Purification of Fully ActivatedClostridium botulinumSerotype B Toxin for Treatment of Patients with Dystonia

Abstract

Clostridium botulinum serotype B toxins 12S and 16S were separated by using a beta-lactose gel column at pH 6.0; toxin 12S passed through the column, whereas toxin 16S bound to the column and eluted with lactose. The fully activated neurotoxin was obtained by applying the trypsin-treated 16S toxin on the same column at pH 8.0; the neurotoxin passed through the column, whereas remaining nontoxic components bound to the column. The toxicity of this purified fully activated neurotoxin was retained for a long period by addition of albumin in the preparation.Clostridium botulinum strains produce immunologically distinct neurotoxins (serotypes A to G). The molecular masses of neurotoxin types A to G are approximately 150 kDa. The neurotoxins are produced as a single form and become dichain-form light (50-kDa) and heavy (100-kDa) chains by cleavage with proteases such as trypsin at about one-third of the distance from the amino terminus, and the toxic activity of the dichain form becomes fully activated (1). In culture fluid and food with acidic conditions, the neurotoxins associate with nontoxic components and form large complexes designated progenitor toxins. Under alkaline conditions, the progenitor toxins dissociate into neurotoxin and nontoxic components (10, 18). The progenitor toxins are found in three forms with molecular masses of 900 kDa (19S), 500 kDa (16S), and 300 kDa (12S) (14). The 12S toxin is composed of a neurotoxin and a nontoxic component having no hemagglutinin (HA) activity (designated nontoxic non-HA [NTNH]), whereas the 16S and 19S toxins are composed of a neurotoxin, NTNH, and HA. The serotype A strain produces three forms of toxins (19S, 16S, and 12S). Type B, C, and D strains produce the 16S and the 12S toxins. We purified different-sized progenitor toxins from serotype A, C, and D cultures (2,4,6,12,13) and demonstrated that (i) the 19S toxin is a dimer of the 16S toxin; (ii) HA consists of four subcomponents with molecular masses of 52 to 53, 33 to 35, 19 to 23, and 15 to 17 kDa, designated here as HA3b, HA1, HA3a, and HA2, respectively; and (iii) NTNH of the 12S toxin has a cleavage site(s) at the N-terminal region.Recently, serotype A and B progenitor toxins have been used for treating patients with strabismus, blepharospasm, nystagmus, facial spasm, spastic aphonia, and many other forms of dystonia (9, 11). In both toxin types, progenitor toxins are used because they are easily obtained and are more stable than neurotoxin. The treatment is very effective but has a serious side effect for some patients in whom antiprogenitor toxins, including antineurotoxin antibodies, are produced after several injections. It seems that using neurotoxin alone is better than using the progenitor toxin (a complex of the neurotoxin and a nontoxic component). Furthermore, it has been reported that serotype B toxin, which is used therapeutically at present, is partially cleaved (16) and therefore the toxin is not fully activated. In this paper, we report a simple procedure for largescale purification of botu...

Efficacy of a Potential Trivalent Vaccine Based on Hc Fragments of Botulinum Toxins A, B, and E Produced in a Cell-Free Expression System

“…3b, right panel). The same antiserum reacted with the toxin A complex that exhibited a multiband profile, in agreement with the presence of hemagglutinins (1,27). Incubation with antipeptide-specific antibody generated, as expected, a single band at 47 kDa representing in vitro-expressed HcA and a single band at 100 kDa for neurotoxin A heavy chain (Tox A).…”

Production of anti-neurotoxin antibody is enhanced by two subcomponents, HA1 and HA3b, of Clostridium botulinum type B 16S toxin–haemagglutinin

“…We believed this to indicate that PTX may have adjuvant activity. As HA1 and HA3b, but not HA2 or HA3a, bind specifically to the glycoprotein or glycolipid of erythrocytes and epithelial cells of the small intestine (Fujinaga et al, 2000(Fujinaga et al, , 2004Inoue et al, 2001Inoue et al, , 2003Nishikawa et al, 2004), we speculated that HA1 and HA3b might have adjuvant activity. In this study, we first observed that when mice were immunized with formalin-detoxified NTX, 12S or 16S, a significantly greater amount of anti-NTX Ab was produced in the mice injected with 16S than in NTX-or 12S-injected lanes 1-4).…”

“…Therefore, NTXs can be separated into two fragments, designated a light chain (L, 50 kDa) and a heavy chain (H, 100 kDa) upon SDS-PAGE under reducing conditions (Sugiyama, 1980). C. botulinum type B culture produces both single (unnicked) and dichain (nicked) forms of NTX (Arimitsu et al, 2003). In culture fluids, NTX is associated with non-toxic components to form progenitor toxin (PTX).…”

“…The 16S toxin contains HA in addition to 12S toxin. HA consists of four subcomponents with molecular masses of 51, 34, 19-23 and 18 kDa, designated HA3b, HA1, HA3a and HA2, respectively (Arimitsu et al, 2003;Fujinaga et al, 1994;Oguma et al, 1999). The NTNH of the 16S toxin is intact (130 kDa), whereas that of the 12S toxin is cleaved at its Nterminal region to form 112 and 15 kDa bands upon SDS-PAGE with a reducing reagent (Arimitsu et al, 2003;Oguma et al, 1999).…”

“…HA consists of four subcomponents with molecular masses of 51, 34, 19-23 and 18 kDa, designated HA3b, HA1, HA3a and HA2, respectively (Arimitsu et al, 2003;Fujinaga et al, 1994;Oguma et al, 1999). The NTNH of the 16S toxin is intact (130 kDa), whereas that of the 12S toxin is cleaved at its Nterminal region to form 112 and 15 kDa bands upon SDS-PAGE with a reducing reagent (Arimitsu et al, 2003;Oguma et al, 1999). In alkaline conditions (above pH 7?2), PTXs dissociate into an NTX and a non-toxic component, the 12S toxin dissociates into an NTX and an NTNH, and the 16S toxin dissociates into an NTX and a non-toxic component that is a complex of NTNH and HA.…”