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City Changes of Clostridium Botulinum Type a Neurotoxin Acta Pharmacologica Sinica 2006 Sep; 27 (9): 1238!1246 Full-length article Disulfide bond reduction corresponds to dimerization and hydrophobi- city changes of Clostridium botulinum type A neurotoxin Jiunn-jye WEY1, Shiao-shek TANG1, Tzong-yuan WU2,3 1Institute of Preventive Medicine, National Defense Center, Taipei, Taiwan 115, China; 2Department of Bioscience Technology, Chung Yuan Christian University, Chungli, Taiwan 320, China Key words Abstract Clostridium botulinum; botulinum neuro- Aim: To determine the structure factors that mediate the intoxication process of toxin A; hydrophobicity; phase partition botulinum neurotoxin type A (BoNT/A). Methods: Triton X-114 phase separation experiments and 1-anilino-8-naphthalene sulfonate binding assay were used to 3 Correspondence to Dr Tzong-yuan WU. study the structural factor that corresponds to the hydrophobicity change of Phn 886-3-265-3520. BoNT/A. In addition, sucrose density gradient centrifugation and a chemical Fax 886-3-265-3599. E-mail [email protected] crosslinking study were employed to determine the quaternary structure of BoNT/ A. Results: Our results demonstrated that in other than acidic conditions, the Received2005-12-22 disulfide reduction is the structural factor that corresponds to the hydrophobicity Accepted2006-04-16 change of BoNT/A. The quaternary structure of BoNT/A exists as a dimmer in doi: 10.1111/j.1745-7254.2006.00372.x acidic solution (pH 4.5), although the monomeric structure of BoNT/A was re- ported based on X-ray crystallography. Conclusion: Disulfide bond reduction is critical for BoNT/A s channel formation and ability to cross endosome membranes. This result implies that compounds that block this disulfide bond reduction may serve as potential therapeutic agents for botulism. protein of 25 kDa (SNAP-25). The receptor binding domain Introduction specifically binds to its neuronal receptor, ganglioside GT1b [8] Botulinum neurotoxins (BoNT) are potent toxins and are or GD1a, and unidentified protein receptors . After receptor also therapeutic agents[1]. These botulism-causing protein binding and receptor-mediated endocytosis of the neuro- toxins are produced by the anaerobic eubacterium, Clostri- toxins, they enter acidic neuronal organelles, synaptic dium botulinum. There are seven serologically and geneti- vesicles, or endosomes[8,9]. It is believed that the transloca- cally different BoNT, named BoNT/A-G, produced by sero- tion domain undergoes a conformational change and forms types A, B, C, D, E, F, and G Clostridium botulinum strains[2]. a protein-conducting channel on the membrane of the endo- BoNT/A is synthesized by serotype A Clostridium botuli- some to translocate the catalytic domain into the cytoplasm num strain as a single-chain protein with a molecular mass of of neuronal cells[10]. The catalytic domain of BoNT/A is a approximately 150 kDa[3,4]. This protein is post-translationally zinc protease[11,12] and is highly specific for the C-terminus of proteolyzed to form a di-chain molecule in which the two SNAP-25, a soluble NSF accessory protein receptor (SNARE) chains, a ~50 kDa light chain and a ~100 kDa heavy chain, protein complex component[13]. Cleavage of the C-terminus remain linked by a disulfide bond and non-covalent bonds[5,6]. of SNAP-25 by the catalytic domain of BoNT/A inhibits The crystal structure of BoNT/A shows a linear arrangement SNARE complex formation as well as neurotransmitter re- of three functional domains named the receptor binding lease[13,14]. Inhibition of the neurotransmitter, eg, domain, translocation domain, and catalytic domain[7]. These acetylcholine, released in the neuromuscular junction ulti- structural characters correspond to the intoxication process mately leads to paralysis and causes botulism[1]. This spe- of BoNT/A and the mechanism of BoNT/A intoxication con- cific action at the neuromuscular junction of BoNT is in- sisting of 4-steps: receptor binding, internalization, creasingly being used to treat various neuromuscular disor- translocation, and cleavage of the synaptosomal associated ders such as strabismus, torticollis, and blepharospasm[15,16]. 1238 2006 CPS and SIMM Http://www.chinaphar.com Wey JJ et al Among the intoxication processes of BoNT/A and other injection, two booster injections were given. Two weeks types of BoNT, the translocation process is the least under- after the booster, the rabbits were exsanguinated from the stood[1,7]. Previous results show that BoNT can form ion cervical artery. Polyclonal antibodies were purified from the channels in artificial planar lipid bilayers under acidic condi- rabbit s serum by protein A Sepharose 4 fast-flow affinity tions[10,17,18] and PC12 cell membrane[19]. But, how can such a chromatography for IgG antibodies[27]. To establish hybri- big soluble protein dramatically change to a hydrophobic doma cell lines that produce BoNT/A monoclonal antibodies, membrane protein? And how BoNT responds to acidic con- BALB/c mice were immunized intraperitoneally with 250 !g ditions to form an ion channel and how the light chain trans- toxoid in Freund s complete adjuvant and were boosted twice locates across the membrane barrier is not clear. Furthermore, with 100 !g toxoid in Freund s incomplete adjuvant at inter- the quaternary structure of BoNT/A in aqueous solution is vals of 2 weeks. Mice were boosted intraperitoneally with 50 not fully resolved. Native gel electrophoresis had showed !g toxoid in PBS 1 month later, and spleen cells were fused that the possible quaternary structure of BoNT/A is trimer or with murine plasmacytoma FO cells for 4 d by the method of tetramer[20,21]. However, the crystal structure of BoNT/A Galfre and Milstein[28]. An enzyme-linked immunosorbent meant that it was initially recognized as a dimeric species[22], assay (ELISA) was used to screen culture supernatants show- but it is now recognized as a monomeric species[23]. It is ing positive binding to BoNT/A proteins. Hybridomas from believed that the quaternary structure derived from X-ray positive-binding wells were cloned by the limiting dilution crystallography is not reliable, because under crystallization method. To obtain monoclonal antibodies, approximately conditions the quaternary structure may change[24, 25]. Thus, 5"106 hybridoma cells were inoculated into the peritoneal a clear solution to the structure of BoNT/A in solution is cavity of Pristance primed BALB/c mice, and ascitic fluid critical, not only because of these conflicting reports but was collected on d 7#14. Monoclonal antibodies were then also because it can clarify the translocation process of BoNT/ purified from the ascitic fluid by 40% saturated ammonium A. In this study, sucrose density gradient centrifugation sulfate precipitation and protein A Sepharose 4 fast-flow af- and chemical crosslinking experiments illustrated that only finity chromatography for IgG antibodies. after disulfide bond reduction did BoNT/A undergo dimer- ELISA assay of BoNT/A The modified ELISA method ization in acidic conditions. And Triton-X 114 phase-sepa- developed by Engvall and Perlman[29] was used to analyze ration experiments showed that acidic conditions and disul- the antibodies against BoNT/A. Microtiter plates (PolySorp fide reduction are factors mediating the change in hydro- surface,Nalge Nunc International, Naperville, IL, USA) were phobicity of BoNT/A. These results thus imply that disul- coated with 100 !L per well of BoNT/A protein (0.1 mg/mL) fide reduction is the structural factor that corresponds to in 0.01 mol/L PBS, pH 7.0, overnight. After blocking with 1% BoNT/A translocation in synaptic vesicles or endosomes. BSA (bovine serum albumin, Sigma Chemical Co, St Louis, MO. USA) in phosphate-buffered saline (PBS) for 1 h, plates were washed, and diluted polyclonal antibodies or hybri- Materials and methods doma supernatant (100 !L) were added to the wells and then Preparation of BoNT/A neurotoxin and toxoid We puri- incubated for 1 h. Plates were next washed and incubated fied BoNT/A neurotoxin from cultures of C botulinum type with alkaline phosphate (AP)-conjugated secondary anti- A (ATCC 7948) according to the method developed by bodies for 1 h. Finally, the alkaline phosphate enzyme activ- DasGupta and Sathyamoorthy[3]. The purity was examined ity was developed with the addition of substrate, and read- by 8% SDS-PAGE and Coomassie blue staining (Figure 1A). ings were taken at an optical density (OD) of 405 nm with a The different batch preparations contained the most nicked Dynatech MR700 microplate reader. For the BoNT/A BoNT/A and traceable un-nicked BoNT/A. To generate an- immunoassay, a sandwich ELISA was developed. The ELISA tibodies against the BoNT/A toxin, we prepared the BoNT/A plate wells were coated with 0.1 !g (1 !g/mL) monoclonal toxoid following the method developed by Kozaki and antibodies overnight and then incubated with 100 !L of the Sakaguchi[26]. BoNT/A (0.2 mg/mL) was toxoided by dialy- BoNT/A protein or BoNT/E (0.007 to 1 !g/mL) or samples sis against 0.4% formalin in 0.1 mol/L phosphate buffer (pH from sucrose density gradient centrifugation fractions for 7.0) for 6 d at room temperature. 1 h. The bound BoNT/A proteins were then labeled with 100 Production of antibodies The toxoid was mixed with an !L polyclonal antibodies (1 !g/mL), probed with AP-conju- equal volume of Freund s complete adjuvant. Three 0.5-mL gated goat anti-rabbit IgG, and quantified using the colori- doses of the toxoid emulsion were injected into two rabbits metric assay. Absorbance at 405 nm was measured with an (2.7 and 3.2 kg) at 7-day intervals. Six weeks after the third ELISA plate reader. 1239 Wey JJ et al Acta Pharmacologica Sinica ISSN 1671-4083 SDS-PAGE and immunoblotting Proteins were separated (2 !g) and incubated at room temperature.
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