Purification and Molecular Characterization of a Mammalian Neurotoxin as a Pharmaceutical Tool from the Venom of Iranian Scorpion Androctonus crassicauda
,
Abstract
Background: Venom of scorpions are complex bioactive polypeptides. To gain greater insights into the structural and functional impacts of toxins from Androctonus crassicauda (Buthidae), a dangerously venomous scorpion species, its venom was isolated, purified, and characterized.
Methods: Long chain toxin with four disulfide bonds purified by size exclusion chromatography and reversed-phase HPLC and characterized by amino acid sequencing and molecular weight determination.
Results: The primary structure analysis exhibits a neurotoxin named AnCra2 with 7302.24 Da molecular weight and 64 amino acid residues that cause paralysis and lead to death in NIH mice. The LD50 of AnCra2 was determined to be 0.61±0.04 μg/mice. Phylogenetic analysis displays the toxin has 97% sequence similarity with alpha toxins reported from north African scorpions that affect voltage-gated sodium channels (VGSC), also proposed that differentiation among the scorpions of family Buthidae is affected by the geographical conditions and efficiency in evolutionary variations. AnCra2 exposed binding residues have a high affinity for receptor residues in site-3 (segment-3) of VGSC that are approved by three-dimensional structure and homology modeling.
Conclusion: Purified AnCra2 seems to be a new putative Alpha neurotoxin in homology with the structure of neurotoxins that act on VGSC as a pharmaceutical tool.
1. Possani LD, Becerril B, Delepierre M, Ty-tgat J (1999) Scorpion toxins specific for Na+-channels. Eur J Biochem. 264 (2): 287–300.
2. Gopalakrishnakone P, Possani LD, Schwartz EF, Rodríguez De La Vega RC (2015) Scorpion Venoms, Toxinol-ogy. Springer Science+Business Media Dordrecht.
3. Cohen L, Lipstein N, Gordon D, Cohen L, Lipstein N GD (2006) Allosteric inter-actions between scorpion toxin receptor sites on voltage-gated Na channels im¬ply a novel role for weakly active com-ponents in arthropod venom. FASEB J. 20(11): 1933–1935.
4. Quintero-Hernández V, Jiménez-Vargas JM, Gurrola GB, Valdivia HH, Possani LD (2013) Scorpion venom components that affect ion-channels function. Toxi-con. 76: 328–342.
5. Froy O, Gurevitz M (2003) New insight on scorpion divergence inferred from com-parative analysis of toxin structure, phar-macology and distribution. Toxi¬con. 42 (5): 549–555.
6. Rodríguez De La Vega RC, Possani LD (2005) Overview of scorpion toxins spe-cific for Na+ channels and related pep-tides: Biodiversity, structure-function re-la¬tionships and evolution. Toxicon. 46 (8): 831–844.
7. Dehghani R, Fathi B (2012) Scorpion sting in Iran: A review. Toxicon. 60(5): 919–933.
8. Possani LD, Merino E, Corona M, Bolivar F, Becerril B (2000) Peptides and genes coding for scorpion toxins that affect ion-channels. Biochimie. 82(9–10): 861–868.
9. Rodríguez de la Vega RC, Schwartz EF, Possani LD (2010) Mining on scorpion venom biodiversity. Toxicon. 56(7): 1155–1161.
10. Huys I, Olamendi-Portugal T, Garcia-Gómez BI, Vandenberghe I, Van Beeu-men J, Dy¬ason K (2004) A subfamily of acidic alpha-K (+) toxins. J Biol Chem. 279(4): 2781–2789.
11. Housley DM, Housley GD, Liddell MJ, Jennings EA (2017) Scorpion toxin pep-tide action at the ion channel subunit lev¬el. Neuropharmacology. 127: 46–78.
12. Caliskan F, García BI, Coronas FIV, Resta¬no-Cassulini R, Korkmaz F, Sahin Y (2012) Purification and cDNA clon-ing of a novel neurotoxic peptide (Acra3) from the scorpion Androctonus crassi¬cauda. Peptides. 37(1): 106–112.
13. Bayatzadeh MA, Mirakabadi AZ, Babaei N, Doulah AH, Doosti A (2020) Charac-terization, molecular modeling and phy-logenetic analysis of a long mammalian neurotoxin from the venom of the Irani-an scorpion Androctonus crassicauda. Bi¬ologia (Bratisl). 75(7): 1029–1041.
14. Lowry OH, Rosebrough NJ, Farr AL, Ran¬dall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem. 193(1): 265–275.
15. Tytgat J, Debont T, Rostoll K, Müller GJ, Verdonck F, Daenens P (1998) Purifica-tion and partial characterization of a “short” insectotoxin-like peptide from the venom of the scorpion Parabuthus schlechteri. FEBS Lett. 441(3): 387–391.
16. Crest M, Jacquet G, Gola M, Zerrouk H, Benslimane A, Rochat H (1992) Kali-otoxin, a novel peptidyl inhibitor of neu-ronal BK-type Ca (2+)-activated K+ chan¬nels characterized from Androcto-nus mauretanicus mauretanicus venom. J Bi¬ol Chem. 267(3): 1640–1647.
17. Spearman C (1908) The method of “right and wrong cases” (Constant Stimuli) with¬out Gauss’s formula. Br J Psychol. 2: 227–242.
18. Kärber G (1931) Beitrag zur kollektiven Behandlung pharmakologischer Reihen-versuche. Arch Exp Path Pharm. 162: 480–483.
19. Bienert S, Heer FT, de Beer TAP, Lepore R, Rempfer C, Gumienny R (2018) SWISS-MODEL: homology modelling of protein structures and complexes. Nucle¬ic Acids Res. 46: 296–303.
20. Källberg M, Wang H, Wang S, Peng J, Wang Z, Lu H (2012) Template-based protein structure modeling using the Rap¬torX web server. Nat Protoc. 7(8): 1511–1522.
21. Yan Y, Huang SY (2018) Protein-Protein Docking with Improved Shape Com¬ple-mentarity. In: Huang DS, Bevilacqua V, Premaratne P, Gupta P (Eds) Intelli¬gent Computing Theories and Applica¬tion. ICIC 2018. Lecture Notes in Com¬puter Science, vol 10954. Springer, Cham.
22. Hall TA (1991) BioEdit: a user-friendly bi¬ological sequence alignment editor and analysis program for Windows 95/98/ NT. Nucleic Acids Symp Ser. 41: 95–98.
23. Kumar S, Stecher G, Li M, Knyaz C, Tamu¬ra K (2018) MEGA X: Molecular evolu¬tionary genetics analysis across compu¬ting platforms. Mol Biol Evol. 35(6): 1547–1549.
24. Batista CVF, Román-González SA, Salas-Castillo SP, Zamudio FZ, Gómez-La-gunas F, Possani LD (2007) Proteo¬mic analysis of the venom from the scorpion Tityus stigmurus: Biochemical and phys¬i-ological comparison with other Tityus spe¬cies. Comp Biochem Physiol - C Tox-i¬col Pharmacol. 146(1–2): 147–57.
25. Leipold E, Lu S, Gordon D, Hansel A, Heinemann SH (2004) Combinatorial in-teraction of scorpion toxins Lqh-2, Lqh-3, and LqhαIT with sodium channel re-ceptor sites-3. Mol Pharmacol. 65(3): 685–691.
26. Fabrichny IP, Mondielli G, Conrod S, Mar¬tin-Eauclaire M-F, Bourne Y, Mar-chot P (2012) Structural insights into antibody sequestering and neutralizing of Na+ chan¬nel α-Type modulator from Old World scorpion venom. J Biol Chem. 287(17): 14136–14148.
27. Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK: a pro¬gram to check the stereochemical quality of protein structures. J Appl Crystallogr. 26(2): 283–291.
28. Gupta M, Wadhwa G, Sharma SK, Jain CK (2013) Homology modeling and valida¬tion of SAS2271 transcriptional regula¬tor of AraC family in Staphylo-coccus au¬reus. Asian Pac J Trop Dis. 3(1): 1–4.
29. Clairfeuille T, Cloake A, Infield DT, Llongueras JP, Arthur CP, Li ZR (2019) Structural basis of α-scorpion toxin ac-tion on Na v channels. Science. 363 (6433): eaav8573.
30. Adi-Bessalem S, Laraba-Djebari F, Bougis PE, Martin-Eauclaire MF, Hammoudi-Triki D (2019) Serotherapy against Volt-age-Gated Sodium Channel-Targeting αTox¬ins from Androctonus Scorpion Veno¬m. Toxins (Basel). 11(2): 63–86.
31. Mouhat S, Jouirou B, Mosbah A, De Waard M, Sabatier JM (2004) Diversity of folds in animal toxins acting on ion channels. Biochem J. 378: 717–726.
32. Dhawan R, Joseph S, Sethi A, Lala AK (2002) Purification and characterization of a short insect toxin from the venom of the scorpion Buthus tamulus. FEBS Lett. 528(1–3): 261–266.
33. de la Vega RCRR, Possani LD (2007) Nov¬el paradigms on scorpion toxins that af¬fects the activating mechanism of so-di¬um channels. Toxicon. 49(2): 171–180.
34. Garcia M, GAO YD, McManus O, Ka-czor¬owski G (2001) Potassium chan¬nels: from scorpion venoms to high-resolu¬tion structure. Toxicon. 39(6): 739–748.
35. Gurevitz M (2012) Mapping of scorpion toxin receptor sites at voltage-gated so-dium channels. Toxicon. 60(4): 502–511.
36. Kahn R, Karbat I, Ilan N, Cohen L, Sokolov S, Catterall WA (2009) Molec-ular requirements for recognition of brain voltage-gated sodium channels by scor¬pion α-toxins. J Biol Chem. 284(31): 20684–20691.
37. Borges A, Alfonzo MJ, García CC, Winand NJ, Leipold E, Heinemann SH (2004) Iso¬lation, molecular cloning and func¬tion¬al characterization of a novel beta-toxin from the Venezuelan scorpi-on, Ti¬tyus zulianus. Toxicon. 43(6): 671–684.
38. Cestèle S, Qu Y, Rogers JC, Rochat H, Scheuer T, Catterall WA (1998) Voltage sensor-trapping: enhanced activation of sodium channels by beta-scorpion toxin bound to the S3-S4 loop in domain II. Neuron. 21(4): 919–931.
39. Catterall WA, Cestèle S, Yarov-Yarovoy V, Yu FH, Konoki K, Scheuer T (2007) Voltage-gated ion channels and gating modifier toxins. Toxicon. 49(2): 124–141.
40. Estrada G, Restano-Cassulini R, Ortiz E, Possani LD, Corzo G (2011) Addition of positive charges at the C-terminal pep¬tide region of CssII, a mammalian scor¬pion peptide toxin, improves its af-finity for sodium channels Nav1.6. Pep-tides. 32(1): 75–79.
41. Yang F, Liu S, Zhang Y, Qin C, Xu L, Li W (2018) Expression of recombinant α-toxin BmKM9 from scorpion Buthus mar¬tensii Karsch and its functional charac¬terization on sodium channels. Peptides. 99: 153–160.
42. Zaharenko AJ, Schiavon E, Ferreira Jr WA, Lecchi M, de Freitas JC, Richard-son M, Wanke E (2012) Characteriza¬tion of se¬lectivity and pharmacophores of type 1 sea anemone toxins by screen¬ing seven Nav sodium channel isoforms. Peptides. 34(1): 158–167.
43. Jalali A, Rahim F (2014) Epidemiological review of scorpion envenomation in Iran. Iran J Pharm Res IJPR. 13(3): 743–756.
44. Lourenço WR (2017) A new species of Physoctonus Mello-leitão, 1934 from the ‘Campos formations’ of southern Ama-zonia (Scorpiones, Buthidae). Zookeys. 711: 67–80.
45. Mourão CBF, Possani LD, Guerrero-Var-gas JA, Schwartz EF, Quintero-Hernán-dez V (2012) Identification and phylogenetic analysis of Tityus pachyurus and Tityus obscurus Novel Putative Na+-Channel Scorpion Toxins. PLoS One. 7 (2): e30478.
46. Sanaei-Zadeh H, Marashi SM, Dehghani R (2017) Epidemiological and clinical characteristics of scorpionism in Shiraz (2012–2016); development of a clinical severity grading for Iranian scorpion envenomation. Med J Islam Repub Iran. 17: 31–27.
2. Gopalakrishnakone P, Possani LD, Schwartz EF, Rodríguez De La Vega RC (2015) Scorpion Venoms, Toxinol-ogy. Springer Science+Business Media Dordrecht.
3. Cohen L, Lipstein N, Gordon D, Cohen L, Lipstein N GD (2006) Allosteric inter-actions between scorpion toxin receptor sites on voltage-gated Na channels im¬ply a novel role for weakly active com-ponents in arthropod venom. FASEB J. 20(11): 1933–1935.
4. Quintero-Hernández V, Jiménez-Vargas JM, Gurrola GB, Valdivia HH, Possani LD (2013) Scorpion venom components that affect ion-channels function. Toxi-con. 76: 328–342.
5. Froy O, Gurevitz M (2003) New insight on scorpion divergence inferred from com-parative analysis of toxin structure, phar-macology and distribution. Toxi¬con. 42 (5): 549–555.
6. Rodríguez De La Vega RC, Possani LD (2005) Overview of scorpion toxins spe-cific for Na+ channels and related pep-tides: Biodiversity, structure-function re-la¬tionships and evolution. Toxicon. 46 (8): 831–844.
7. Dehghani R, Fathi B (2012) Scorpion sting in Iran: A review. Toxicon. 60(5): 919–933.
8. Possani LD, Merino E, Corona M, Bolivar F, Becerril B (2000) Peptides and genes coding for scorpion toxins that affect ion-channels. Biochimie. 82(9–10): 861–868.
9. Rodríguez de la Vega RC, Schwartz EF, Possani LD (2010) Mining on scorpion venom biodiversity. Toxicon. 56(7): 1155–1161.
10. Huys I, Olamendi-Portugal T, Garcia-Gómez BI, Vandenberghe I, Van Beeu-men J, Dy¬ason K (2004) A subfamily of acidic alpha-K (+) toxins. J Biol Chem. 279(4): 2781–2789.
11. Housley DM, Housley GD, Liddell MJ, Jennings EA (2017) Scorpion toxin pep-tide action at the ion channel subunit lev¬el. Neuropharmacology. 127: 46–78.
12. Caliskan F, García BI, Coronas FIV, Resta¬no-Cassulini R, Korkmaz F, Sahin Y (2012) Purification and cDNA clon-ing of a novel neurotoxic peptide (Acra3) from the scorpion Androctonus crassi¬cauda. Peptides. 37(1): 106–112.
13. Bayatzadeh MA, Mirakabadi AZ, Babaei N, Doulah AH, Doosti A (2020) Charac-terization, molecular modeling and phy-logenetic analysis of a long mammalian neurotoxin from the venom of the Irani-an scorpion Androctonus crassicauda. Bi¬ologia (Bratisl). 75(7): 1029–1041.
14. Lowry OH, Rosebrough NJ, Farr AL, Ran¬dall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem. 193(1): 265–275.
15. Tytgat J, Debont T, Rostoll K, Müller GJ, Verdonck F, Daenens P (1998) Purifica-tion and partial characterization of a “short” insectotoxin-like peptide from the venom of the scorpion Parabuthus schlechteri. FEBS Lett. 441(3): 387–391.
16. Crest M, Jacquet G, Gola M, Zerrouk H, Benslimane A, Rochat H (1992) Kali-otoxin, a novel peptidyl inhibitor of neu-ronal BK-type Ca (2+)-activated K+ chan¬nels characterized from Androcto-nus mauretanicus mauretanicus venom. J Bi¬ol Chem. 267(3): 1640–1647.
17. Spearman C (1908) The method of “right and wrong cases” (Constant Stimuli) with¬out Gauss’s formula. Br J Psychol. 2: 227–242.
18. Kärber G (1931) Beitrag zur kollektiven Behandlung pharmakologischer Reihen-versuche. Arch Exp Path Pharm. 162: 480–483.
19. Bienert S, Heer FT, de Beer TAP, Lepore R, Rempfer C, Gumienny R (2018) SWISS-MODEL: homology modelling of protein structures and complexes. Nucle¬ic Acids Res. 46: 296–303.
20. Källberg M, Wang H, Wang S, Peng J, Wang Z, Lu H (2012) Template-based protein structure modeling using the Rap¬torX web server. Nat Protoc. 7(8): 1511–1522.
21. Yan Y, Huang SY (2018) Protein-Protein Docking with Improved Shape Com¬ple-mentarity. In: Huang DS, Bevilacqua V, Premaratne P, Gupta P (Eds) Intelli¬gent Computing Theories and Applica¬tion. ICIC 2018. Lecture Notes in Com¬puter Science, vol 10954. Springer, Cham.
22. Hall TA (1991) BioEdit: a user-friendly bi¬ological sequence alignment editor and analysis program for Windows 95/98/ NT. Nucleic Acids Symp Ser. 41: 95–98.
23. Kumar S, Stecher G, Li M, Knyaz C, Tamu¬ra K (2018) MEGA X: Molecular evolu¬tionary genetics analysis across compu¬ting platforms. Mol Biol Evol. 35(6): 1547–1549.
24. Batista CVF, Román-González SA, Salas-Castillo SP, Zamudio FZ, Gómez-La-gunas F, Possani LD (2007) Proteo¬mic analysis of the venom from the scorpion Tityus stigmurus: Biochemical and phys¬i-ological comparison with other Tityus spe¬cies. Comp Biochem Physiol - C Tox-i¬col Pharmacol. 146(1–2): 147–57.
25. Leipold E, Lu S, Gordon D, Hansel A, Heinemann SH (2004) Combinatorial in-teraction of scorpion toxins Lqh-2, Lqh-3, and LqhαIT with sodium channel re-ceptor sites-3. Mol Pharmacol. 65(3): 685–691.
26. Fabrichny IP, Mondielli G, Conrod S, Mar¬tin-Eauclaire M-F, Bourne Y, Mar-chot P (2012) Structural insights into antibody sequestering and neutralizing of Na+ chan¬nel α-Type modulator from Old World scorpion venom. J Biol Chem. 287(17): 14136–14148.
27. Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK: a pro¬gram to check the stereochemical quality of protein structures. J Appl Crystallogr. 26(2): 283–291.
28. Gupta M, Wadhwa G, Sharma SK, Jain CK (2013) Homology modeling and valida¬tion of SAS2271 transcriptional regula¬tor of AraC family in Staphylo-coccus au¬reus. Asian Pac J Trop Dis. 3(1): 1–4.
29. Clairfeuille T, Cloake A, Infield DT, Llongueras JP, Arthur CP, Li ZR (2019) Structural basis of α-scorpion toxin ac-tion on Na v channels. Science. 363 (6433): eaav8573.
30. Adi-Bessalem S, Laraba-Djebari F, Bougis PE, Martin-Eauclaire MF, Hammoudi-Triki D (2019) Serotherapy against Volt-age-Gated Sodium Channel-Targeting αTox¬ins from Androctonus Scorpion Veno¬m. Toxins (Basel). 11(2): 63–86.
31. Mouhat S, Jouirou B, Mosbah A, De Waard M, Sabatier JM (2004) Diversity of folds in animal toxins acting on ion channels. Biochem J. 378: 717–726.
32. Dhawan R, Joseph S, Sethi A, Lala AK (2002) Purification and characterization of a short insect toxin from the venom of the scorpion Buthus tamulus. FEBS Lett. 528(1–3): 261–266.
33. de la Vega RCRR, Possani LD (2007) Nov¬el paradigms on scorpion toxins that af¬fects the activating mechanism of so-di¬um channels. Toxicon. 49(2): 171–180.
34. Garcia M, GAO YD, McManus O, Ka-czor¬owski G (2001) Potassium chan¬nels: from scorpion venoms to high-resolu¬tion structure. Toxicon. 39(6): 739–748.
35. Gurevitz M (2012) Mapping of scorpion toxin receptor sites at voltage-gated so-dium channels. Toxicon. 60(4): 502–511.
36. Kahn R, Karbat I, Ilan N, Cohen L, Sokolov S, Catterall WA (2009) Molec-ular requirements for recognition of brain voltage-gated sodium channels by scor¬pion α-toxins. J Biol Chem. 284(31): 20684–20691.
37. Borges A, Alfonzo MJ, García CC, Winand NJ, Leipold E, Heinemann SH (2004) Iso¬lation, molecular cloning and func¬tion¬al characterization of a novel beta-toxin from the Venezuelan scorpi-on, Ti¬tyus zulianus. Toxicon. 43(6): 671–684.
38. Cestèle S, Qu Y, Rogers JC, Rochat H, Scheuer T, Catterall WA (1998) Voltage sensor-trapping: enhanced activation of sodium channels by beta-scorpion toxin bound to the S3-S4 loop in domain II. Neuron. 21(4): 919–931.
39. Catterall WA, Cestèle S, Yarov-Yarovoy V, Yu FH, Konoki K, Scheuer T (2007) Voltage-gated ion channels and gating modifier toxins. Toxicon. 49(2): 124–141.
40. Estrada G, Restano-Cassulini R, Ortiz E, Possani LD, Corzo G (2011) Addition of positive charges at the C-terminal pep¬tide region of CssII, a mammalian scor¬pion peptide toxin, improves its af-finity for sodium channels Nav1.6. Pep-tides. 32(1): 75–79.
41. Yang F, Liu S, Zhang Y, Qin C, Xu L, Li W (2018) Expression of recombinant α-toxin BmKM9 from scorpion Buthus mar¬tensii Karsch and its functional charac¬terization on sodium channels. Peptides. 99: 153–160.
42. Zaharenko AJ, Schiavon E, Ferreira Jr WA, Lecchi M, de Freitas JC, Richard-son M, Wanke E (2012) Characteriza¬tion of se¬lectivity and pharmacophores of type 1 sea anemone toxins by screen¬ing seven Nav sodium channel isoforms. Peptides. 34(1): 158–167.
43. Jalali A, Rahim F (2014) Epidemiological review of scorpion envenomation in Iran. Iran J Pharm Res IJPR. 13(3): 743–756.
44. Lourenço WR (2017) A new species of Physoctonus Mello-leitão, 1934 from the ‘Campos formations’ of southern Ama-zonia (Scorpiones, Buthidae). Zookeys. 711: 67–80.
45. Mourão CBF, Possani LD, Guerrero-Var-gas JA, Schwartz EF, Quintero-Hernán-dez V (2012) Identification and phylogenetic analysis of Tityus pachyurus and Tityus obscurus Novel Putative Na+-Channel Scorpion Toxins. PLoS One. 7 (2): e30478.
46. Sanaei-Zadeh H, Marashi SM, Dehghani R (2017) Epidemiological and clinical characteristics of scorpionism in Shiraz (2012–2016); development of a clinical severity grading for Iranian scorpion envenomation. Med J Islam Repub Iran. 17: 31–27.
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| Section | Original Article | |
| Keywords | ||
| Androctonus crassicauda; Phylogenetic analysis; Molecular modeling | ||
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How to Cite
1.
Rabiei H, Zare Mirakabadi A, Mohtat B, Mirza B. Purification and Molecular Characterization of a Mammalian Neurotoxin as a Pharmaceutical Tool from the Venom of Iranian Scorpion Androctonus crassicauda. J Arthropod Borne Dis. 2025;:238-252.
