Turk J Biochem 2018; 43(6): 651–661

Research Article

Xichao Xia*, Yang Liu, Jianxin Huang, Xiaozhu Yu, Zhiguo Chen, Xinhua Zheng, Fuan Wang, Junfeng Zhang, Shipeng Xue and Zhaofei Cheng Molecular cloning and in silico characterization of two alpha-like neurotoxins and one metalloproteinase from the maxilllipeds of the subspinipes mutilans İki alfa benzeri nörotoksinlerin ve bir adet metalloproteinazın silico karakterizasyonunda kırkayak klonlanması ve kırkayak ekstraktlarının maksilet spp. https://doi.org/10.1515/tjb-2018-0009 divergence and independent evolution of SsuTA1 and Received November 27, 2017; accepted February 13, 2018; previously SsuTA2 from other α-neurotoxins. published online March 28, 2018 Conclusion: The results suggested that centipede S. sub- Abstract spinipes mutilans is an ancient member of venomous , but its venom exhibits novel scenario. Aims: In order to shed light of characterizations of cen- Keywords: Deduced protein sequences; Alpha-like-neuro- tipede Scolopendra subspinipes mutilans venom, a two toxin; Metalloproteinase; Scolopendra subspinipes muti- novel full-lengths of alpha-like-neurotoxin and one met- lans; Neglect group. alloproteinase cDNAs derived from the maxilllipeds RNA of centipede S. subspinipes mutilans were isolated, and, respectively, named as SsuTA1, SsuTA2 and SsuMPs. Özet Materials and methods: The SsuTA1, SsuTA2 and SsuMPs were cloned from the S. subspinipes mutilans using the Amaçlar: Kırkayak çağı scolopendra subspinipes muti- rapid amplification of cDNA ends methods. lans venomunun karakterizasyonlarının ışığında, iki adet Results: In the current study, SsuTA1 and SsuTA2 were, tam uzunlukta alfa-benzeri-nörotoksin ve santrifüj S. respectively, composed of 82 amino acid residues and subspinipes mutilansının maksilllipre RNA’sından türetil- 106 amino acid residues. Deduced protein sequence of miş bir metalloproteinaz cDNA’sı izole edildi ve sırasıyla SsuTA1 shared high homology with that of SsuTA2, one SsuTA1, SsuTA2 ve SsuMPs. major difference was the C-terminal 24-residue exten- Gereç ve Yöntemler: SsuTA1, SsuTA2 ve SsuMP’ler, S. sion in SsuTA2. An abundance of cysteine residues and subspinipes mutilanslarından cDNA uçlarının hızlı amp- several adjacent beta-sheets were observed in SsuTA1 lifikasyonu kullanılarak klonlanmıştır. and SsuTA2. SsuMPs had 594 amino acid residues con- Bulgular: Mevcut çalışmada SsuTA1 ve SsuTA2 sırasıyla 82 taining with a molecular mass of 68.29 kDa. The primary amino asit artığı ve 106 amino asit kalıntısından oluşmak- sequence analysis indicated that the SsuMPs contains a tadır. SsuTA1’in indirgenmiş protein sekansı, SsuTA2’ninki zinc-­binding motif (HEIGHSLGLAHS) and methionine- ile yüksek benzerlik paylaştı, önemli bir fark SsuTA2’deki turn motif (YIM). Phylogenetic analysis revealed early C-terminali 24-artık uzantısıydı. SsuTA1 ve SsuTA2’de çok sayıda sistein kalıntısı ve birkaç bitişik p-tabaka gözlen- miştir. SsuMPs, 68.29 kDa’lık bir moleküler kütle içeren 594 *Corresponding author: Xichao Xia, Medical College of Pingdingshan University, Pingdingshan 467000, Henan Province, China; and Basic amino asit tortusuna sahipti. Birincil dizi analizi SsuMP’le- Medicine College of Nanyang Medical University, Nanyang 473041, rin bir çinko bağlayıcı motif (HEIGHSLGLAHS) ve metiyo- Henan Province, China, e-mail: [email protected]. nin dönüş motifi (YIM) içerdiğini göstermiştir. Filogenetik http://orcid.org/0000-0001-7836-8110 analizler SsuTA1 ve SsuTA2’nin diğer α-nörotoksinlerden Yang Liu, Jianxin Huang, Xiaozhu Yu, Zhiguo Chen, Xinhua Zheng, erken ayrışmasını ve bağımsız evrimini ortaya çıkardı. Fuan Wang and Zhaofei Cheng: Medical College of Pingdingshan University, Pingdingshan 467000, Henan Province, China Sonuç: Sonuçlar sentipede S. subspinipes mutilans’ın Junfeng Zhang and Shipeng Xue: Basic Medicine College of Nanyang eski bir zehirli artropod üyesi olduğunu, ancak zehirinin Medical University, Nanyang 473041, Henan Province, China yeni bir senaryo sergilediğini ortaya koymuştur. 652 Xichao Xia et al.: Characterization of two alpha-like neurotoxins and one metalloproteinase

Anahtar Kelimeler: Ortaya çıkan protein dizileri; Alfa characterize these proteins and small polypeptides from benzeri nörotoksin; Metalloproteinaz; Scolopendra subs- different organisms [18, 19]. In the previous study, we have pinipes mutilans; İhmal grubu. isolated metalloprotease and hyaluronidase from venom of Buthus martensi [20, 21]. Centipede Scolopen- dra subspinipes mutilans is a traditional Chinese medicine and has been successfully used to treat immune-related Introduction diseases, especially rheumatoid arthritis [22–24]. In the current study, two novel sequences of alpha-like-neuro- Venomous are an important group of toxin and one metalloprotease sequence were cloned from kingdom. Their venomous glands can generate a variety the centipede S. subspinipes mutilans venom for the first of toxins composed of small polypeptides which can act time, and molecular structures of these sequences were on multiple sites of preys and/or predators [1, 2]. In addi- elucidated by bioinformatic methods. tion, these glands produce few water-soluble enzymes which are contribute to digested of food and facilitate the action of other toxic components of the venom [3]. Inter- estingly, these small polypeptides and enzymes also func- Materials and methods tion as important role on the neuromuscular system, the vascular system, the blood coagulation cascade and mem- Venom brane integrity and so on [4, 5]. Therefore, some enzymes and polypeptides have also been attained great concern were brought from special animal farm of and their functions are gradually been shed light [6, 7]. Nanyang and identified as S. subspinipes mutilans which Centipedes, one important group of arthropods, is also named as S. subspinipes subspinipes by current are belong to the chilopod class. The body is composed ­. The fresh centipede maxillipeds were dis- of head and thorax. Each thorax segment has one pair sected, snap frozen in liquid nitrogen and stored at −80°C of ambulatory legs besides of the one in the hindmost until use. segment where these structures are involved into mechan- ical defense and/or sensory purposes [8]. Another pair of modified legs is named the forcipules and sited at the Total RNA isolation and reverse transcription post-cephalic segment. A venom gland is located at the femoral part of the forcipule. The venom is used to sub- Total RNA was isolated from maxillipeds using TRIzol jugate prey and defense against predators [9]. Unlike (Takara, Dalian, China) according to the manufacturer’s , and insect, venom systems of them are protocol. The integrity of RNA was monitored by 1.2% extensively studied, a few studies are reported about the agarose gel electrophoresis. The concentration of RNA ancient venom system of centipedes. was accurately calculated by the ratio of the OD260/ Alpha-neurotoxins are classified into different groups OD280. The reverse transcription was carried out based on based on the preferential toxicity to mammals or insects M-MLV First-Strand cDNA synthesis Kit (Takara, Dalian, as well as their differential binding properties (classi- China) instructions using the DNase I (Takara, Dalian, cal alpha-mammal, alpha-like neurotoxins and alpha- China)-treated total RNA as template and Oligo (dT18)- insect neurotoxins) [10]. A number of alpha-neurotoxin adaptor as primer (Takara, Dalian, China). The reaction sequences had been cloned and characterized from , mixtures were treated at 72°C for15 min, next incubated scorpion and spider [11–13]. Venom metalloprotease is an at 42°C for 1 h, and last terminated by heating at 72°C for important part of venom and generally regarded as a hem- 10 min. The reverse transcription product was diluted to orrhagic factor that is associated with degrading extracel- 1:50 and stored at −80°C for following use. lular matrix and preventing blood clot formation [14, 15]. Now, metalloproteases have been reported on various venomous animals, these enzymes seems to act primarily Cloning of S. subspinipes mutilans as haemorrhagins [16, 17]. As to out known, the full alpha- alpha-like-neurotoxin and metalloproteinase neurotoxin and metalloprotease sequences from centi- pede venom are not reported [8]. Degenerate primers of Ssu1 and Ssu2, Sp1 and Sp2 An important way in understanding the increased (Table 1) were designed according to alpha-neurotoxin functional diversity of secretions is to identify and gene sequences and used to amplify alpha-like-neurotoxin Xichao Xia et al.: Characterization of two alpha-like neurotoxins and one metalloproteinase 653

Table 1: Sequences of PCR primers. Sequence and phylogenetic analysis

Primer Sequence (5′–3′) The alpha-like-neurotoxin and metalloproteinase gene Ssu1 CTTCNBCTNATGANAGGTNTGG sequences from S. subspinipes mutilans were analyzed Ssu2 GCACCANCAGGCNTTNCNGTAT and compared using the BLASTX and BLASTP programs SP1 GANGAAGACGGTCCTCTAAC with a GenBank database search (www. ncbi.nlm.nih. SP2 ATGNGCCGNTTCGTGAGC 5′ Race Innerprimer CATGGCTACATGCTGACAGCCTA gov/blast). The signal peptide was predicted by SignalP 5′ Race Outerprimer CGCGGATCCACAGCCTACTGATGATCAGTCGATG program (http://www.cbs.dtu.dk/services/SignalP). The Ssu5-1 GTAACCTTCGTCCATCGTCTGC multiple sequence alignment was performed using the Ssu5-2 CAAACCGAACCGAGCGAGA DANMEN analysis program. The theoretical amino acid MP5-1 G CAAACCGAACCGAGCGAGA composition, isoelectric point and molecular weight (Mw) MP5-2 TAACCTTCGTCCATCGTCTGC 3′ Race Outerprimer TACCGTCGTTCCACTAGTGATTT were computed using the Expasy ProtParam Tool (http:// 3′ Race Innerprimer CGCGGATCCTCCACTAGTGATTTCACTATAGG web.expasy.org/protparam/). N-glycosylation sites (N-X-S/ Ssu3-1 GAGAACGGTGCTGATAGTGG T) were predicted with the NetNGlyc1.0 Server (http://www. Ssu3-2 ACAGGTGTGGTGAGTGGAC cbs.dtu.dk/services/NetNGlyc/). Phosphorylation sites MP3-1 GATGGATTCTCGGGAAACG were performed with the NetPhos 2.0 Server (http://www. MP3-2 GTATGCGATTGTGAAGGAAG cbs.dtu.dk/services/NetPhos/). Prediction of secondary structure was fulfilled by CLC Protein Workbench 6 software as well as SMART research tool (http://smart.embl.de/). and metalloproteinases derived from snake Trimeresurus The tertiary structure was predicted by SWISS-MODEL soft- stejnegeri and Agkistrodon piscivorus leucostoma, spider ware (http://www.swissmodel.expasy.org/). The phyloge- Loxosceles intermedia and scorpion Buthus martensii and netic tree constructed from the alignment was generated Mesobuthus eupeus, respectively. PCR were performed in by the neighbor-joining method using MEGA5.0 software.

25 μL using 3 μL buffer (1.5 mM MgCl2), 0.5 U Taq polymer- The reliability of the tree obtained was assessed by boot- ase (Takara, Dalian, China), 1 μL primer (100 nM, each), strapping, using 1000 bootstrap replications. 1 μL of reverse transcription products, 2 μL dNTPs (250 μM each), and 17.5 μL water. The cycle parameters were as follows: an initial denaturation at 94°C for 4 min, 35 cycles of 94°C for 40 s, 48°C for 30 s, 72°C for 45 s and Results 72°C for 8 min. The PCR product was subcloned into the pMDT-19 vector (Takara, Dalian, China), sequenced from cDNA and the deduced protein both directions (Takara, Dalian, China) and identified ­characterizations of alpha-like-neurotoxin alpha-like-neurotoxin and metalloproteinases partial and metalloproteinase cDNA sequences. Highly stringent primers were designed from the Two novel alpha-like-neurotoxins from S. subspinipes partial cDNA sequences and used to characterize the 5′ mutilans were cloned and, respectively, named as SsuTA1 and 3′ regions of the alpha-like-neurotoxin and metallo- (accession number AAF2075864) and SsuTA2 (acces- proteinase cDNAs by RACE-PCR (Takara, Dalian, China) sion number AAF2075860). Full length cDNA of SsuTA1 following the manufacturer’s protocol. 5′ Raceouter was 491 bp in length containing 20 bp of 5′ untrans- primer and Ssu5-1 (Table 1) were used for the first-round lated region (UTR), 246 bp of open reading frame (ORF) PCR of 5′ RACE, 3′ Raceouter primer and Ssu3-1 (Table 1) contained 82 amino acids with a calculated molecular for the first-round PCR of 3′ RACE, respectively. Next, the mass of 9.16 kDa and a theoretical pI of 8.75, and 225 bp first-round PCR products were used as the template to of 3′ UTR (Figure 1). A termination signal (AATAAA) was perform the nested PCR using Raceinner primers (5′ iner, located at the positions 439–441 in the 3′ UTR. SsuTA2 3′ iner) included in the kit, and gene specific primers was 566 bp cDNA sequence including 318 bp of ORF con- Ssu5-2, Ssu3-2 (Table 1). Last, the 5′ RACE and 3′ RACE tained 106 amino acids with a calculated molecular mass PCR products were cloned and five clones were sequenced of 11.9 kDa and a theoretical pI of 9.0, and a termination­ using the method described above. Based on mentioned signal (AATAAA) in 3′ UTR (Figure 1). Characterization methods, MP5-1 and MP5-2 as well as MP3-1 and MP3-2 were of cysteine-rich was observed in both of them, in which used to isolate the 5′ RACE and 3′ RACE PCR products of seven Cys residues in SsuTA1 account for 21% of the metalloproteinase. sequence and seven Cys residues in SsuTA2 for 13% of 654 Xichao Xia et al.: Characterization of two alpha-like neurotoxins and one metalloproteinase

Figure 1: Complete cDNA and deduced amino acid sequences of SsuTA1 and SsuTA2. (A) shows the sequence of SsuTA1, (B) the sequence of SsuTA2. Start and stop codons are marked with bold. The signal region is indicated with single underlined. Putative polyadenylation signal “AATAAA” is shown with wavy line. Putative phosphorylation sites are marked with blue and underline. the sequence. A signal peptide composed of 33 deduced amino acids in 5′ UTR were, respectively, observed in the SsuTA1 and SsuTA2. No N-glycosylation site was observed in SsuTA1 and SsuTA2. Analysis of potential phospho- rylation sites with NetPhos 2.0 showed that SsuTA1 and SsuTA2 contained five serine phosphorylation sites, two threonine phosphorylation sites, and four tyrosine phos- phorylation sites (Figure 1). The full length metalloproteinase was isolated from S. subspinipes mutilans and named SsuMPs. cDNA of Figure 2: Complete cDNA and deduced amino acid sequences ′ SsuMPs was 2015 bp in length comprising 134 bp of 5 of ScsuMPs. untranslated region (UTR), 1782 bp of open reading frame Start and stop codons are marked with bold. The signal region is encoded a protein of 594 amino acids, and 99 bp of 3′ UTR indicated with single underline. The zinc-binding motif is marked (accession number AAF2075872) (Figure 2). A termination with yellow and boxed. The methionine-turn motifs are marked by a signal (AATAAA) was detected at the positions 1986–1991 wavy underline. Putative polyadenylation signal “AATAAA” is shown with with dot. Putative phosphorylation sites are marked with bold in the 3′ UTR (Figure 2). After analysis of deduced protein and underline. sequence by SignalP program, it consists of a 15-residue signal region containing the initial ATG cordon was detected in the 5′ UTR (Figure 2). Conserved motif HEXX- residue, Gly residue, Ser residue, Ala and 2 Leu residues HXXGXXHS in metalloproteinase was also observed in (Figure 2). Five N-glycosylation sites was observed in SsuMPs, in which variable amino acid X including Ile SsuMPs. Analysis of potential phosphorylation sites with Xichao Xia et al.: Characterization of two alpha-like neurotoxins and one metalloproteinase 655

NetPhos 2.0 showed that SsuMPs contained 27 serine occurred in the C-terminal (Figure 3). Conserved resides phosphorylation sites, 41 threonine phosphorylation of amino acids Leu25, Gly29, Val30, Ser31, Asp35, Ile38 in sites, and 10 tyrosine phosphorylation sites (Figure 2). N-­terminal was observed and showed a high homology with that of ­scorpions (Figure 3). Our previous study showed that methionine-turn Multiple sequence alignment of alpha-like- motif of metalloproteinase in scorpion venom is YIM neurotoxin and metalloproteinase not CIM. Here, similar phenomenon was also observed. Like metalloproteinase of B. martensi, YIM was also Deduced protein sequence of SsuTA1 showed 75% located at HEXXHXXGXXHS downstream in where a turn identity with that of SsuTA2, the major difference was motif was formed from secondary structure (Figure 4).

Figure 3: Comparison of deduced amino acid sequences of SsuTA1 and SsuTA2 with other members of alpha-neurotoxins. The result of homology with less than or equal to 75% is showed with pink. The result of homology with less than or equal to 50% is marked with green. Mesobuthus eupeus (accession number ABR21068), Mesobuthus martensii (accession number AAA69557), Hottentotta judaicus (accession number CBW45614), Buthus occitanus israelis (accession number ACJ23095), Apis mellifera (accession number NP_001011612.1), Haplopelma schmidti (accession number AAP33077.1), corallinus (accession number AAF13252.1), Micrurus laticollaris (accession number AFU76493.1). 656 Xichao Xia et al.: Characterization of two alpha-like neurotoxins and one metalloproteinase

Figure 4: Comparison of the deduced amino acid sequence of ScsuMPs with other members of metalloproteinase. The result of homology with less than or equal to 75% is showed with pink. The result of homology with less than or equal to 50% is marked with green. Buthus martensii (accession number KF492696.1), Mesobuthus eupeus (accession number ABR20110.1), Hottentotta judaicus (accession number ADY39479.1), Nasonia vitripennis (accession number XP_008212814.1), Acromyrmex echinatior (accession number ACV83935.1). Xichao Xia et al.: Characterization of two alpha-like neurotoxins and one metalloproteinase 657

A Strand Strand Strand 20 40

Strand Strand Strand 60 80

B Strand Strand Strand 20 40

Strand Strand Strand 60 80

Strand Helix 100

Figure 5: Predicted secondary structures of SsuTA1 and SsuTA2 from S. subspinipes mutilans. (A) shows the predicted secondary structure of SsuTA1 by CLC Protein Workbench 6 software. (B) shows the predicted secondary structure of SsuTA2 by SMART research tool.

Second and 3D structures of alpha-like-­ neurotoxin and metalloproteinase

Results of predicted secondary structure showed that six beta-sheets are successively formed in SsuTA1 and six beta-sheets in SsuTA2 (Figure 5), assuming SsuTA1 and SsuTA2 possesses the three-finger feature of alpha-toxins. Notably, one alpha-helix was observed in the predicted secondary structure of SsuTA2. The alignments of SsuTA1 Figure 6: Predicted 3D structures of SsuTA1 and SsuTA2. and SsuTA2 3D structure of showed a potential similarity (A) shows the predicted 3D structure of SsuTA1 by Swiss-model, with that of alpha-neurotoxins (Figure 6). The alignments (B) shows the predicted 3D structure of SsuTA2 by Swiss-model. of SsuMPs secondary and 3D structures of showed a simi- Formation of 3D structures of SsuTA1 and SsuTA2 is mainly based on larity with that of metalloproteinases and collagenases the insecticidal alpha scorpion toxin (PDB ID:1LQH). (Figures 7 and 8).

ScsuMPs

Sequence homology and phylogenetic Homo sapiens analysis of alpha-like-neurotoxin and ScsuMPs metalloproteinase Homo sapiens Similarity analysis by NCBI BLAST program showed that the deduced amino acid sequence of SsuTA2 share ScsuMPs a high similarities with alpha-neurotoxin of scorpion, Homo sapiens such as 64% Mesobuthus martensii (accession number

AAA69557), 59% Buthus occitanus Israelis (accession ScsuMPs number ACJ23095), 57% Mesobuthus eupeus (accession Homo sapiens number ABR21068), and 52% Hottentotta judaicusthe (accession number CBW45614). Similar phenomenon Figure 7: Predicted secondary structure of ScsuMPs. was also detected in SsuTA1. It was showed that 65% of The secondary structure is predicted by SMART research tool. 658 Xichao Xia et al.: Characterization of two alpha-like neurotoxins and one metalloproteinase

48 Hottentotta judaicus 63 Mesobuthus martensii 65 Mesobuthus eupeus 90 SsuTA2 28 75 SsuTA1 Buthus occitanus israelis 35 Rhodnius prolixus Haplopelma schmidti Phoneutria nigriventer Apis mellifera 37 Micrurus corallinus Micrurus laticollaris 97 Aipysurus laevis 25 Laticauda semifasciata 23 Micrurus altirostris

0.1

Figure 9: A molecular phylogenic tree of different species alpha- neurotoxin based on the neighbor-joining method. The α-toxins of S. subspinipes mutilans were underlined. Buthus occitanus israelis (accession number ACJ23095), Mesobuthus mar- tensii (accession number AAA69557), Mesobuthus eupeus (acces- Figure 8: Predicted 3D structure of ScsuMPs from S. subspinipes. sion number ABR21068), Hottentotta judaicus (accession number 3D structure is predicted by Swiss-model. Formation of 3D struc- CBW45614), Apis mellifera (accession number NP_001011612.1), ture of ScsuMPs is mainly based on the metalloproteinase-9 Laticauda semifasciata (accession number CAA26373.1), Aipysurus (PDB ID:5UE3). laevis (accession number CAA31747 ), Micrurus altirostris (acces- sion number AED89566.1), Micrurus laticollaris (accession number AFU76493.1), Micrurus corallinus (accession number AAF13252.1), Haplopelma schmidti (accession number AAP33077.1), Rhodnius bootstrap value is observed between SsuTA1 and SsuTA2, prolixus (accession number JAA77161.1), Phoneutria nigriventer and scorpion such as M. martensii (accession number (accession number AAC26166). AAA69557), H. judaicusthe (accession number CBW45614) and M. eupeus (accession number ABR21068), 90% boot- strap values find between B. occitanus Israelis (accession number XP_698601.6) and Bos taurus (accession number number ACJ23095) and species above mentioned. These XP_590696.3), third to scorpion Hottentotta judaicus species among of centipede and scorpion have only 28% (accession number ADY39479.1) and B. martensii (acces- of bootstrap value with others including of insect, snake sion number KF492696), last to snake Trimeresurus and spider. The results suggest that a high probability stejnegeri (accession number ABC73079.1) and Agkistro- of monophyly is supported by available data presented don piscivorus (accession number ACV83935.1) (Figure 10). from SsuTA1 and SsuTA2, and scorpion. Phylogenetic However, the bootstrap is only 54% between SsuMPs analysis of sequences provides evidence that SsuTA1 and and others of metalloproteinases (Figure 10). The result SsuTA2 were closest to the alpha-neurotoxin of scorpion reflects early divergence and independent evolution of (Figure 9). SsuMPs from other metalloproteinases. Notably, SsuMPs Similarity analysis of NCBI BLAST showed the deduced only shares 8.44% and 9.82% similarities with scorpion B. amino acid sequence of SsuMPs has different-degree simi- martensii (accession number KF492696) and H. judaicus larities with various metalloproteinases families, such (accession number ADY39479.1), and 12.19% with insects as 42% homology with matrix metalloproteinase-14 of Acromyrmex echinatior (accession number EGI70348.1), Apis dorsata (accession number XP_006610526.1), 41% respectively (Figure 10). with matrix metalloproteinase-24 of Nasonia vitripennis (accession number XP_008212814.1), 38% with matrix metalloproteinase-16 of Ceratitis capitata (accession Discussion number XP_004520956.1), 40% with matrix metallopro- teinase-17 of Danio rerio (accession number XP_698601.6). Close inspection of SsuTA1 and SsuTA2 sequences showed Available data from phylogenetic analysis revealed that characterizations of cysteine-rich was observed in both SsuMPs sequence is most closed to insect Culex quinque- of them, it greeting with that of spider and snake alpha- 1 2 fasciatus (accession number XP_001843240) and Zooter- neurotoxins [13, 25]. Generally, the profile of C X6C X3­ 3 4 5 6 7 8 9 10 mopsis nevadensis (accession number KDR20064), next C XC C X4C XC XmC XC XnC derived from three-terminal to metalloproteinase Danio rerio (accession is considers as one common folding pattern in snake Xichao Xia et al.: Characterization of two alpha-like neurotoxins and one metalloproteinase 659

100 Apis dorsata 100 Nasonia vitripennis in scorpion venom may reflect the positive selectivity and 80 Ceratitis capitata adaptive evolution process of scorpion species under envi- Bos taurus ronmental pressure. Toxins of centipede are beneficial for Danio rerio 100 100 catching their prey and defending against their predators. 99 Lepisosteus oculatus ScsuMPs Base on apparent difference of structural properties, these 99 54 Zootermopsis nevadensis findings suggest that SsuTA1 and SsuTA2 are likely evolu- 97 Culex quinquefasciatus tionary intermediate neurotoxins for alpha-toxin. Buthus martensii In the SsuTA1 and SsuTA2, the amino acids Leu25, 100 Hottentotta judaicus Ixodes scapularis Gly29, Val30, Ser31, Asp35, Ile38 of N-Terminal showed Agkistrodon piscivorus a high homology with that of scorpions. In , it is 100 Tr imeresurus stejnegeri defined that conserved residues of Gln7, Ser8, Gln10, Trp29, Asp31, His32, Val47 and Lys48 in the N-terminal 0.2 derived from alpha-neurotoxin function as a crucial player Figure 10: A molecular phylogenic tree of different species metal- to bind the acetylcholine receptor [13, 25, 29, 30]. Replace- loprotease based on the neighbor-joining method. ment of these amino acids results in decline of affinity of The ScsuMPs were underlined. Zootermopsis nevadensis (acces- toxin to the acetylcholine receptor. It is speculated that sion number KDR20064), Culex quinquefasciatus (accession number XP_001843240), Nasonia vitripennis (accession number these conserved residues in SsuTA1 and SsuTA1 likely play XP_008212814.1), Apis dorsata (accession number XP_006610526.1), a potential role in binding acetylcholine receptor, but this Ceratitis capitata (accession number XP_004520956.1), Ixodes hypothesis need be elucidated in the future. scapularis (accession number XM_002413437), Danio rerio (acces- Secondary structure showed six beta-sheets are succes- sion number XP_698601.6), Lepisosteus oculatus (accession number sively formed in SsuTA1 and seven beta-sheets in SsuTA2, XP_006640454.1), Bos taurus (accession number XP_590696.3), assuming SsuTA1 and SsuTA2 possesses the three-finger Trimeresurus stejnegeri (accession number ABC73079.1), Agkistrodon piscivorus leucostoma (accession number ACV83935.1), Hotten- feature of alpha-toxins. It is suggested that the three-finger totta judaicus (accession number ADY39479.1), Buthus martensii alpha-toxins share a similar structural scaffold formed by (KF492696). three adjacent (generally referred to as fingers) beta-sheets stabilized by four conserved disulfide bridges [31, 32]. Amazingly, SsuTA2 had eight Cys residues which contrib- alpha-neurotoxins [26]. Notably, distributions of Cys ute to form four conserved disulfide bridges except of adja- residue in SsuTA1 and SsuTA2 was consistent with alpha- cent beta-sheets. However, the deduced protein sequence neurotoxins of scorpionida Buthus occitanus Israelis, of SsuTA1 only contain seven Cys residues and does not Mesobuthus martensii, Mesobuthus eupeus, and Hot- equate to the mature peptide. Undoubetdly, this phenom- tentotta judaicus, but not with snake Micrurus altirostris ena is need be elucidated in the next study. An additional and spider Phoneutria nigriventer, reveals that is likely feature of resemblance to the alpha-toxins is revealed by associated with the adaptive evolution. Scorpion alpha- SsuTA1 and SsuTA2 spatial arrangement, as obtained using neurotoxins are classified into three major groups based a 3D structure. They possess the basic formation composed on the preferential toxicity to mammals or insects as well of a alpha-helix located on one face of the molecule and as their differential binding properties (classical alpha- antiparallel beta-strands on the opposite face that is similar mammal, alpha-insect neurotoxins and alpha-like neu- with those of alpha-toxins from scorpion [28, 33]. rotoxins). Classical alpha-mammal neurotoxins bind with As to , venom of scorpions, and high affinity to rat brain (voltage-gated sodium channels, centipedes are beneficial for catching their prey and VGSCs) and are highly toxic to mammals, while they are defending against their predators [34]. Our previous study practically non-toxic to insects. Alpha-insect Neurotoxins­ showed that the protein sequence of B. martensi metal- bind to insect VGSCs with high affinity and are very active loproteinase share a high identify with snakes, insects in insects but less potent in mammals. Alpha-like Neuro- and spiders [20]. In the present work, it is expected that toxins, which could not bind to rat brain synaptosomes, the centipede metalloproteinase sequences would resem- are active in both mammal and insect nervous systems ble sequences from other venomous arthropods, such as [27]. Adaptive evolution of scorpion neurotoxins and (spiders and scorpions) and insects. However, ion channels have also been constructed on the basis of this was proven otherwise. Notably, SsuMPs only shares genomic organization, structure and pharmacology of 8.44% and 9.82% similarities with scorpion B. martensii toxin and ion channels, as well as scorpion species distri- and H. judaicus, and 12.19% with insects Acromyrmex bution [27, 28]. The difference of function and components echinatior, respectively. The lack of observed similarities 660 Xichao Xia et al.: Characterization of two alpha-like neurotoxins and one metalloproteinase between SsuMPs and metalloproteinases from other Acknowledgement: This research was funded by the arthropods might be partly explained the ancient and National Natural Science Foundation of Henan (No. independent evolutionary history of the centipede venom 18A330004, PXY-BSQD-2018009, 17A180010, PXY- system [35]. It has been estimated that the myriapod group PYJJ-2018005) and China Postdoctoral Science Founda- diverged from the chelicerata at 642 ± 63 million years ago tion Funded Project (2016M590143). [24]. 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