Molecular characterization of a novel secretory phospholipase A2 as coding gene from venom glands of Iranian Scorpio maurus (Arachnida: Scorpionidae)


 Background

Scorpions as a venomous arthropod can defend and capture the prey by secretion of venom into their venomic sacs. The venom is a mixture of a variety of compounds with specific biological activities, such as phospholipase A2 (PLA2) enzyme to be present in scorpion venom. This study is the first investigation of structural and molecular characterization of PLA2 from Iranian scorpion, S. maurus that can be added to the literature when targeting molecular characterization of PLA2 coding gene from venom glands of Iranian S. maurus.
Results

 S. maurus PLA2 with 432 base pair (bp) nucleotide length encoded a protein of 144 amino acid residues and 16.34 kilodaltons (kDa). According to DiANNA 1.1 web server prediction, this protein had 5 disulfide bridges. The deposited PLA2 in GenBank (MW241004) was very similar to S. maurus (MF347455.1) with 95.14% identity. The phylogenetic analysis indicated that our PLA2 had a close relationship with insect PLA2. Our PLA2 had 38.83% identity with bee venom PLA2 according to SWISS-MODEL prediction and 100% confidence and 39% identity with insect phospholipase A2 family, which was predicted by Phyre2. Our PLA2 was similar to chain A of bee venom PLA2. Conserved domains on target PLA2 contained 17–118 amino acid in length and 45–52 amino acids in the active site. There was no signal peptide.
Conclusions

In the light of our results, the coding sequence of characterized PLA2 was a member of the PLA2 superfamily. According to the three-dimensional structure prediction, the target PLA2 with five disulfide bonds has a very high similarity to the structure of phospholipase A2 belonged to the group III subfamily. Interestingly, the active site residue of target PLA2 also has the His-Asp acid pair and the effective amino acid in catalytic domain of it was\({\text{H}\text{i}\text{s}}^{49}\).

Iran as a country located in the Middle East, due to its climate, has a good potential for scorpions living [25]. Fars province which is located in the southwest of Iran has hot and humid weather and scorpions are important public health problem in this region [26]. Scorpio maurus as a species of Scorpionidae family is a considerable species in this province [27]. The aim of this study was to identify PLA2 from the venom glands of Iranian scorpion, S. maurus based on a molecular characterization.

Characterization of the PLA2 coding gene
To decide the PLA2 coding sequence, polymerase chain reaction (PCR) reaction, which was performed by speci c primers on the synthesized complementary DNA (cDNA), appeared as a fragment close to the awaited size of 432 base pair (bp) (Figure1). The PLA2 sequence was deposited in GenBank under accession number (MW241004).
Analysis of cDNA and protein sequence of S. maurus PLA2 S. maurus PLA2 protein 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 (Figure 2 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 (0.2). The signi cant relationship between insect PLA2 was quite obvious. In addition, the phylogenetic analyses showed that the enzyme detected in human differed signi cantly from that of insects ( Figure 3). There was no signal peptide in target PLA2. The comparison of our PLA2 sequence with similarly characterized related proteins displayed a conserved catalytic site (active site), which was common in PLA2 superfamily of secretory and cytosolic enzymes. Conserved domains on target PLA2 contained 17-118 amino acids length ( Figure 5). According to ExPASy-PROSITE online tool, the active site of target PLA2 contained 45-52 amino acids length which was conserved among different species of scorpions ( Figure 2). The catalytic domain included "CCRTHDXC motif," which was the binding domain that supported the active site in PLA2 superfamily.

Prediction of protease cleavage sites
Based on PROSPER, three types of protease families cleavage the target PLA2 sequence were determined and shown in Table 3. According to PeptideCutter web server, the prediction enzymes including Caspase-1 to -10, Enterokinase, Factor Xa, GranzymeB, and Thrombin were demonstrated not to cut the target PLA2.

Discussion And Conclusion
The current study was the rst investigation of structural and molecular characterization of PLA2 from venom glands of Iranian S. maurus that can be added to the literature when targeting molecular characterization of PLA2 coding gene from venom glands of Iranian S. maurus. Phospholipase A2 (PLA2) is functionaries of the in ammatory process which catalyzes the hydrolyze phospholipids at the sn-2 position of the glycerol backbone and releases fatty acid and lysophospholipids [28,29]. Group III subfamily of PLA2s have been identi ed from various sources such as reptiles [30,31], mammals [32], parasites [33], and arthropods including scorpion [18,21]. The coding sequence of PLA2 was detected from S. maurus venom glands for the rst time in Iran. The detected PLA2 has encoded a protein of 144 amino acid residue to be 16.34 kDa. Till now, several studies reported the genes encoding phospholipases A2 from different species of scorpions including Hemilipin from H. lepturus [21], Imperatoxin I and Phospholipin from P. imperator [19,20] 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 Tunesian S. maurus (GeneBank: MF347455.1) [37], while this level of similarity is very low compared to other species of scorpions. Comparing the coding sequence of target PLA2 with other characterized PLA2 from different species of scorpions, was shown that our PLA2 was a member of the PLA2 superfamily.
Phylogenetically, there is a distant relationship between group I, II, and III of phospholipase A2, but at the site of calcium-binding and the region of active site, they are quite similar [38,39]. In the current study, the result of likelihood analysis showed that the characterized PLA2 from Iranian S. maurus and other arthropods, scorpions and Apis mellifera (FE373554.1), are well clustered, whereas this one from Homo sapience (M86400.1) is distantly located. similar to the phylogenetic relationship of phospholipases A2 from A. phaiodactylus [39].
The difference in 13 amino acids residue in the gene encoding sequence of phospholipase A2 enzyme between the Iranian and Tunisian S. maurus is most likely due to differences in the geographical condition in which they live. The residues , , , belonged to group III subfamily, but similarity to vertebrates' phospholipase A2 was smaller. Phylogenetic analysis also con rms these results. Similar to characterized PLA2 of A. phaiodactylus [39], Imperatoxin I of P. imperator [19], our PLA2 is closely related to genomic structure of A. mellifera, the only known representative structure in group III PLA2. Evaluation of the 3D structure of the target gene revealed that the identi ed protein was very similar to the chain A of characterized PLA2 from bee venom. Four disul de bonds were predicted for phospholipase A2 of M. tamulus at the position of eight cysteines (Cys8-Cys30), (Cys29-Cys68), (Cys35-Cys61) and (Cys59-Cys96) [35]. It is similar to the position of the human group III PLA2 disul de bonds in ten cysteines (Cys8-Cys30), (Cys29-Cys68), (Cys35-Cys61), (Cys59-Cys91), and (Cys101-Cys113) [40], while different positions were predicted to the ve disul de bonds in target PLA2. The histidine-aspartic (His-Asp) acid pair has been suggested to be necessary for the catalytic mechanism of the phospholipase A2 [41]. In this study, the active site residue of target PLA2 also has the His-Asp acid pair and the effective amino acid in catalytic domain of target PLA2 was while played a major role in the active site residue of bee's venom and Tunisian S. maurus [37].

Scorpion collection
The scorpions were collected from Fars Province, Zarrin Dasht County, south-west of Iran, and transferred alive to the laboratory of Medical Entomology in Shiraz University of Medical Sciences, Shiraz, Iran. Samples were identi ed via a valid key [42]. Before RNA extraction, venom of the collected scorpions was milked manually to release the venom. Three days after venom milking, the telson of the scorpions were 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 the Shiraz University of Medical Sciences.

Preparation of cDNA
Total RNA was extracted from the venom glands of S. maurus telson by High Pure RNA Isolation Kit, Roche®. RNA sample was treated enzymatically by DNase based on manufacturer's manual. Extracted RNA concentration was measured using a Nanodrop (analytikjena®).
H i s 4 9 , H i s 3 4 Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) According to 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 lled up to the 20 μL volume with Diethylpyrocarbonate-treated water (DEPC DW) and was dissolved by vortexing. cDNA synthesis reaction was performed in four steps according to 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 PCR ampli cation steps as a template.

Polymerase Chain Reaction (PCR)
The desired DNA sequences were ampli ed in total 20 μL volume containing 10 μL Taq DNA Polymerase Master Mix RED (2X), 1 μL forward primer, 1 μL of reverse and nally 1 μL of synthesized cDNA. It was performed to 35 cycles of 30 s at 94°C as denaturation, 30 sec at 50°C as annealing temperature, 30 sec at 72°C as extension and 10 min at 72°C for a nal extension.
DNA gel puri cation and sequencing PCR products were run onto 2% tris borate EDTA (TBE) agarose gel with appropriate DNA ladder. Our speci c band was observed in the gel documentation instrument. Selected band was puri ed according to protocol of Gene All kit. Expected size bands were sequenced using GSPs forward and reverse.

Phylogenetic analysis
To evaluate the target PLA2 protein, a phylogenetic tree was created based on different characterized related protein sequences including scorpion species, Apis mellifera, and Homo sapiens by utilizing the maximum likelihood method. Multiple sequences alignment for evolutionary analyses were conducted in MEGA7 software based on the Clustal W method [43]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) were shown next to the branches [44].
Structural characteristics of S. maurus PLA2 MEGA software (Version 7.0) was used for alignments. All primers were designed by the GeneRunner (Version 4) and Oligo 7 software. The designed primers speci city was determined using Primer-BLAST on NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). To identify the structural features of the protein coded by PLA2 and guarantee that obtained sequence could be a part of phospholipase family proteins, the S. maurus PLA2 was translated by GeneRunner software (Version 4.0), and the concluded sequence was evaluated by diverse instruments. Protein BLAST was performed to decide proteins with great closeness to recognize and record their characterization. To compare and classify protein structure, similar proteins were chosen for alignment with Clustal Omaga (https://www.ebi.ac.uk/Tools/msa/clustalo/). Amino acid compounds of S.maurus PLA2 were analyzed by utilizing ProtParam online tool (https://web.expasy.org/protparam/). The PLA2 coding gene was translated by GeneRunner software (version 4). A protein BLAST was also carried out for more comparative relation. To disul de bridge prediction of the target protein, DiANNA 1.1 web server was used [45,46]. Disul de bridges formation are essential for biological activity in many proteins [47]. To predict disul de bonds, DiANNA 1.1 web server (http://clavius.bc.edu/~clotelab/DiANNA/) was used.

Three-Dimensional structure prediction
In order to predict the 3D structure, the SWISS-MODEL online tool (https://swissmodel.expasy.org/) and Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index ) were used, which predicted 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).
Active site structure Several online tools were used to evaluate the signal peptide of the gene in question. PLA2 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) [8,9]. Superimposition of active site was carried out by UCSF Chimera (Version 1.14) and DeepView/Swiss-pdbviewer (Version 4.10) software ( Figure 3) (34). The root-mean-square deviation (RMSD) of bee venom PLA2 and target protein 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 ( Table 2). Active-site residues were universally conserved within protein families, displaying their key role for substrate catalysis [48]. The active site prediction was undertaken by ExPASy-PROSITE (https://prosite.expasy.org/). Several online devices were used for signal peptide analysis such as     Phylogenetic relationship between different species of scorpion, Apis mellifera, and Homo sapiens based on PLA2 sequences. The accession number of each sequence has been shown in front of its name. The percentage of replicate trees in which the related taxa clustered together in the bootstrap test (1000 replicates) has been displayed next to the branches. The scale bar corresponds to 0.2 substitutions per nucleotide Figure 6 Nucleotides and predicted amino acid sequence of S. maurus PLA2. Nucleotide and amino acid numbers appeared on the left. The middle-letter at above the nucleotide sequence is coded the amino acid sequence. Coding sequence and predicted amino acid sequence. Coding sequence and predicted amino acid sequence of PLA2. The disulphide bonds between cysteine residues in the protein structure have been signed by black boxes. The conserved domines of target PLA2 amino acid length with PLA2 superfamily have been shown by black line.