Isolation and molecular size of Clostridium botulinum type C toxin.

A procedure is described for the purification of hemagglutinin-free Clostridium botulinum type C toxin. The toxin was purified approximately 1,000-fold from the original culture supernatant in an overall yield of 60% to a final specific toxicity of 4.4 x 10(7) minimal lethal doses/mg of protein. The toxin had a molecular weight of 141,000 and consisted of a heavy and a light chain. The molecular weights of the subunits were approximately 98,000 and 53,000. When comparing the molecular size and composition of type C toxin to that of botulinum toxins of different types, some common features may be suggested; i.e., the toxin has a molecular weight between 141,000 to 160,000 and is comprised of a heavy and a light chain linked by disulfide bonds (or bond).     

Presumptive identification of common adenovirus serotypes by the development of differential cytopathic effects in the human lung carcinoma (A549) cell culture

Abstract The neurotoxin gene from Clostridium barati ATCC43756 was cloned as a series of overlapping polymerase chain reaction (PCR) generated fragments using primers designed to conserve toxin sequences previously published. The toxin gene has an open reading frame (ORF) of 1268 amino acids giving a calculated molecular mass of 141049 Da. The sequence identity between the C. barati ATCC43756 and non-proteolytic C. botulinum 202F neurotoxins is 64.2% for the light chain and 73.6% for the heavy chain. This is much lower than reported identities for the type E neurotoxins from C. botulinum and C. butyricum (96% identity between light chains and 98.8% between the heavy chains). Previously identified conserved regions in other botulinal neurotoxins were also conserved in that of C. barati . An ORF upstream of the toxin coding region was revealed. This shows strong homology to the 3' end of the gene coding for the nontoxic-nonhemagglutinin (NTNH) component of the progenitor toxin from C. botulinum type C neurotoxin.     

Comparison of antigenicity of toxins produced by Clostridium botulinum type C and D strains

C1 neurotoxin of Clostridium botulinum strains C-Stockholm (C-ST), C beta-Yoichi, C-468, CD6F, and C-CB19 and type D toxin of strains D-1873 and D-CB16 were purified by gel filtration, ion exchange, and affinity chromatographies. The purified toxins had di-chain structure made of heavy and light chains. The toxins of C beta-Yoichi, C-468, CD6F, and C-CB19 reacted with anti-C-ST heavy chain and anti-C-ST light chain in immunodiffusion tests and enzyme-linked immunosorbent assay, whereas D-CB16 toxin reacted with anti-D-1873 heavy chain and anti-D-1873 light chain. However, C-6813 toxin reacted with anti-D-1873 heavy chain and anti-C-ST light chain but not with anti-C-ST heavy chain or anti-D-1873 light chain immunoglobulin G. These results indicate common antigens in the heavy chains of C-6813 and D-1873 toxins and in the light chains of C-6813 and C-ST toxins. Further, they provide evidence for heterogeneity within type C1 toxin subunits.     

Comparison of antigenicity of toxins produced by Clostridium botulinum type C and D strains.

C1 neurotoxin of Clostridium botulinum strains C-Stockholm (C-ST), C beta-Yoichi, C-468, CD6F, and C-CB19 and type D toxin of strains D-1873 and D-CB16 were purified by gel filtration, ion exchange, and affinity chromatographies. The purified toxins had di-chain structure made of heavy and light chains. The toxins of C beta-Yoichi, C-468, CD6F, and C-CB19 reacted with anti-C-ST heavy chain and anti-C-ST light chain in immunodiffusion tests and enzyme-linked immunosorbent assay, whereas D-CB16 toxin reacted with anti-D-1873 heavy chain and anti-D-1873 light chain. However, C-6813 toxin reacted with anti-D-1873 heavy chain and anti-C-ST light chain but not with anti-C-ST heavy chain or anti-D-1873 light chain immunoglobulin G. These results indicate common antigens in the heavy chains of C-6813 and D-1873 toxins and in the light chains of C-6813 and C-ST toxins. Further, they provide evidence for heterogeneity within type C1 toxin subunits.     

Monoclonal antibody to type F Clostridium botulinum toxin

Hybridomas synthesizing monoclonal antibodies (MAbs) against type F Clostridium botulinum toxin were developed. MAb from one stable hybridoma, hybridoma 223, consisted of kappa light chains and an immunoglobulin G subclass 2a heavy chain. This MAb was used in a double-sandwich enzyme-linked immunosorbent assay to detect type F toxin in foods, culture fluids, and purified toxin preparations. The sensitivity of the double-sandwich enzyme-linked immunosorbent assay was approximately 10 mouse lethal doses of toxin per ml of toxic fluid.     

Comparison of Clostridium botulinum toxins type D and C1 in molecular property, antigenicity and binding ability to rat-brain synaptosomes

Botulinum type D neurotoxin was purified 950-fold from the culture supernatant with an overall yield of 32%. The purified toxin had a specific toxicity of 5.8 X 10(7) mouse minimal lethal dose per mg of protein and a relative molecular mass of 140000. The purified toxin had a di-chain structure consisting of heavy and light chains with relative molecular masses of 85000 and 55000, respectively, linked by one disulfide bond. These subunits had different amino acid compositions and antigenicities. A similarity in molecular constructions and amino acid compositions was observed between type D and type C1 toxins as well as between their subunits. Among the seven kinds of monoclonal antibodies against type D toxin, six reacted with the heavy chain of type D toxin, while one of the six also reacted with the heavy chain of type C1 toxin and neutralized the toxicities of the two toxins. The other one of monoclonal antibodies reacted with the light chains of both toxins. This evidence indicates that both toxins have common antigenic sites on their heavy and light chains and that the antigenic site on the heavy chain may contribute to the neutralization of both toxins by antibody. The binding of type D toxin to rat brain synaptosomes was examined by use of 125I-labelled type D toxin. The binding was competitively inhibited not only by unlabelled type D and C1 toxins, but also by the heavy chains of both toxins, however, it was not inhibited by the light chain of type D toxin. These results suggest that the toxin receptors on synaptosomal membrane are common for type D and C1 toxins, and that the heavy chain contributes to the binding of toxin to synaptosomes and the structure of the binding sites on the heavy chains of both toxins is quite similar.     

Binding of Clostridium botulinum type B toxin to rat brain synaptosome

Abstract Purified toxin and its subunits from Clostridium botulinum type B were labeled with ¹²⁵iodine and binding of them to rat brain synaptosomes was studied. Labeled toxin and heavy chain were shown to bind to synaptosomes and there was no significant difference in the molar quantity of bound toxin and heavy chain at several concentrations of synaptosomes, whereas labeled light chain did not bind to synaptosomes. The binding of labeled heavy chain to synaptosomes was inhibited by unlabeled toxin and heavy chain to a similar degree as that of labeled toxin. The binding of labeled toxin and heavy chain to synaptosomes were inhibited by a monoclonal antibody which is specific for the heavy chain.     

Clostridium botulinum type C toxin

Summary The purification and crystallization of type C botulinum toxin along with its physical characteristics are described. The shape of Clostridium botulinum type C toxin molecule is globular like a pressed ball with a 7.4 nm diameter and a 4.3 urn thickness. The molecular volume is approximately 185 nl and the molecular weight is 141 000. The toxin molecule is composed of two parts, which are separable under appropriate conditions. These parts have some differences in the electrophoretic properties, amino acid distribution, immunological, and functional characteristics. The toxin molecule can be reconstituted by association of S-S bond between the two chains. The expression of the toxicity requires that the fragments of the polypeptide chain carrying the necessary information be functionally organized for the proper development of the specific tertiary structure for active conformation.     

Cloning and whole nucleotide sequence of the gene for the light chain component of botulinum type E toxin from Clostridium butyricum strain BL6340 and Clostridium botulinum type E strain Mashike.

Chromosomal DNAs were extracted from Clostridium butyricum strain BL6340 and Clostridium botulinum type E strain Mashike. The 6.0 Kbp fragment coding for the entire light chain (L) component and the N-terminus of heavy chain (H) component of botulinum type E toxin was obtained from each extracted DNAs after digestion with HindIII. The entire nucleotide sequences for the light chain components of these cloned genes were determined, and the derived amino acid sequences were compared to each other, and with those of botulinum type A, C1, D, and tetanus toxins reported previously. The cleavage site of L and H components of type E toxin was presumed to be Arg-422. In a total of 422 amino acid residues of L component, 17 residues were different between butyricum and type E toxins, and all these differences were found within 200 residues of N-terminus of L component. On the contrary, five regions showing highly homologous sequences were found in L components among these six toxins, and one more region between botulinum type E and tetanus toxins.     

Cloning of the structural gene for Clostridium botulinum type C1 toxin and whole nucleotide sequence of its light chain component

The toxigenicity of Clostridium botulinum type C1 is mediated by specific bacteriophages. DNA was extracted from one of these phages. Two DNA fragments, 3 and 7.8 kb, which produced the protein reacting with antitoxin serum were cloned by using bacteriophage lambda gt11 and Escherichia coli. Both DNA fragments were then subcloned into pUC118 plasmids and transferred into E. coli cells. The nucleotide sequences of the cloned DNA fragments were analyzed by the dideoxy chain termination method, and their gene products were analyzed by Western immunoblot. The 7.8-kb fragment coded for the entire light chain component and the N terminus of the heavy chain component of the toxin, whereas the 3-kb fragment coded for the remaining heavy chain component. The entire nucleotide sequence for the light chain component was determined, and the derived amino acid sequence was compared with that of tetanus toxin. It was found that the light chain component of C1 toxin possessed several amino acid regions, in addition to the N terminus, that were homologous to tetanus toxin.     

Cloning of the structural gene for Clostridium botulinum type C1 toxin and whole nucleotide sequence of its light chain component.

The toxigenicity of Clostridium botulinum type C1 is mediated by specific bacteriophages. DNA was extracted from one of these phages. Two DNA fragments, 3 and 7.8 kb, which produced the protein reacting with antitoxin serum were cloned by using bacteriophage lambda gt11 and Escherichia coli. Both DNA fragments were then subcloned into pUC118 plasmids and transferred into E. coli cells. The nucleotide sequences of the cloned DNA fragments were analyzed by the dideoxy chain termination method, and their gene products were analyzed by Western immunoblot. The 7.8-kb fragment coded for the entire light chain component and the N terminus of the heavy chain component of the toxin, whereas the 3-kb fragment coded for the remaining heavy chain component. The entire nucleotide sequence for the light chain component was determined, and the derived amino acid sequence was compared with that of tetanus toxin. It was found that the light chain component of C1 toxin possessed several amino acid regions, in addition to the N terminus, that were homologous to tetanus toxin.     

Isolated light chains of botulinum neurotoxins inhibit exocytosis. Studies in digitonin-permeabilized chromaffin cells

The effects of botulinum neurotoxins or their light and heavy chain subunits were investigated in digitonin-permeabilized adrenal chromaffin cells. Because these cells are permeable to proteins, the toxin had direct access to the cell interior. Botulinum type A neurotoxin and its light chain subunit inhibited Ca2+-dependent catecholamine secretion in a dose-dependent manner. The heavy chain subunit had no effect. Inhibition required introduction of the neurotoxin or light chain into the cell and was not seen when intact cells were incubated with these proteins. The inhibition of secretion by type A neurotoxin and light chain was incomplete, the maximal response being 65%. The inhibition was not overcome by increasing Ca2+ concentrations. The action of the light chain was irreversible and rapid. Botulinum type E neurotoxin also inhibited secretion in a dose-dependent manner. Its potency was increased 30-fold following mild trypsinization, which nicked the single chain protein to the dichain form. In contrast to the results seen with types A and E, botulinum type B neurotoxin did not inhibit secretion, while its light chain totally abolished secretion. Trypsinization of the neurotoxin produced the dichain form, which did not inhibit secretion. Reduction of the trypsinized neurotoxin with dithiothreitol produced inhibition equivalent to that seen with the purified light chain subunit. Isolated type A heavy chain had no effect on the inhibitory action of type A or B light chains. The data demonstrate that the ability of botulinum neurotoxins to inhibit secretion is confined to the light chain region of these proteins. Furthermore, while the botulinum neurotoxin types A, B, and E have similar macrostructures, they are not identical with respect to their biological activities.     

A型肉毒毒素轻链胞内持留模型的建立

[目的] 建立A型肉毒毒素轻链胞内持留模型来模拟A型肉毒毒素引起的长期中毒。[方法] 设计构建pcDNA3.1-ALC-GFP重组质粒,提取质粒进行PCR验证,并将其转染进Nureo-2a细胞中表达,利用Western Blot与细胞免疫荧光分析验证ALC-GFP的表达情况及其在细胞内的长时间持留,建立A型肉毒毒素轻链的胞内持留模型,并将该模型用于抗肉毒药物的筛选。[结果] 成功构建pcDNA3.1-ALC-GFP重组质粒并将其转染进Nureo-2a细胞中,验证了ALC-GFP在细胞内具有长时间持留性,成功建立了A型肉毒毒素轻链胞内持留模型,并利用该模型筛选出潜在抗肉毒药物CB3。[结论] 成功建立了A型肉毒毒素轻链胞内持留模型,并初步应用于CS1,CE2和CB3药物筛选,为A型肉毒毒素长期中毒的解毒研究奠定了基础。     

Specific detection of Clostridium botulinum type B by using the polymerase chain reaction.

The polymerase chain reaction (PCR) and a radiolabeled oligonucleotide probe were used to specifically detect proteolytic and nonproteolytic Clostridium botulinum type B. Two synthetic primers deduced from the amino acid sequence data of type B neurotoxin were used to amplify a 1.5-kbp fragment corresponding to the light chain of the toxin. Although, nonspecific priming was observed when the PCR protocol was tested with other clostridial species, only the PCR product from C. botulinum type B isolates reacted with the radiolabeled internal probe. As little as 100 fg of DNA (approximately 35 clostridial cells) could be detected after only 25 amplification cycles.     

Specific detection of Clostridium botulinum type B by using the polymerase chain reaction.

The polymerase chain reaction (PCR) and a radiolabeled oligonucleotide probe were used to specifically detect proteolytic and nonproteolytic Clostridium botulinum type B. Two synthetic primers deduced from the amino acid sequence data of type B neurotoxin were used to amplify a 1.5-kbp fragment corresponding to the light chain of the toxin. Although, nonspecific priming was observed when the PCR protocol was tested with other clostridial species, only the PCR product from C. botulinum type B isolates reacted with the radiolabeled internal probe. As little as 100 fg of DNA (approximately 35 clostridial cells) could be detected after only 25 amplification cycles.     

A 50-kDa fragment from the NH2-terminus of the heavy subunit of Clostridium botulinum type A neurotoxin forms channels in lipid vesicles

1. A 50-kDa fragment representing the NH2-terminus of the heavy subunit of botulinum type A neurotoxin was found, at low pH, to evoke the release of K+ from lipid vesicles loaded with potassium phosphate. Similar K+ release was also observed with the intact neurotoxin, its heavy chain and a fragment consisting of the light subunit linked the 50-kDa NH2-terminal heavy chain fragment. The light subunit alone, however, was inactive. 2. In addition to K+, the channels formed in lipid bilayers by botulinum neurotoxin type A or the NH2-terminal heavy chain fragment were found to be large enough to permit the release of NAD (Mr 665). 3. The optimum pH for the release of K+ was found to be 4.5. Above this value K+ release rapidly decreased and was undetectable above pH 6.0. 4. The binding of radiolabelled botulinum toxin to a variety of phospholipids was assessed. High levels of toxin binding were only observed to lipid vesicles with an overall negative charge; much weaker binding occurred to lipid vesicles composed of electrically neutral phospholipids. 5. A positive correlation between the efficiency of toxin-binding and the efficiency of K+ release from lipid vesicles was not observed. Whereas lipid vesicles containing the lipids cardiolipin or dicetyl phosphate bound the highest levels of neurotoxin, the toxin-evoked release of K+ was low compared to vesicles containing either phosphatidyl glycerol, phosphatidyl serine or phosphatidyl inositol. 6. The implications of these observations to the mechanism by which the toxin molecule is translocated into the nerve ending are discussed.     

Structure of tetanus toxin. I. Breakdown of the toxin molecule and discrimination between polypeptide fragments.

Tetanus toxin was digested with papain, yielding one major polypeptide (Fragment C) with a molecular weight corresponding to 47,000 +/- 5%, thus comprising about one-third of the toxin molecule. Fragment C was antigenically active, atoxic, and stimulated the formation of antibodies neutralizing the lethal action of tetanus toxin in vivo. Furthermore, a second split product (Fragment B) was isolated from the papain digest, containing two polypeptide chains linked together via a disulfide bond. Fragment B (Mr = 95,000 +/- 5%) was atoxic and showed a reaction of nonidentity with Fragment C on immunodiffusion analysis against tetanus antitoxin. The basic two-chain structure (heavy and light chain polypeptide, cf. Matsuda, M., and Yoneda, M. (1975) Infect. Immun. 12, 1147-1153) of tetanus toxin has been confirmed and the relationship between Fragments B and C within this framework has been established. Fragment C was distinguished from the light chain by electrophoresis in sodium dodecyl sulfate and by immunodiffusion analysis, indicating that this fragment constitutes a portion of the heavy chain polypeptide. Fragment B showed a reaction of partial identity with the light as well as the heavy chain from tetanus toxin. Reduction of Fragment B with dithiothreitol followed by gel chromatography yielded a fraction which was indistinguishable from the light chain portion of the toxin molecule. It is concluded that Fragment B comprises the complementary portion of the heavy chain (remaining after scission of the polypeptide bond(s) releasing Fragment C) linked to the light chain by a disulfide bond.     

Chains and fragments of tetanus toxin, and their contribution to toxicity

1. Single-chain toxin is enzymatically converted into two-chain isotoxins which differ from the precursor by their higher pharmacological activity, acidity and hydrophilicity. The interchain disulfide bridge and the disulfide loop within fragment C have been located at the amino acid level. 2. Independent of the enzymes used, the nicking sites are positioned within a region spanning no more than 17 amino acids. The N- and C-termini of the primary gene product are preserved in the two-chain toxin. The chains have been separated by isoelectric focussing and can be reconstituted to functionally intact toxin. 3. Light chain inhibits neurotransmitter release on different systems. First, permeabilized bovine adrenal chromaffin cells and rat pheochromocytoma (PC 12) cells release catecholamines when exposed to micromolar [Ca2+]. Inhibition is achieved with light chain or reduced two-chain toxin, but not with single-chain toxin or heavy chain. Washing away the light chain does not restitute the Ca2(+)-evoked release. The light chains of tetanus and botulinum A toxin act in a apparently similar, however not identical manner. Second, light but not heavy chain inhibits the release of acetylcholine when injected into Aplysia neurones. 4. The pharmacology of heavy chain is quite different. Ganglioside binding is mediated by its fragment C moiety, and modulated by the adjoining beta 2 piece and by light chain. Heavy chain and to a lesser degree its N-terminal beta 2-fragment promote the loss of calcein from liposomes indicating pore formation. Its C-terminal fragment C is inactive in this respect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunochemical identification of the ADP-ribosyltransferase in botulinum C1 neurotoxin as C3 exoenzyme-like molecule

Botulinum C1 neurotoxin and C3 exoenzyme were purified to apparent homogeneity from the culture filtrate of Clostridium botulinum type C strain 003-9. Both preparations catalyzed ADP-ribosylation of the same substrate, the Mr 22,000 rho gene product (Gb). When the light and heavy chains of C1 toxin were separated, ADP-ribosyltransferase activity in the toxin was quantitatively recovered in the light chain fraction. Anti-C1 toxin antiserum precipitated the ADP-ribosyltransferase activity and the neurotoxicity of C1 toxin in parallel, whereas it had no effect on C3 exoenzyme. On the other hand, anti-C3 exoenzyme antiserum precipitated the ADP-ribosyltransferase activities of both C3 exoenzyme and C1 toxin. This antibody, however, did not precipitate the neurotoxicity of C1 toxin. The ADP-ribosyltransferase in C1 toxin was quantitatively adsorbed onto the anti-C3 antibody column and separated from the majority of C1 toxin protein. The enzyme was then eluted with acidic urea and Western blotting analysis of this eluate revealed the appearance of a protein band positively stained with anti-C3 antibody at a position similar to that of C3 exoenzyme. Quantitative determination by enzyme-linked immunosorbent assay showed that the C3-like immunoreactivity is present in the C1 toxin molecules at the molecular ratio of 1 to 1,000. These results suggest that the ADP-ribosyltransferase activity in C1 toxin is expressed by a C3-like molecule which is present in a small amount in the toxin preparation and appears to bind to the toxin component(s). The above results also indicate that the ADP-ribosyltransferase in C1 toxin is not related to its neurotoxin action.     

Cloning of a Clostridium botulinum type B toxin gene fragment encoding the N-terminus of the heavy chain

Two lambda gt11 clones of the toxin gene of Clostridium botulinum type B were identified by the monoclonal antibody specific to the heavy chain of type B toxin. Neither of the expressed fusion proteins from the lysates of lysogenic E. coli Y1089 showed any botulinal toxic activity. One of the clones hybridized to the oligonucleotide probe which was synthesized according to the amino acid sequence of N-terminus of heavy chain. The sequence analysis revealed that highly homologous regions in N-terminus of heavy chain exist among botulinum neurotoxins (type A, B) and tetanus toxin on the amino acid sequence level.