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Title: Two novel chloride channel found in the transcriptome the Iranian eupeus venom gland

Masoumeh Baradaran

Toxicology Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz,

Email: [email protected]

Amir Jalali

Email:[email protected]

Hamid Galehdari*

Email:[email protected]

Abstract

Scorpion venom is an important source of bioactive peptides including of toxins, enzymes, and antimicrobial peptides. Most of them have pharmacological value. Accordingly, molecular characterization of venom components can be a necessary platform for more investigations about them. Here we characterized two new chloride-channel toxins founded in cDNA library of venom Iranian scorpion, . cDNA was constructed from RNA extracted from venom gland of Mesobuthus eupeus. With the cloning of cDNA in pSMART2IFD vector and transformation of vectors to E.coli host cells, cDNA library was constructed. By screening the cDNA library sequences of venom, we have identified the cDNA of two chloride channel toxins, meuCl14 and meuCl15. We deposited them in NCBI database and characterized the peptides that they encode. They are 56 and 60 amino-acid peptides. The primary amino acid structures show considerable homology to previously described chloride channel modifiers. A 20-amino-acid signal peptide for meuCl14 and a 24-amino-acid signal peptide for meuCl15 were identified. A transmembrane domain was predicted for both toxins. The secondary structure and some physiochemical properties of both toxins were also presented in this study.

Keywords: Mesobuthus eupeus, chloride channel , cDNA library,scorpion venom gland

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Interoduction

Mammalian Chloride channels are major group membrane proteins that regulate the passive flow of anion across the plasma membrane or in membranes of intracellular organelles(Jentsch, Stein, Weinreich, & Zdebik, 2002). They were classified based on their regulation mainly to five classes including: cystic fibrosis transmembrane conductance regulator (CFTR), which is activated by cyclic AMP-dependent phosphorylation; calcium-activated chloride channels (CaCCs); voltage-gated chloride channels (ClCs); ligand-gated chloride channels (GABA (γ-aminobutyric acid)and glycine- activated); and volume-regulated chloride channels(Verkman & Galietta, 2009). Chloride channels are important proteins because they involved in many physiologically processes including the regulation of the excitability of neurones, skeletal, cardiac and smooth muscle, cell volume regulation, transepithelial salt transport, the acidification of internal and extracellular compartments, the cell cycle and apoptosis("Chloride channels," 2009). Thereby targeting the chloride channel by modulator molecules is the mechanisms of some currently used clinical drugs and some other chloride channel modulators in preclinical development and clinical trials(Verkman & Galietta, 2009).

The mammalian ClC family according to localization divided to three groups that totally contains nine members; ClC-1, ClC-2, hClC-Ka (rClC-K1) and hClC-Kb (rClC-K2); ClC-3 to ClC-5, and ClC-6 and -7. ClC-1 and ClC-2 are plasma membrane chloride channels as are ClC-Ka and ClC-Kb (Estevez et al., 2001). There is some chloride channel toxins associated with other ion channel toxins in venomous specially scorpion and snake. The amino acid sequences of 18 chloride channel toxins have been deposited in UniProt database (http://venomzone.expasy.org/by_species/2119.html). The currently identified chloride channel toxins in scorpion venom have been derived from Leiurus quinquestriatus hebraeus, Leiurus quinquestriatus quinquestriatus, , Mesobuthus tamulus, tamulus sindicus, Androctonus mauritanicus mauritanicus, Parabuthus schlechteri, Androctonus australis. All of them are modifiers of ClC-2 or ClC-3.

Scorpion venoms contains of ion channel-modifying peptides which affect on sodium, potassium and chloride channels(Quintero-Hernández, Jiménez-Vargas, Gurrola, Valdivia, & Possani, 2013). Although there has been much activity in drug development for modifiers of channel targets (sodium, potassium and calcium), considerably less attention has been given to chloride channels. There is some chloride channel toxins associated with other ion channel toxins in venomous animals specially scorpion and snake. Thereby identifying the functional and molecular characterization of chloride channel toxins is valuable and can be resulted to drug discovery or be a platform for drug design. The results of analysis the cDNA library of Iranian scorpion, Mesobuthus eupeus venom gland demonstrate the presence of chloride-channel toxins in the venom of this scorpion. In this study we have molecularly characterized two of new chloride-channel toxins from Iranian M. eupeus.

Material and methods

Double stranded cDNA was synthesized from total RNA extracted from the venom of Mesobuthus eupeus. After cloning the dscDNA in to appropriate vector, it was transformed in to bacteria host cells for constructing the cDNA library. Transcriptome of cDNA library were sequenced and analyzed for finding the antimicrobial peptides. Nucleotide BLAST (BLASTn) was done with online BLAST tool in the NCBI website (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Open reading frames (ORFs) were found with online ORF Finder tool (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). BLAST tool in UniProt website was used for Protein BLAST (http://www.uniprot.org/). Molecular weight, theoretical pI and net charge of peptides (at neutral pH) were predicted using peptide property calculator online tool at INNOVAGEN (http://www.innovagen.com/proteomics-tools). SignalP4.1 (http://www.cbs.dtu.dk/services/SignalP/) was used to determine the presence of a signal peptide (http://www.cbs.dtu.dk/services/SignalP/). Hydrophobicity of each peptide was calculated with

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GPMAW lite online tool available on http://www.alphalyse.com/gpmaw_lite.html. Secondary structure of peptides are predicted using the GOR secondary structure prediction method software(Garnier, Gibrat, & Robson, 1996) (http://npsa-pbil.ibcp.fr/cgi- bin/npsa_automat.pl?page=npsa_gor4.html). Alignment of every peptide was done using Alignment tool in UniProt. Transmembrane domains were predicted by the TMHMM tool (http://www.cbs.dtu.dk/services/TMHMM/).

Results

In analysis of cDNA library of M. eupeus we found two transcripts (named meuCl14 and meuCl15) that their primary amino acid structures show considerable homology (e-value <10−3) to previously described chloride channel modifiers of scorpion in BLASTn analysis and protein BLAST using Uniprot. The cDNA sequences (figure 1) were deposited in NCBI under the accession numbers KU316183 and KU316184. Alignment of the amino acids sequences of meuCl14 and meuCl15 with similar peptides are shown in figure 2. Both peptides classified in modifiers of ClC-2 family of mammalian chloride channel toxin.

According to alignment, amino acid sequences of meuCl14, meuCl15 and their homologous are very conserved. All of them contain eight conserved cystein residues. In addition to the cysteins, meuCl14, meuCl15 and their homologous are severely conserved in 4 amino acids series including: MCMPCFTT, QQCR, GKCFG, QCLC (figure2). A 20 amino-acids signal peptide for meuCl14 and a 20 amino-acids signal peptide for meuCl15 were predicted (figure 2). Secondary structure and some physiochemical properties of both founded toxins are illustrated in table1. A transmembrane domain was predicted for both toxins. These domains are highlighted with green in amino acid sequences in table 1. Based on existence of signal peptide, (Nothwehr & Gordon, 1990) we suggest that meuCl14 and meuCl15 are secretary toxins that when insertion the completed peptide in Endoplasmic Reticulum, the signal peptides are removed by a signal peptidase enzyme and mature peptide will be released in Endoplasmic Reticulum. Transmembrane predicted for them consist of a large part of signal peptide plus 1 or 7 (1 in meuCl15 and, 7 in meuCl14) beginning amino acids of mature peptide. It is proposed that the peptides attach to membrane of Endoplasmic Reticulum trough these domains.

The molecular information of this paper can be a window for more steps of research in chloride channel toxins of M. eupeus with the aim of more finding about drug design.

Tables and figures

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Figure.1 cDNA and translated open-reading frame amino acid sequences encoding the (A) meuCl14 and (B) meuCl15. Nucleotides of open-reading frame are single underlined; and stop codons are indicated by asterisks.

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Figure 2. Amino acid sequences alignment of meuCl14 and meuCl15 with homologues. Conserved residues are represented

in dark gray. Residues with similar physicochemical properties are shown in light gray. Dash showes that there is not any

residue in that position. The percent of similarity with meuCl14 are brought in the right of alignment. Signal peptides are

highlighted in red and mature peptide in blak.

Table 1. Predicted Secondary structure and some physiochemical properties of peptides described in this study. The random

coil is represented by "c", the extended strand by "e". Amino acids of transmembrane domains are highlighted with green.

Toxin Secondary structure Molecular Iso- Net Water name weight(g/m electr char solubili ol) ic ge at ty point pH 7 meuCl MESFLLALFLTVMIATHTEAMCMPCFTTRPDMAQQCRACCKGRGKCFG 6231.48 0.6 poor 14 PQCLCGYD 7.31 cccceeeeeeeeeeeeecccccceeeeeccccccceeeeccccccccccceeeeec

meuCl MKFLYGIVFIALFLTVMIATHTEAMCMPCFTTRPDMAQQCRACCKGRGK 6723.15 7.9 2.6 poor 15 CFGPQCLCGYD ccccccceeeeeeeeeeeeeecccccceeeeeccccccceeeeccccccccccceeeeec

References

Chloride channels. (2009). Br J Pharmacol, 158(Suppl 1), S130-S134. doi: 10.1111/j.1476- 5381.2009.00503_6.x

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Estevez, R., Boettger, T., Stein, V., Birkenhager, R., Otto, E., Hildebrandt, F., & Jentsch, T. J. (2001). Barttin is a Cl- channel beta-subunit crucial for renal Cl- reabsorption and inner ear K+ secretion. Nature, 414(6863), 558-561. doi: 10.1038/35107099 Garnier, J., Gibrat, J. F., & Robson, B. (1996). GOR method for predicting protein secondary structure from amino acid sequence. Methods Enzymol, 266, 540-553. Jentsch, T. J., Stein, V., Weinreich, F., & Zdebik, A. A. (2002). Molecular structure and physiological function of chloride channels. Physiol Rev, 82(2), 503-568. doi: 10.1152/physrev.00029.2001 Nothwehr, S. F., & Gordon, J. I. (1990). Targeting of proteins into the eukaryotic secretory pathway: signal peptide structure/function relationships. Bioessays, 12(10), 479-484. doi: 10.1002/bies.950121005 Quintero-Hernández, V., Jiménez-Vargas, J. M., Gurrola, G. B., Valdivia, H. H. F., & Possani, L. D. (2013). Scorpion venom components that affect ion-channels function. Toxicon, 76, 328-342. doi: 10.1016/j.toxicon.2013.07.012 Verkman, A. S., & Galietta, L. J. V. (2009). Chloride channels as drug targets. Nat Rev Drug Discov, 8(2), 153- 171. doi: 10.1038/nrd2780

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