[39 Tetanus and Botulism Neurotoxins Isolation and Assay

By Giampietro Schiavo and Cesare Montecucco Introduction

The paralysis associated with tetanus and botulism is caused by neurotoxins produced by bacteria of the genus Clostridium. These are the most powerful toxins known, the LD50 in mice being in the range 0.1-1 ng/kg. Tetanus neurotoxin (TeNT) is produced by toxigenic strains of Clostridium tetani in a single type, whereas seven different serotypes (A, B, C, D, E, F, and G) of botulism neurotoxin (BoNT) are made by Clostridium botuli-num and other Clostridia.1"3

The neurotoxins have a similar structural organization. They are synthesized as a single polypeptide chain of 150 kDa, which accumulates in the cytosol until bacterial lysis. When released the toxin is rapidly cleaved by various proteinases, at a single exposed loop.4 This generates the two-chain neurotoxin, composed of a heavy chain (H, 100 kDa) and a light chain (L, 50 kDa), held together by a single disulfide bond, as depicted in Fig. 1.

These toxins, as well as all bacterial protein toxins with intracellular targets, penetrate cells with a four-step mechanism: (a) binding, (b) internalization, (c) membrane translocation, and (d) cytosolic target modification. The 50-kDa carboxyl-terminal part of the H chain appears to be responsible for the neurospecific binding at the presynaptic membrane of the neuromuscular junction.1"3 Neuronal receptors involved in such a specific and high-affinity binding have not been identified, but different receptors are involved in the binding of the different neurotoxins.5'6 After binding, the neurotoxins are internalized into endosome-like vesicular organelles, and at this stage they cannot be neutralized by antitoxin antibodies.6-7 For the toxins to reach their cytosolic targets, they must move across the intracellular vesicle membrane, and there is evidence that this step requires acidifica' L. L. Simpson (ed.), "Botulinum Neurotoxins and Tetanus Toxin." Academic Press, New York, 1989.

2 C. Montecucco, E. Papini, and G. Schiavo, FEBS Lett. 346, 92 (1994).

3 C. Montecucco and G. Schiavo, Mol. Microbiol. 13, 1 (1994).

4 K. G. Krieglstein, A. H. Henschen, U. Weller, and E. Habermann, Eur. J. Biochem. 202, 41 (1991).

5 J. D. Black and J. O. Dolly, J. Cell Biol. 103, 521 (1986).

6 C. Montecucco, Trends Biochem. Sci. 11, 314 (1986).

7 J. D. Black and J. O. Dolly, J. Cell Biol. 103, 535 (1986).

Fig. 1. Scheme of the structure and activation of tetanus and botulism neurotoxins. The single polypeptide chain of 150 kDa is folded in three distinct 50-kDa domains: L is the zinc endopeptidase, HN is responsible for cell penetration, and Hc is responsible for the neurospe-cific binding. The proteinase activity of each toxin is revealed only after the generation of free L chain by selective proteolysis and reduction.

Fig. 1. Scheme of the structure and activation of tetanus and botulism neurotoxins. The single polypeptide chain of 150 kDa is folded in three distinct 50-kDa domains: L is the zinc endopeptidase, HN is responsible for cell penetration, and Hc is responsible for the neurospe-cific binding. The proteinase activity of each toxin is revealed only after the generation of free L chain by selective proteolysis and reduction.

tion of the vesicle lumen.8 The 50 kDa amino-terminal part of the H chain is mainly responsible for the translocation of the L chain.910 Finally, once inside the neuronal cytosol, each clostridial neurotoxin L chain recognizes and cleaves at a unique site a single protein component of the neuroexo-cytosis apparatus.311"18

The L chains of TeNT and BoNT represent a new group of zinc-dependent endopeptidases.3 A single molecule of L chain is capable of catalyzing the proteolysis of many substrate molecules before being neutralized by the cell.3 This property, together with the neurospecificity, accounts for the high potency of the neurotoxins. This chapter deals with the isolation of

8 L. C. Williamson and E. A. Neale,./. Neurochem. 63, 2342 (1994).

9 D. H. Hoch, M. Romero-Mira, B. E. Ehrlich, A. Finkelstein, B. R. DasGupta, and L. L. Simpson, Proc. Natl. Acad. Sci. U.S.A. 82, 1692 (1985).

10 C. C. Shone, P. Hambleton, and J. Melling, Eur. J. Biochem. 167, 175 (1987).

11 G. Schiavo, B. Poulain, O. Rossetto, F. Benfenati, L. Tauc, and C. Montecucco, EMBO J. 11, 3577 (1992).

12 G. Schiavo, F. Benfenati, B. Poulain, O. Rossetto, P. Polverino de Laureto, B. R. DasGupta, and C. Montecucco, Nature (London) 359, 832 (1992).

13 Deleted in press.

14 G. Schiavo, C. C. Shone, O. Rossetto, F. C. G. Alexander, and C. Montecucco, J. Biol. Chem. 268, 11516 (1993).

15 J. Blasi, E. R. Chapman, E. Link, T. Binz, S. Yamasaki, P. De Camilli, T. Sudhof, H. Nieman, and R. Jahn, Nature (London) 365, 160 (1993).

16 G. Schiavo, O. Rossetto, S. Catsicas, P. Polverino De Laureto, B. R. DasGupta, F. Benfenati, and C. Montecucco, J. Biol. Chem. 268, 23784 (1993).

17 G. Schiavo, A. Santucci, B. R. DasGupta, P. P. Metha, J. Jontes, F. Benfenati, M. C. Wilson, and C. Montecucco, FEBS Lett. 335, 99 (1993).

18 J. Blasi, E. R. Chapman, S. Yamasaki, T. Binz, H. Nieman, and R. Jahn, EMBO J. 12, 4821 (1993).

18a G. Schiavo, C. Malizio, W. S. Trimble, P. Polverino de Laureto, H. Sugiyama, E. Johnson, and C. Montecucco, J. Biol. Chem. 269, 20213 (1994).

neurotoxins free from contaminant proteinase activities, their characterization, and assay of the zinc endopeptidase activities.

Safety Precautions

Clostridial neurotoxins are very toxic. However, they do not affect individuals immunized with the corresponding toxoids (toxins detoxified by treatment with paraformaldehyde). In most countries children are vaccinated with tetanus toxoid, and this is sufficient to provide full protection against tetanus for decades. A booster injection of tetanus toxoid (available from pharmacies and health authorities) before starting research with tetanus toxin is advisable. One dose is sufficient to bring the serum antitetanus toxin titer to a full protection level.

In contrast, the vaccine for BoNT serotypes A, B, C, D, and E is not commercially available and can be obtained from the Centers for Disease Control (CDC, Atlanta, GA). Only after the third injection (usually performed 2 months after the first) is a protective serum anti-BoNT titer generally, but not always, achieved. This can be checked by incubating various dilutions of the serum with the toxin and then injecting into mice. Human anti-TeNT antibodies and horse anti-BoNT antibodies are also available from health authorities, and their injection immediately after accidental penetration of the toxin into the circulatory system is sufficient to prevent the disease.

Work with the toxins should be performed in a contained space. Every tool should be washed at the end of the experiment with dilute sodium hypochlorite (the toxins are extremely sensitive to oxidants).

Purification of Tetanus Neurotoxin

Principle. Tetanus neurotoxin is an abundant protein of the supernatants obtained from cultures of toxigenic strains of Clostridium tetani. The TeNT gene is contained in a plasmid and codes for a single polypeptide chain protein (s-TeNT), composed of 1315 amino acids (MT 150,700).3 s-TeNT is released by bacterial lysis and is rapidly converted by bacterial proteinases into the two-chain TeNT form.4 The most suitable growth medium for the preparation of TeNT does not contain proteins, thus simplifying the purification procedures.19 Different growth conditions and extraction procedures are followed for the preparation of the single-chain TeNT (s-TeNT), two-chain TeNT (TeNT), and the L chain.

19 W. C. Latham, D. F. Bent, and L. Levine. Appl. Microbiol. 10, 146 (1962).

Reagents

Buffer A: 2 mM Sodium citrate, 100 mM sodium potassium phosphate,

1 mM EDTA, 1 mM sodium azide, 1 mM benzamidine, pH 7.5 Buffer B: 10 mM Sodium phosphate, pH 7.4 Buffer C: 10 mM Sodium HEPES, 150 mM NaCl, pH 7.4 Buffer D: 320 mM Sucrose, 4 mM sodium HEPES, pH 7.3 Buffer E: 4 mM Sodium HEPES, 300 mM glycine, 0.02% w/w sodium azide, pH 7.3

Single-Chain Tetanus Neurotoxin. Clostridium tetani (Harvard strain) is harvested before the end of the exponential growth phase20 by centrifuga-tion for 10 min at 10,000 g, and the resulting bacterial pellet is washed twice with 10 mM sodium phosphate, 145 mM NaCl, pH 7.5. The cells are lysed by overnight extraction under stirring at 4° with 100 mM sodium citrate, 1 M NaCl, 2 mM benzamidine, 1 mM diisopropyl fluorophosphate (DFP), pH 7.5. The supernatant, clarified by centrifugation at 10,000 g for 30 min, is fractionated by the addition of a saturated ammonium sulfate solution to 43% saturation at 4°; the solution is kept at pH 7.0 by the addition of 1 M NaH2P04. The toxin precipitate is centrifuged at 22,000 g for 30 min and washed with 40% saturated ammonium sulfate in buffer A. After centrifugation as above, the pellet is dissolved in 50 ml of buffer A containing 1 mM diisopropyl fluorophosphate, dialyzed against 10 mM sodium phosphate, pH 6.8, and then clarified by centrifugation at 40,000 g for 30 min. The resulting supernatant, containing 80% pure single-chain TeNT, is processed with DEAE-cellulose and Aca 34 chromatography as described for two-chain TeNT.20'21

Two-Chain Tetanus Neurotoxin and L Chain. A 6- to 8-day-old sterile culture supernatant of Clostridium tetani (Harvard strain), obtained from a vaccine company (Sclavo, Siena, Italy), is precipitated with 250 g/liter of finely ground solid ammonium sulfate at 4°. The pellet is recovered by centrifugation at 16,000 g for 10 min at 4° and dissolved in 10 mM sodium phosphate, pH 7.4 (buffer B). The precipitation step is repeated with the final percentage of ammonium sulfate increased to 46% of saturation. The resulting pellet is dissolved in buffer B and dialyzed extensively against the same buffer. After clarification of the dialyzed toxin solution by centrifugation (16,000 g for 10 min at 4°), the dark-brown supernatant is applied to a diethylaminoethyl-derivatized cellulose (DEAE-cellulose) column (DE-52; Whatman, Clifton, NJ; 15 mg protein/ml resin), previously equilibrated with buffer B. The toxin is eluted with a linear gradient of 10-100 mM

20 K. Ozutsumi, N. Sugimoto, and M. Matsuda, Appl. Environ. Microbiol. 49, 939 (1985).

21 U. Weller, F. Mauler, and E. Habermann, Naunyn-Schmiedeberg's Arch. Pharmacol. 338, 99 (1988).

sodium phosphate, pH 7.4, and fractions of 4 ml are collected. After checking the protein composition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% polyacrylamide gels, the toxin-containing fractions are pooled and precipitated with 60% saturated ammonium sulfate.

The ammonium sulfate suspension is spun at 27,000 g for 15 min at 4°, and the resulting pellet is dissolved in a minimum volume of buffer B and applied to an Aca 34 gel-filtration column (3 X 130 cm), equilibrated with the same buffer and eluted at a 25 ml/hr constant flow. Fractions of 8 ml are collected. Two peaks, corresponding to TeNT (95% pure) and L chain (70% pure), are obtained. The proportion of L chain increases with the aging of the bacterial culture. The fractions corresponding to TeNT and L chain are pooled from the eluate, precipitated with 60% saturated ammonium sulfate at pH 7.0, and stored at 4° until further purification is performed.

Nicking of Single-Chain Tetanus Neurotoxin. The percentage of single-chain and two-chain TeNT in the final toxin preparation may vary depending on the growth conditions and the age of the bacterial culture. To obtain the two-chain toxin, the solution containing s-TeNT is treated with a proteinase to promote cleavage at a single site located within an exposed loop.4 It is advisable to perform an analytical test to optimize nicking conditions first. s-TeNT is cleaved with tosylsulfonyl phenylalanyl chlo-romethyl ketone-treated trypsin (Serva, Heidelberg, Germany) at 25° for 60 min using a toxin/proteinase ratio of 1000:1 (w/w). Proteolysis is terminated by the addition of soybean trypsin inhibitor at a proteinase-to-inhibi-tor ratio of 1:4 (w/w). The procedure is also applicable with minor modifications to the nicking of single-chain BoNTs.22

Immobilized Metal Ion Affinity Chromatography. The TeNT, isolated as above, and BoNT serotypes A, B, C, E, and F isolated as described elsewhere22 contain traces of contaminant clostridial proteinases. The neurotoxins commercially available from Calbiochem (La Jolla, CA), Sigma (St. Louis, MO), or Wako (Neuss, Germany) also contain additional proteins. The neurotoxins can be rapidly freed from contaminant proteases using an immobilized metal ion affinity chromatography (IMAC) step, which exploits the coordination between electron donor groups of the protein surface and the chelated metal ions bound to the iminodiacetic acid groups attached to the agar-based matrix.23-24 Among the protein

22 L. L. Simpson, J. J. Schmidt, and J. L. Middlebrook, this series, Vol. 165, p. 76.

24 E. S. Hemdan, Y. Zhao, E. Sulkowski, and J. Porath, Proc. Natl. Acad. Sci. U.S.A. 86, 1881 (1989).

surface residues able to interact with the transition metal ions in IMAC separations, histidines and cysteines are the predominant ligands.23'24 The experimental procedure may be divided into two sections: preparation of the metal-bound column and purification of the toxin.25

A chelating Superose HR 10/2 column (Pharmacia, Piscataway, NJ), connected with a Beckman (Palo Alto, CA) System Gold high-performance liquid chromatography (HPLC) apparatus, is washed at a constant flow rate of 0.5 ml/min with 7.5 ml of 50 mM EDTA, 500 mM NaCl, pH 6.0, and equilibrated with 10 ml of 50 mM sodium acetate, 200 mM NaCl, pH 6.0. The column is loaded with zinc ions by flowing 7.5 ml of 100 mM zinc chloride through it. Unbound metal ions are removed with 10 ml of 50 mM sodium acetate, 200 mM NaCl, pH 6.0. Finally, the column is equilibrated with 10 mM sodium HEPES, 150 mM NaCl, pH 7.4 (buffer C). The TeNT and BoNTs (0.1-10 mg), previously dialyzed into buffer C, are loaded onto the column at room temperature, and unbound material is washed away with the same buffer. Toxins are eluted with a linear gradient of 0-25 mM imidazole in buffer C at a constant flow rate of 0.5 ml/min (Fig. 2A).

This procedure is also useful for the purification of Hc, the 50-kDa carboxy-terminal part of the heavy chain of TeNT, which shows an identical retention time. This is due to the fact that histidine residues located in Hc are responsible for TeNT binding to the immobilized zinc. The same procedure is followed to purify BoNTs, as depicted for BoNT/A in Fig. 2A. Purified TeNT, its Hc fragment, and BoNTs are dialyzed against 10 mM sodium HEPES, 50 mM NaCl, pH 7.2, and, after freezing in liquid nitrogen, are stored at -80°.

This procedure cannot be used with BoNT/D because that serotype is not retained by the zinc-IMAC column. To obtain a proteinase-free BoNT/D preparation, a commercially available source (0.5-10 mg; Wako) is used. The material is dialyzed against 20 mM Tris-Cl, pH 8.0, and loaded onto a Mono Q HR 5/5 column, previously equilibrated with the same buffer. Proteins are eluted with a step gradient of NaCl, as shown in Fig. 2B: BoNT/D is present in peak 1, as determined by SDS-PAGE (Fig. 2C); peak 2 contains a nontoxic unidentified protein.

Purification of L Chain of Tetanus Toxin. The L chain, obtained as above, is contaminated by TeNT and small molecular weight peptides. A three-step HPLC purification procedure is followed to remove the contaminants. The L chain ammonium sulfate precipitate is dissolved in 100 mM sodium phosphate, pH 6.8, and loaded in 20-mg aliquots onto a preparative G 3000 SW column (21.5 X 600 mm, Pharmacia). The peak enriched in L

25 O. Rossetto, G. Schiavo, P. Polverino de Laureto, S. Fabbiani, and C. Montecucco, Biochem.

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