498.90	5.55	41.66	94.75	-0.350
H. saulcyi	PLD1 ScoTox-betaI isoform x1	378	43689.21	6.41	32.41	83.07	-0.499
MW: Molecular weight; GRAVY: Grand average of hydropathicity

Table 5

Physiochemical properties of PLD2 ScoTox-alpha genes.
		Molecular weight	Theoretical pI	Instability index	Aliphatic index		GRAVY		Signal Peptide (Sec/SPI)
A. crassicauda	128307.65		9.07	44.89	91.88	-0.348		0
H. saulcyi	128165.45		9.10	44.38	91.27	-0.347		0

No competing interests reported.

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Identification, classification and characterization of the dermonecrotic toxins in venom glands of Hottentotta saulcyi, Androctonus crassicauda and Hemiscorpius lepturus using transcriptome analysis

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