Purification Activation and Endocytosis of Botulinum C2 Toxin

I. OHISHI

9.1 Introduction

Clostridium botulinum type C was firstly isolated by Bengston in 1922 identification of C2 toxin (Bengston, 1922). In 1935, Mason and Robinson reported that C. botulinum type C produced three different toxic factors, CI, C2 and D (Mason and Robinson, 1935), although it had been considered at that time that other types of C. botulinum, types A and B, produced only one antigenic type of the toxin. This was the first use of the term C2 toxin in the literature. Later on, Jansen applied this notion to the toxins produced by C. botulinum Ca and Cp strains, which had been classified by immunological cross-neutralization; Ca produces CI, C2 and D toxins and Cp only C2 toxin (Jansen, 1971). Thus, C2 toxin had been thought of as a botulinum neurotoxin until it was purified and characterized in 1980 (Ohishi etal., 1980).

Botulinum C2 toxin is produced by certain strains of C. botulinum structure of C2 toxin types C and D (Ohishi and Sakaguchi, 1982). The toxin is constructed with two unlinked protein components, neither covalently nor non-covalently linked, designated component I and II. These were named after the elution sequence from a column of cation exchanger, e.g., CM-Sephadex (Iwasaki et a/., 1980, Ohishi et a/., 1980). Because of this unique molecular construction, the purification of the toxin had encountered difficulties, especially in assaying the activity; when the toxin preparation was chromatographed on either ion exchanger or gel-filtration column, the two components of the toxin were eluted separately due to their different charges or their different molecular weights (Iwasaki etal., 1980; Ohishi etal., 1980). This was resolved by the finding that the activity of C2 toxin requires two separate protein components; only a mixture of these components manifests the toxicity (Ohishi etal., 1980).

9.2 Assay Method for the Toxin

One of the important points for purification of proteins is that an assay for biological activity can be done rapidly within a short period. After C2 toxin had been purified, it was found that the toxin, in addition to biological activities of C2 toxin lethality, has various biological activities, i.e. increase in vascular permeability, fluid accumulation in ligated intestine, rounding of tissue-cultured cells (Miyake and Ohishi, 1987; Ohishi et a/., 1980; Ohishi, 1983). Of these activities, however, the appropriate assay method for C2 toxin is the determination of "a time-to-death" after intravenous injection of toxin preparation into mice (Iwasaki et a/., 1980; Ohishi et al., 1980). This is because the assay can be done within about 120 min, if it contains 5-100 ipLD50/ml, which roughly corresponds to 0.2-5 ¡xg of C2/ml as a mixture of components I and II in a ratio of 1:2. The assay by the time-to-death method is as follows.

assay by "time-to-death"

activation of component II

1. Prepare a culture of a C2 toxin-producing strain of C botulinum: it is preferable to use strains, that do not produce botulinum neurotoxin, because the neurotoxin interferes the determination of toxicity of C2 toxin. Otherwise, use anti-neurotoxin serum to neutralize the neurotoxic activity.

2. Concentrate the culture supernatant by adding solid ammonium sulfate at 65 % saturation. Collect the precipitate by centrifuga-tion at 10000 g for 10 min at 4 °C, suspend it in 10 mM K, Na-phosphate buffer (PB), pH 7.3, containing 150mM NaCI (PBS) and dialyze the suspension against the buffer at 4 °C.

3. Before assaying the activity, C2 toxin must be trypsinized (Ohishi et al., 1980; Ohishi, 1987). To prepare the activated toxin, incubate the dialyzed preparation with trypsin at a final concentration of 200 [xg/ml in 50 mM PB, pH 8.0, for 30 min at 37 °C. Terminate the trypsinization by adding twice the weight of soybean trypsin inhibitor into the reaction mixture.

To activate purified component II, a ratio of trypsin to purified component II of 1:10 is required; trypsinization is required only for component II but not for component I (Ohishi eta/., 1980; Ohishi, 1987) and the activity of the toxin can be assayed by mixing untrypsinized component I and trypsinized component II at a protein ratio of 1:2.

4. Determine the intraperitoneal 50 % lethal dose (ipLD50)/ml of the trypsinized toxin sample in mice.

5. Dilute the toxin sample with PBS in an ice bath, inject intravenously into three or four mice, and determine the time-to-death (survival time) in min.

6. Plot the time-to-death (30 to 120 min) against the toxin doses (5 to 100 ipLD50/ml) both in logarithms; this results in a linear relationship between them. From this curve, the time-to-death in min after intravenous injection of a toxin sample can be converted into the lethal activity (ipLD50).

Toxin preparations, whether activated or not activated, should be detoxified by boiling in a water bath for 3 to 5 min. If it might possibly contain culture or culture supernatant of C2 toxin producing bacteria, all materials used for the experiments, e.g. toxin solution, injectors, test tubes, tips etc., should be autoclaved at 1 kg/cm2 for at least 15min or soaked in 1 N NaOH overnight, as they may be possibly contaminated with heat-resistant spores.

decontaminate all materials and equipment after use

9.3 Purification Procedures for the Two Components of C2 Toxin

The flow sheet for purification steps is shown in Table 1.

Table 1. Flow sheet for purification of the two components of C2 toxin

Culture fluid of C. botulinum type C strain 92-13

Ammonium sulfate fractionation

Calcium phosphate gel (Batchwise fractionation)

CM-Sephadex chromatography

Pass-through fraction (component I)

DEAE-Sephadex chromatography

Hydroxyapatite chromatography

Gel filtration on Sephacryl S-300

Component I

Adsorbed and eluted fraction (Component II)

Gel filtration on Sephacryl S-300

Component II

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