The effect of pH on recombinant C-terminal domain of Botulinum Neurotoxin type E (rBoNT/E-HCC)

Document Type: Original article

Authors

1 Department of Biology, Faculty of Basic Sciences, Imam Hussein University, Tehran, Iran.

2 Department of Biology, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz, Iran.

Abstract

Recombinant proteins are tending to be the most favorable vaccine-candidates against botulism. Recombinant Carboxy-terminal of botulinum neurotoxin serotype E (rBoNT/E-HCC) has been introduced as an efficient vaccine against botulism type E. In this report, we made an effort to investigate the effect of different pH on protein structure to assess if rBoNT/E-HCC could be used as a vaccine for oral administration. Initially, rBoNT/E-HCC was expressed and purified. Structural changes of rBoNT/E-HCC at several pH conditions were studied by various techniques including circular dichroism (CD), fluorescence, aggregation and UV-Vis spectroscopy. The results showed the more compact and more stable structure for rBoNT/E-HCC at acidic pH, and loosely folded structure at alkaline pH. Our finding as the first step of rBoNT/E-HCC evaluation, hopefully introduce it as a suitable vaccine candidate for oral administration.

Keywords


1.Simpson LL. Botulinum Neurotoxin and Tetanus Toxin; Academic Press: San Diego CA 1989.

2.Hatheway CL. Toxigenic clostridia. Clin Microbiol Rev 1990;3:66-98.

3.Simpson LL. The origin, structure, and pharmacological activity of botulinum toxin. Pharmacol Rev 1981;33:155-188.

4.Maselli RA. Pathogenesis of human botulism. Ann N Y Acad Sci 1998;841:122-139.

5.Johnson EA, Goodnough MC. Botulism. In: Topley and Wilson’s microbiology and microbial infections: bacterial infections; Coliter, L., Ballows, A., and Sussman, M., eds.; Arnold: London, 1998;3:723-741.

6.Dasgupta BR, Antharavally BS, Tepp W, Evenson ML. Botulinum neurotoxin types A, B, and E: fragmentations by autoproteolysis and other mechanisms including by O-phenanthroline-dithiothreitol, and association of the dinucleotides NAD(+)/NADH with the heavy chain of the three neurotoxins. Protein J 2005;24:337-368.

7.Ahmed SA, Byrne MP, Jensen M, Hines HB, Brueggemann E, Smith LA. Enzymatic autocatalysis of botulinum A neurotoxin light chain. J Protein Chem 2001;20:221-231.

8.Schiavo G, Montecucco C. Tetanus and botulism neurotoxins: isolation and assay. Meth Enzymol 1995;248:643-652.

9.Maruta T, Dolimbek BZ, Aoki KR, Steward LE, Atassi MZ. Mapping of the synaptosome-binding regions on the heavy chain of botulinum neurotoxin A by synthetic overlapping peptides encompassing the entire chain. Protein J 2004;23:539-552.

10.Tonello F, Morante S, Rossetto O, Schiavo G, Montecucco C. Tetanus and botulism neurotoxins: a novel group of zinc-endopeptidases. Adv Exp Med Biol 1996;389:251-260.

11.Cai S, Kukreja R, Shoesmith S, Chang TW, Singh BR. Botulinum neurotoxin light chain refolds at endosomal pH for its translocation. Protein J 2006;25:455-462.

12.Parikh S, Singh BR. Comparative membrane channel size and activity of botulinum neurotoxins A and E. Protein J 2007;26:19-28.

13.Schiavo G, Santucci A, Dasgupta BR, Mehta PP, Jontes J, Benfenati F, Wilson MC, Montecucco C. Botulinum neurotoxins serotypes A and E cleave SNAP-25 at distinct COOH-terminal peptide bonds. FEBS Lett 1993;335:99-103.

14.Schiavo G, Malizio C, Trimble WS, Polverino de Laureto P, Milan G, Sugiyama H, Johnson EA, Montecucco C. Botulinum G neurotoxin cleaves VAMP/synaptobrevin at a single Ala-Ala peptide bond. J Biol Chem 1994;269:20213-20216.

15.Blasi J, Chapman ER, Yamaski S, Binz T, Niemann H, Jahn R. Botulinum neurotoxin C1 blocks neurotransmitter release by means of cleaving HPC1/syntaxin. EMBO J 1993;12:4821-4828.

16.Ahmed SA, Olson MA, Ludivico ML, Gilsdorf J, Smith LA. Identification of residues surrounding the active site of type A botulinum neurotoxin important for substrate recognition and catalytic activity. Protein J 2008;27:151-162.

17.Middlebrook JL. Protection strategies against botulinum toxin. Adv Exp Med Biol 1995;383:93-98.

18.Robinson RF, Nahata MC. Management of botulism. Ann Pharmacother 2003;37:127-131.

19.Byrne MP, Smith LA. Development of vaccines for prevention of botulism. Biochimie  2000;82:955-966.

20.Kubota  T, Watanabe  T, Yokosawa N, Tsuzuki K, Indoh T, Moriishi K, Sanda K, Maki Y, Inoue K, Fujii N. Epitope regions in the heavy chain of Clostridium botulinum type E neurotoxin recognized by monoclonal antibodies. Appl Environ Microbiol 1997;63:1214-1218.

21.Atassi MZ, Dolimbek BZ. Mapping of the antibody-binding regions on the HN-domain (residues 449-859) of botulinum neurotoxin A with antitoxin antibodies from four host species. Full profile of the continuous antigenic regions of the H-chain of botulinum neurotoxin A. Protein J 2004;23:39-52.

22.Agheli-Mansour A, Mousavi SL, Rasooli I, Nazarian S, Amani J, Farhadi N. Cloning, high level expression and immunogenicity of 1163-1256 residues of C-terminal heavy chain of C. botulinum neurotoxin type E. Biologicals 2010;38:260-264.

23.Rostamian M, Mousavy SJ, Ebrahimi F, Ghadami SA, Sheibani N, Minaei ME, Arefpour Torabi MA. Comparative study of immunological and structural properties of two recombinant vaccine-candidates against Botulinum Neurotoxin type E. Iranian Biomed J 2012;16:185-192.

24.Mestecky J. The common mucosal immune system and current strategies for induction of immune response in external secretions. J Clin Immunol 1987;7:265-276.

 25.Wingfield PT, Palmer I, Liang S. Folding and purification of insoluble (inclusion-body) proteins from Escherichia coli. Curr Protoc Protein Sci 2001;6:6.5.

26.Rezaei-Ghaleh N,  Ramshini H,  Ebrahim-Habibi A,  Moosavi-Movahedi AA,  Nemat-Gorgani M. Thermal aggregation of alpha-chymotrypsin: role of hydrophobic and electrostatic interactions. Biophys Chem 2008; 132:23-32.

27.Rudolph R, Bohm G, Lilie H, Jaenicke R. In Protein Function. A Practical Approach, edn 2. Edited by Creighton T.E. New York: IRL Press 1997.

28.LaVallie, ER, DiBlasio EA, Kovacic S, Grant KL, Schendel PF, McCoy JM. A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Biotechnology 1993;11:187-193.

29.Lakowicz JR. In: Principles of fluorescence spectroscopy; Springer: Berlin 2006: 3rd ed,  Vol. 1, protein  fluorescence chapter.

30.Ruddock LW, Hirst TR, Freedman RB. pH-dependence of the dithiol-oxidizing activity of DsbA (a periplasmic protein thiol:disulphide oxidoreductase) and protein disulphide-isomerase: studies with a novel simple peptide substrate. Biochem J 1996; 315:1001-1005.

31.Won CM, Molnar TE, McKean RE, Spenlehauer GA. Stabilizers against heat induced aggregation of RPR, 114849, an acidic fibroblast growth factor (aFGF). Int J Pharm 1998;167:25-36.

32.Wang W. Protein aggregation and its inhibition in biopharmaceutics. Int J Pharm 2005; 289:1-30.   

33.Bedu-Addo FK, Johnson C, Jeyarajah S, Henderson I, Advant SJ. Use of biophysical characterization in preformulation development of a heavy-chain fragment of botulinum serotype B: evaluation of suitable purification process conditions. Pharm Res 2004;21:1353-1361.

34.Cromwell ME, Hilario E, Jacobson F. Protein aggregation and bioprocessing. AAPS J 2006;8:E572-E579.