Abstract
The venom is a mixture of various compounds with specific biological activities, such as the phospholipase () enzyme present in scorpion venom. plays a key role in inhibiting ryanodine receptor channels and has neurotoxic activity. This study is the first investigation of molecular characterization, cloning, and in silico analyses of from Iranian Scorpio maurus, named Maurolipin. After RNA extraction from S. maurus venom glands, cDNA was synthesized and amplified through RT-PCR using specific primers. Amplified Maurolipin was cloned in TA cloning vector, pTG19. For in silico analyses, the characterized gene was analyzed utilizing different software. Maurolipin coding gene with 432 base pair nucleotide length encoded a protein of 144 amino acid residues and 16.34 kilodaltons. Comparing the coding sequence of Maurolipin with other characterized from different species of scorpions showed that this protein was a member of the superfamily. According to SWISS-MODEL prediction, Maurolipin had 38.83% identity with bee venom with 100% confidence and 39% identity with insect phospholipase family, which Phyre2 predicted. According to the three-dimensional structure prediction, Maurolipin with five disulfide bonds has a very high similarity to the structure of that belonged to the group III subfamily. The in silico analyses showed that phospholipase coding gene and protein structure is different based on scorpion species and geographical condition in which they live.
1. Background
The order of scorpions is the oldest one of venomous arthropods living over 400 million years ago [1, 2]. So far, over 2360 scorpion species have been identified and classified as 19 families [3]. The scorpion species have venom glands used to secrete venom into venom sacs to defend and capture the prey [4]. There are various scorpion species, which the stings cause respiratory dysfunctions, cardiotoxicity, and neurotoxicity [5]. The secreted compounds from the scorpion venom gland are a complex mixture of proteins, nonproteins, and some unknown compounds with unknown functions, including inorganic salts, mucoproteins, carbohydrates, peptides, lipids, free amino acids, nucleotides, water, and enzymes such as phospholipase () [6–8]. plays a vital role in inhibiting ryanodine receptor channels and has neurotoxic activity [9].
is an enzyme that hydrolyzes glycerophospholipids at the position of sn‐2 of phospholipids and releases free fatty acids (oleic acid (OA) and arachidonic acid (AA)) and lysophospholipids [10, 11]. This superfamily is classified into 15 groups and many subgroups [12]. superfamily has six principal types, including cytosolic (), platelet-activating factor cetyl hydrolases (PAF-AH), -independent (), lysosomal , adipose-specific (AdPLA2), and secreted phospholipase () [12–14].
as a calcium-dependent enzyme is a typical enzyme in the venom of a snake, lizard, bee, and scorpion that has been identified till now [10, 15–17]. This low-molecular weight enzyme is 13-15 kilodaltons (kDa) [18]. So far, 17 isoforms of have been identified and divided into groups I-III, V, and IX-XIV [19]. from the venom glands of scorpions is classified as a group III subfamily [20]. has been identified from different species of scorpions till now, including Pandinus imperator [21, 22], Hemiscorpius lepturus [23–25], Anuroctonus phaiodactylus [20], Heterometrus fulvipes [26], Heterometrus laoticus [10], and Tunisian Scorpio maurus [27].
As a country located in the Middle East, due to its climate, Iran has a good potential for scorpions’ life [28]. According to previous studies, Iran has one of the highest ranks in terms of scorpion envenomation over the world [29]. The last research has shown that Iran possesses 68 scorpion species that comprise 19 genera and four families [30]. The data estimated that the south and southwest of Iran possess about 95% of scorpion species, so were known as densely populated areas [31]. Fars Province, located in the southwest of Iran, has hot and humid weather and scorpions are important public health problem in this region [32]. Scorpio maurus, as a species of the Scorpionidae family, is a notable species in this province [33].
2. Objectives
This study aimed to identify from the venom glands of Iranian scorpion, S. maurus, based on molecular characterization and in silico analyses.
3. Methods
3.1. Scorpion Collection
The scorpions were collected from Fars Province, Zarrin Dasht County, in the southwest of Iran and transferred alive to the laboratory of Medical Entomology in Shiraz University of Medical Sciences, Shiraz, Iran. The morphological characteristics of this species are shown in Figures 1(a)-1(c). Samples were identified via a valid key [30]. Before RNA extraction, the venom of the collected scorpions was milked manually to release the venom. Three days after venom milking, the telson of one scorpion was separated and stored at −70°C. Other parts of the body were stored at ethanol 96% and were kept in the archives of the Museum of the Department of Medical Entomology in Shiraz University of Medical Sciences.
3.2. Primer Designing
To design the gene-specific primers (GSPs), researchers obtained the mRNA sequences of from different species of scorpions such as Tunisian S. maurus (GenBank : MF347455), H. lepturus (GenBank : KX924472), A. phaiodactylus (GenBank : AY571967.1), and Opisthacanthus cayaporum (GenBank : FM998793.1) from the National Center for Biotechnology Information (NCBI). The standard size band was 432 base pair (bp). The specific primers were 5′-TCCAAAGAAGAAATGGA (forward primer) and 5′-GTCTTTGTAGCTCTTTTTCCAGG (reverse primer).
3.3. RNA Extraction
Total RNA was extracted from the venom glands of one S. maurus telson by using a High Pure RNA Isolation Kit, Roche®. The RNA sample was treated enzymatically by DNase based on the manufacturer’s manual. Extracted RNA concentration was measured using a Nanodrop (Analytik Jena®).
3.4. Reverse Transcription Polymerase Chain Reaction (RT-PCR)
According to the manufacturer’s instruction, 3 μL of total RNA was used as a template for cDNA synthesis by AccuPower® CycleScript RT Premix with (d) (Bioneer Company, Korea). 0.1 to 1 μg of RNA template was filled up to the 20 μL volume with sterile water and was dissolved by vortexing. cDNA synthesis reaction was performed in four steps according to the manufacturer’s manual, including 30 sec at 25°C for primer annealing, 4 min at 45°C for cDNA synthesis, 30 sec at 55°C for melting secondary structure and cDNA synthesis, and 5 min at 95°C for heat inactivation. The synthesized cDNA was kept at −70°C for amplification steps as a template. Researchers amplified the desired DNA sequences in total 20 μL volume containing 10 μL Taq DNA Polymerase Master Mix RED (2X), 1 μL of forward primer, 1 μL of reverse primer, 1 μL of synthesized cDNA, and finally 7 μL of sterile water. It was performed to 35 cycles of 30 sec at 94°C as denaturation, 30 sec at 50°C as annealing temperature, 30 sec at 72°C as an extension, and 10 min at 72°C for a final extension.
3.5. TA Cloning
PCR products were run onto 1% tris borate EDTA (TBE) agarose gel with an appropriate DNA ladder (100 bp). Our specific band was observed in the gel documentation instrument, and the selected band was purified according to the protocol of GeneAll® kit.
The purified PCR product was ligated to the linearized pTG19 vector with a TA cloning kit (Vivantis®) according to the manufacturer’s instructions in 1 : 3 ratio. Escherichia coli strain DH5α competent cells were prepared before ligation reaction and stored at −70°C. Ligation mixtures were transformed in prepared competent cells by heat shock method. Blue-white screening technique was used on Luria-Bertani (LB) agar plates containing ampicillin (100 μg/ml), 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) (40 μg/ml), and isopropyl ß-D-1-thiogalactopyranoside (IPTG) (40 μg/ml) for detection of recombinant colonies . Each white colony was suspended in 30 μl of sterile water and boiled for 10 min. 1 μl of that has been used as a DNA template in PCR reaction for the characterization of recombinant colonies containing target insert using specific and universal M13 primers. After detecting the recombinant colonies based on PCR technique, plasmid DNA was extracted using the GeneAll® plasmid isolation kit according to manufacturer’s manual. Maurolipin coding gene was sequenced using universal M13 forward and reverse primers.
3.6. Phylogenetic Analysis
To evaluate Maurolipin coding sequence, researchers created a phylogenetic tree based on deposited protein-coding sequences in GenBank, including Heterometrus fulvipes (GenBank : DQ146998.1), Mesobuthus tamulus (GenBank : AY443497.1), Pandinus cavimanus (GenBank : JN315724.1), Hadrurus spadix (GenBank : GFAH01000435.1), Mesobuthus gibbosus (GenBank : KF770818.1), Anuroctonus phaiodactylus (GenBank : EF364043.1), Hemiscorpius lepturus (GenBank : KX924472.1), Hemiscorpius lepturus (GenBank : BK059885.1), Opisthacanthus cayaporum (GenBank : FM998793.1), Scorpio maurus (GenBank : MF347455.1), Apis mellifera (GenBank : EF373554.1), and Homo sapiens (GenBank : M86400.1) by utilizing the maximum likelihood method [34]. Multiple sequence alignment for evolutionary analyses was conducted in MEGA7 software based on the Clustal W method [35]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) was shown next to the branches [36].
3.7. In Silico Analyses
3.7.1. Structural Characteristics of Maurolipin
MEGA software (Version 7.0) was used for the sequence alignments. All primers were designed by the Gene Runner (Version 4) and Oligo 7 software. The designed primer specificity was determined using Primer-BLAST on NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi). To identify the structural feature of Maurolipin and guarantee that the obtained sequence could be a part of phospholipase family proteins, Maurolipin coding sequence was translated by Gene Runner software (Version 4.0), and diverse instruments evaluated the concluded sequence. Protein BLAST (https://www.ncbi.nlm.nih.gov) was performed to identify proteins with great closeness to recognize and record their characteristics. To compare and classify protein structure, researchers chose similar proteins for alignment with Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). Amino acid compounds of Maurolipin were analyzed by utilizing ProtParam online tool (https://web.expasy.org/protparam/). To disulfide bridge prediction of the target protein, DiANNA 1.1 web server (https://clavius.bc.edu/∼clotelab/DiANNA/) was used [37, 38]. Disulfide bridge formation is essential for biological activity in many proteins [39].
3.7.2. Three-Dimensional Structure Prediction
In order to predict the three-dimensional (3D) structure, the SWISS-MODEL online tool (https://swissmodel.expasy.org/) and Phyre2 (https://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=indexfulvip) were used to predict the 3D structure of a query protein through the sequence alignment of template proteins. This model was chosen for superimposition. The predicted 3D structure was evaluated by UCSF Chimera software (Version 1.14).
3.7.3. Active Site Structure
breaks the sn-2 position of the glycerol backbone of phospholipids, mainly in a metal-dependent reaction, to produce lysophospholipid (LysoPL) and a free fatty acid (FA) [10, 11]. Superimposition of active site was carried out by UCSF Chimera (Version 1.14) and DeepView/Swiss-PdbViewer (Version 4.10) software. The root-mean-square deviation (RMSD) of bee venom and Maurolipin active-site residue was calculated by Chimera software (Version 1.14) to measure the average distance between corresponding atoms in two protein chains based on carbon alpha atoms. The active site prediction was undertaken by ExPASy-PROSITE (https://prosite.expasy.org/).
3.7.4. Prediction of Protease Cleavage Sites
PROSPER (Protease Specificity Prediction Server) (https://prosper.erc.monash.edu.au/webserver.html) and PeptideCutter (https://web.expasy.org/peptide_cutter/) servers were used for prediction of the activated form of target protein.
4. Results
4.1. Characterization of Maurolipin Coding Sequence
To identify Maurolipin coding sequence, RT-PCR was performed by specific primers on the synthesized complementary DNA (cDNA), which appeared as a fragment close to the expected size of 432 (bp) (Figure 1(d)). About colony PCR, the expected size of PCR product amplified using universal M13 primers was 580 bp (Figure 2). Maurolipin coding sequence was deposited in GenBank under accession number (MW241004).
4.2. Analysis of Maurolipin Coding Sequence
Maurolipin coding sequence contained a 432 bp open reading frame (ORF) that encoded a protein of 144 amino acid residues with a predicted molecular mass of 16.34 kDa. The amino acid sequence of Maurolipin was evaluated by Gene Runner software. The BLAST result showed that Maurolipin coding protein sequence was similar to Tunisian S. maurus (Sm-PLVG) (GenBank : MF347455.1) with 95.14% identity. Other listed proteins with high identity levels in BLAST search were 74.07% similar to Hemilipin from H. lepturus (GenBank : KX924472.1), 78.12% to from O. cayaporum (GenBank : FM998793.1), and 74.12% to from A. phaiodactylus (GenBank : EF364043.1). Maurolipin protein-coding sequence was assessed using protein BLAST alignment.
4.3. Phylogenetic Analysis
Initial tree(s) for the heuristic search were received automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach and then selecting the topology with higher level logarithm likelihood value. Instructive branch lengths were typically drawn to scale and showed the number of substitutions per site (0.1). The significant relationship between insect was quite evident. In addition, the phylogenetic analyses showed that the enzyme detected in humans differed significantly from that of insects (Figure 3).
4.4. In Silico Analyses
4.4.1. Structural Characteristics of Maurolipin
The consequences indicated that all selected proteins were associated with insect . Above all, Maurolipin is high similar to Tunisian S. maurus (Sm-PLVG) (GenBank : AVD99009.1) with 90.28% identity. Sequence alignment of Maurolipin coding gene with three reported from different scorpion species including S. maurus (GenBank : AVD99009.1), H. fulvipes (GenBank : Q3YAU5.1), and H. lepturus (GenBank : A0A1L4BJ46.1) was performed by Clustal Omega and conserved amino acids were highlighted (Figure 4). The conserved amino acids between the two S. maurus species sequences of Maurolipin and Sm-PLVG (GenBank : AVD99009.1) were analyzed. Maurolipin did not resemble Sm-PLVG (GenBank : AVD99009.1) in 13 amino acids. The amino acid compounds of Maurolipin are shown in Table 1. The predicted disulfide bonds in five positions were between (Cys8 and Cys45), (Cys25 and Cys46), (Cys52 and Cys75), (Cys77 and Cys84), and (Cys115 and Cys131) (Figure 5).
4.4.2. Three-Dimensional Structure Prediction
The three-dimensional (3D) structure of Maurolipin by SWISS-MODEL demonstrated that Maurolipin was similar to chain A of bee venom , group III subfamily, with 38.83% identity. The predicted 3D structure demonstrated that Maurolipin resembled chain A of bee venom (Figure 6). Superimposition of 3D structure of bee venom and S. maurus is shown in Figure 7. The 3D structure of Maurolipin by Phyre2 displayed that the target sequence was close to the insect family with 100% confidence and 39% identity. On the other hand, Maurolipin was similar to vertebrate with 97.3% confidence and 33% identity.
4.4.3. Active Site Structure
The comparison of Maurolipin sequence with similarly characterized related proteins displayed a conserved catalytic site (active site), which was common in the superfamily of secretory and cytosolic enzymes. Conserved domains on Maurolipin were in the position of 17 to 118 amino acids length. According to the ExPASy-PROSITE online tool, the Maurolipin active site was in the position of 45 to 52 of amino acids length, which was conserved among different species of scorpions (Figure 4). The comparison of Maurolipin and bee venom active site was done by calculating the root mean square deviation (RMSD), which is reported in Table 2. The catalytic domain included the “CCRTHDXC motif” and the binding domain that supported the active site in the superfamily.
4.4.4. Prediction of Protease Cleavage Sites
Based on PROSPER, three types of protease family cleavage Maurolipin coding sequences were determined and shown in Table 3. According to the PeptideCutter web server, the prediction enzymes including caspase-1 to −10, enterokinase, factor Xa, granzymeB, and thrombin cannot cut Maurolipin coding sequence.
5. Discussion and Conclusion
Prediction of protein structure is the focus of interest of many investigators. The current study was the first investigation of molecular characterization and in silico analyses of Maurolipin structure from venom glands of Iranian S. maurus that can be added to the literature when targeting molecular characterization of coding gene from venom glands of Iranian S. maurus. Phospholipase () has a relevant role in of the inflammatory process, which catalyzes the hydrolyze phospholipids at the sn-2 position of the glycerol backbone and releases fatty acid and lysophospholipids [40, 41]. Group III subfamily of has been identified from various sources such as reptiles [42, 43], mammals [44], parasites [45], and arthropods including scorpion [20, 23]. The coding sequence of was detected from S. maurus venom glands for the first time in Iran. The detected has encoded a protein of 144 amino acid residue named Maurolipin. Till now, several studies reported the genes encoding phospholipases from different species of scorpions, including Hemilipin from H. lepturus [23], Leptulipin from H. lepturus [25], Imperatoxin I and Phospholipin from P. imperator [21, 22], Phaiodactylipin from A. phaiodactylus [20], Heteromtoxin from Heterometrus laoticus [10], Phospholipase from H. fulvipes [46], MtPL from Mesobuthus tumulus [47], Sm-PLVG from Tunisian S. maurus [27], and Phospholipase from Hadrurus gertschi [48].
The results of BLAST in NCBI showed that the detected sequence from the Iranian Scorpio maurus has a very high similarity to the same sequence of Tunisian S. maurus (GenBank : MF347455.1) [49]. However, this level of similarity is very low compared to other species of scorpions. Comparing the coding sequence of Maurolipin with other characterized from different species of scorpions showed that Maurolipin was a member of the superfamily.
Phylogenetically, there is a weak relationship between groups I, II, and III of phospholipase , but at calcium-binding site and the active site region, they are pretty similar [50, 51]. In the current study, the result of the likelihood analysis showed that the characterized from Iranian S. maurus and other arthropods, scorpions, and Apis mellifera (GenBank : FE373554.1) are well clustered. In contrast, the one from Homo sapiens (GenBank : M86400.1) is distantly located similar to the phylogenetic analyses of A. phaiodactylus [51].
The difference in 13 amino acids in the gene encoding sequence of Maurolipin and Sm-PLVG is most likely due to differences in the geographical condition in which they live, because the structure of a protein reflects its genetic sequence. The residues , , , , , , , , , , , and of Maurolipin were Val, Ser, Leu, Val, Lys, Phe, Phe, Thr, Asn, Asp, Asp, Asp, and Asn in Sm-PLVG (GenBank : AVD99009.1), respectively. Indeed, the difference in the amino acid residue is more significant among different species of scorpions.
Based on the three-dimensional structure prediction results, Maurolipin is highly similar to the structure of phospholipase that belonged to the group III subfamily; however, it was slightly similar to vertebrates’ phospholipase . Phylogenetic analysis also confirms these results. Similar to characterized of A. phaiodactylus [51] and Imperatoxin I of P. imperator [21], Maurolipin is closely related to the genomic structure of A. mellifera, the only known representative structure in group III . Evaluation of the 3D structure of the target gene revealed that the identified protein was very similar to the chain A of characterized from bee venom. Four disulfide bonds were predicted for phospholipase of M. tamulus at the position of eight cysteines (Cys8-Cys30), (Cys29-Cys68), (Cys35-Cys61), and (Cys59-Cys96) [47]. It is similar to the position of the human group III disulfide bonds in ten cysteines (Cys8-Cys30), (Cys29-Cys68), (Cys35-Cys61), (Cys59-Cys91), and (Cys101-Cys113) while different positions were predicted to the five disulfide bonds in Maurolipin [52]. The histidine-aspartic (His-Asp) acid pair is stated necessary for the catalytic mechanism of the phospholipase . Active-site residues were universally conserved within protein families, displaying their key role for substrate catalysis [53]. In this study, RMSD of Maurolipin and bee venom was calculated. The RMSD value computes the average deviation between the equivalent atoms of two proteins depending on conformational differences and structural dimensions [54, 55]. The smaller the RMSD, the further similar the two structures. In this study, the active site residue of Maurolipin also has the His-Asp acid pair, and the effective amino acid in the catalytic domain was while played a significant role in the active site residue of bee’s venom and Sm-PLVG [49].
Abbreviations
| : | Phospholipase |
| c: | Cytosolic |
| PAF-AH: | Platelet-activating factor cetyl hydrolases |
| : | Independent |
| Ad: | Adipose-specific |
| s: | Secreted phospholipase |
| OA: | Oleic acid |
| AA: | Arachidonic acid |
| cDNA: | Complementary DNA |
| PCR: | Polymerase chain reaction |
| bp: | Base pair |
| kDa: | Kilodaltons |
| ORF: | Open reading frame |
| X-Gal: | 5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranoside |
| IPTG: | Isopropyl ß-D-1-thiogalactopyranoside |
| LB: | Luria-Bertani |
| MCL: | Maximum composite likelihood |
| 3D: | Three-dimensional structure |
| NCBI: | National Center for Biotechnology Information |
| RT-PCR: | Reverse Transcription Polymerase Chain Reaction |
| μL: | Microliter |
| Met: | Methionine |
| TBE: | Tris borate EDTA |
| GSPs: | Gene-specific primers |
| RMSD: | Root-mean-square deviation |
| Thr: | Threonine |
| Asn: | Asparagine |
| Lys: | Lysine |
| Tyr: | Tyrosine |
| Asp: | Aspartic acid |
| Cys: | Cysteines |
| His: | Histidine |
| Val: | Valine |
| Leu: | Leucine |
| Phe: | Phenylalanine |
| Glu: | Glutamic acid |
| Ser: | Serine |
| Pro: | Proline. |
Data Availability
All data generated or analyzed during this study are available upon reasonable request to the corresponding author.
Ethical Approval
No human or animal data or tissue was used in this study. All experiments were conducted in accordance with the ethical principles and the national norms and standards for conducting Medical Research in Iran. The study was approved by Iran National Committee for Ethics in Biomedical Research (Approval ID : IR. SUMS. REC.1399.87). Specimens (Scorpions) were not client-owned animals. The authors collected them from the natural habitats (Zarrin Dasht County, Fars Province, South of Iran) with the permission of Shiraz University of Medical Sciences following the Fars Environmental Protection Organization.
Consent
No written consent has been obtained from the patients as there is no patient identifiable data included.
Conflicts of Interest
The authors declare that there are no conflicts of interest.
Authors’ Contributions
PS-A and KA conceived and designed the study and contributed to the discussion of results. HA, AS, QA, and AR conceived the study and analyzed data; PS-A, KA, and DM drafted the manuscript. All authors have read and approved the final manuscript.
Acknowledgments
The authors are appreciative of the financial support provided by the Shiraz University of Medical Sciences. The authors are very grateful to Mr. Ali Dianat for collecting scorpions and Mrs. Sorna Dabbaghmanesh and Mr. Amin Hosseinpour, the Medical Entomology Laboratory staff at the School of Health, for their kind support. This article is part of the results of the first author’s Ph.D. thesis in Medical Entomology and Vector Control at the Department of Medical Entomology and Vector Control, School of Public Health, Shiraz University of Medical Sciences (SUMS), Shiraz, Iran. This investigation was funded by Shiraz University of Medical Sciences (SUMS), Shiraz, Iran (Grant No: 98-01-04–20786). This article previously had been located, online, as preprint in the Research Gate entitled “Molecular characterization of a novel secretory phospholipase as coding gene from venom glands of Iranian Scorpio maurus (Arachnida: Scorpionidae)” according to the following link https://www.researchsquare.com/article/rs-549530/v1 [56].