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.     

Isolated Light Chain of Tetanus Toxin Inhibits Exocytosis: Studies in Digitonin-Permeabilized Cells

Previous work indicates that the heavy chain of tetanus toxin is responsible for the binding of the toxin to the neuronal membrane and its subsequent internalization. In the present study, the light chain of tetanus toxin mimicked the holotoxin in inhibiting Ca2+-dependent secretion of [3H]norepinephrine from digitonin-permeabilized adrenal chromaffin cells. Preincubation of tetanus toxin with monoclonal antibodies to the light chain prevented the inhibition by tetanus toxin. Preincubation of tetanus toxin with nonimmune ascites fluid or with monoclonal antibodies directed against the C fragment (the C-terminal of the heavy chain) or the heavy-chain portion of the B fragment did not prevent inhibition by tetanus toxin. The data indicate that the light chain is responsible for the intracellular blockade of exocytosis.     

Identification of a binding site for ganglioside on the receptor binding domain of tetanus toxin

The carboxyl-terminal region of the tetanus toxin heavy chain (H(C) fragment) binds to di- and trisialylgangliosides on neuronal cell membranes. To determine which amino acids in tetanus toxin are involved in ganglioside binding, homology modeling was performed using recently resolved X-ray crystallographic structures of the tetanus toxin H(C) fragment. On the basis of these analyses, two regions in tetanus toxin that are structurally homologous with the binding domains of other sialic acid and galactose-binding proteins were targeted for mutagenesis. Specific amino acids within these regions were altered using site-directed mutagenesis. The amino acid residue tryptophan 1288 was found to be critical for binding of the H(C) fragment to ganglioside GT1b. Docking of GD1b within this region of the toxin suggested that histidine 1270 and aspartate 1221 were within hydrogen bonding distance of the ganglioside. These two residues were mutagenized and found also to be important for the binding of the tetanus toxin H(C) fragment to ganglioside GT1b. In addition, the H(C) fragments mutagenized at these residues have reduced levels of binding to neurites of differentiated PC-12 cells. These studies indicate that the amino acids tryptophan 1288, histidine 1270, and aspartate 1221 are components of the GT1b binding site on the tetanus toxin H(C) fragment.     

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.     

On the role of polysialoglycosphingolipids as tetanus toxin receptors. A study with lipid monolayers

Lipid monolayers of different compositions were used to study the interaction of tetanus toxin with membrane lipids and to evaluate the role of polysialoglycosphingolipids as membrane receptors. At neutral pH, the toxin binds to dioleoylglycerophosphocholine monolayers and inserts into the phospholipid layer. This effect is potentiated by acidic phospholipids without an apparent preference for a single class of phospholipids. Polysialoglycosphingolipids further increase the fixation and penetration of tetanus toxin in lipid monolayers, but no specific requirement for a particular ganglioside was identified. The ganglioside effect is abolished in the presence of other nervous tissue lipids: cerebrosides and glycosphingolipid sulfates are partially responsible for this effect. The penetration of tetanus toxin in the lipid monolayer is pH dependent. It increases with lowering pH, it is facilitated by acidic phospholipids and by glycosphingolipid sulfates and it is mediated both by hydrophobic and electrostatic interactions as deduced from an analysis of the effect of ionic strength. Fragment B of tetanus toxin the low-pH-driven lipid interaction of the toxin. On the basis of the present findings, the possible role of polysialoglycosphingolipids in the neurospecific binding of tetanus toxin is discussed.     

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.     

Tetanus toxin: primary structure, expression in E. coli, and homology with botulinum toxins.

A pool of synthetic oligonucleotides was used to identify the gene encoding tetanus toxin on a 75-kbp plasmid from a toxigenic non-sporulating strain of Clostridium tetani. The nucleotide sequence contained a single open reading frame coding for 1315 amino acids corresponding to a polypeptide with a mol. wt of 150,700. In the mature toxin molecule, proline (2) and serine (458) formed the N termini of the 52,288 mol. wt light chain and the 98,300 mol. wt heavy chain, respectively. Cysteine (467) was involved in the disulfide linkage between the two subchains. The amino acid sequences of the tetanus toxin revealed striking homologies with the partial amino acid sequences of botulinum toxins A, B, and E, indicating that the neurotoxins from C. tetani and C. botulinum are derived from a common ancestral gene. Overlapping peptides together covering the entire tetanus toxin molecule were synthesized in Escherichia coli and identified by monoclonal antibodies. The promoter of the toxin gene was localized in a region extending 322 bp upstream from the ATG codon and was shown to be functional in E. coli.     

The structures of the H(C) fragment of tetanus toxin with carbohydrate subunit complexes provide insight into ganglioside binding

The entry of tetanus neurotoxin into neuronal cells proceeds through the initial binding of the toxin to gangliosides on the cell surface. The carboxyl-terminal fragment of the heavy chain of tetanus neurotoxin contains the ganglioside-binding site, which has not yet been fully characterized. The crystal structures of native H(C) and of H(C) soaked with carbohydrates reveal a number of binding sites and provide insight into the possible mode of ganglioside binding.     

Neuronal binding of tetanus toxin compared to its ganglioside binding fragment (H(c))

The non-toxin 50 kD C-terminus peptide of the heavy chain of tetanus H(c) contains the ganglioside binding domain of tetanus toxin (TTX). H(c) retains much of the capacity of tetanus toxin for binding internalization and transport by neurons. For this reason tetanus H(c) has been studied as a vector for delivery of therapeutic proteins to neurons. We directly compared H(c) and TTX in the capacity to bind and be internalized by neurons by ELISA. Primary cultures of dissociated fetal cortical neurons were incubated with equimolar amounts of TTX or H(c). Neuronal associated tetanus protein was 4-8 fold greater on a molar basis with tetanus toxin compared to H(c) (1 h incubation). This increase in neuronal tetanus protein was evident with incubation in concentrations from 0.1 microM to 2 microM. There were greater amounts of TTX delivered to the cultured cells at both 0 degrees C (representing membrane bound tetanus protein) and 37 degrees C (bound and internalized tetanus protein). Unlike H(c), TTX showed significant continued accumulation of protein with increasing incubation durations. Neuronal associated TTX increased 2-3 fold over incubation times ranging from 1 to 8 h. Tetanus toxin appears to be clearly superior to the ganglioside binding fragment (H(c)) in the capacity for neuronal binding and internalization. Atoxic tetanus proteins containing additional molecular domains as well as H(c) may be more suitable vectors for linkage with therapeutic proteins and delivery to neurons.     

Myosin degradation fragments in skeletal muscle

Myosin heavy chain degradation fragments produced in vivo have been identified in chicken pectoralis muscle. The fragments were identified by electrophoresis of unfractionated extracts of chicken pectoralis muscle on sodium dodecyl sulfate/polyacrylamide gels followed by immunoblotting on nitrocellulose sheets. Monoclonal antibodies directed against the S2 and light meromyosin subfragments as well as type II myosin-specific polyclonal antibodies directed against the entire myosin heavy chain were used to characterize the fragments, which range in molecular weight from approximately 80,000 to 180,000. All fragments contain the extreme carboxy-terminal portion of the molecule and are distinct from the classical proteolytic fragments such as heavy and light meromyosin, S1, S2 or rod. These fragments appear to be produced in vivo by proteolytic cleavage of peptides from the amino-terminal (S1) end of the heavy chain while the myosin molecule is still embedded in the thick filament. Fragment concentrations are estimated to be approximately 5 to 10% of that of the intact myosin heavy chain. These fragments are not the result of artifactual damage to myosin, e.g. proteolysis or hydrodynamic shear. The techniques described in this paper provide a probe into the early stages of myosin and thick filament degradation in vivo.     

Interaction of diphtheria toxin fragments A, B and protein crm 45 with liposomes

Diphtheria toxin, its fragments A, B and the protein serologically related to toxin, crm 45, have been studied for their hydrophobicity using the method of charge shift electrophoresis. These molecules were then assayed for liposome interaction. The results have shown that the diphtheria toxin B fragment behaves as an amphiphatic protein because it contains a hydrophobic domain located in that portion of the B chain which remains in protein crm 45. Toxin fragment A is hydrophilic. Incubation of protein crm 45 or toxin fragment B with preformed liposomes leads to association of these proteins with lipid vesicles. Fragment A does not interact with liposomes. Binding of protein crm 45 with lipid vesicles is dependent on time and temperature. Protein crm 45 is unidirectionally associated with liposomes, its enzymic fragment A directed outside the liposome. Fragment B or protein crm 45, upon binding with liposomes, does not affect the permeability of the vesicles.     

Characterization of tetanus toxins and toxin components by amino terminal analyses

Extract tetanus toxin, filtrate tetanus toxin, and the heavy and light chains of filtrate toxin were analyzed for their amino termini with 4-N,N-dimethylaminoazobenzene-4′isothiocyanate and phenylisothiocyanate. Extract toxin (intracellular toxin) is a single-chain polypeptide with proline as the amino terminus. Filtrate toxin (extracellular toxin) is a mixture of species produced by endogenous proteases, and showed three major amino terminal residues, proline, asparagine, and serine. Cleavage points in the filtrate toxin molecule appear to be on either side of a disulfide bond. Reductive and nonreductive preparative electrophoresis of filtrate toxin produce different species of light and heavy chains. The light chains have a single amino terminus of proline, indicating that the light chain is the amino terminal portion of the toxin molecule. The heavy chains showed no proline but rather asparagine and serine as the major amino termini. Small amounts of other amino terminal residues were present, indicating microheterogenity at the cleavage sites in the toxin. The results permit the construction of a model of tetanus toxin which is consistent with the fragments obtained from either reductive or nonreductive preparative electrophoresis of filtrate toxin.     

The complete amino acid sequence of the Clostridium botulinum type A neurotoxin, deduced by nucleotide sequence analysis of the encoding gene

A 26-mer oligonucleotide probe was synthesized (based on the determined amino acid sequence of the N-terminus of the Clostridium botulinum type A neurotoxin, BoNT/A) and used in Southern blot analysis to construct a restriction map of the region of the clostridial genome encompassing BoNT/A. The detailed information obtained enabled the cloning of the structural gene as three distinct fragments, none of which were capable of directing the expression of a toxic molecule. The central portion was cloned as a 2-kb PvuII-TaqI fragment and the remaining regions of the light chain and heavy chain as a 2.4-kb ScaI-TaqI fragment and a 3.4-kb HpaI-PvuII fragment, respectively. The nucleotide sequence of all three fragments was determined and an open reading frame identified, composed of 1296 codons corresponding to a polypeptide of 149 502 Da. The deduced amino acid sequence exhibited 33% similarity to tetanus toxin, with the most highly conserved regions occurring between the N-termini of the respective heavy chains. Conservation of Cys residues flanking the position at which the toxins are cleaved to yield the heavy chain and light chain allowed the tentative identification of those residues which probably form the disulphide bridges linking the two toxin subfragments.     

Production of neutralizing human monoclonal antibody directed to tetanus toxin in CHO cell.

By the fusion of lymphocytes from hyperimmunized people with heteromyeloma cells, 600 human hybridoma cell lines were generated. Even though seven cell lines produced antibodies against tetanus toxoid, only two antibodies from hybrid CH8 and CH5 only neutralized the tetanus toxin and completely protected the mice that had been challenged with the toxin even at the level of 90 mean lethal dose. The cDNA of light (L) chain and heavy (H) chain variable region was isolated, and then inserted into expression vectors containing human IgG constant regions. After transfection of the recombinant human IgG gene into Chinese Hamster Ovary (CHO) cells, transformants secreting the complete human antibody were selected. The recombinant human antibodies produced from CHO cells possessed neutralizing activity against tetanus toxin just like the original human antibodies produced from human hybridoma cell lines. Western blot analysis showed that rCH8 and rCH5 antibodies recognized the H chain of tetanus toxin and did not bind to its L chain. The neutralizing test showed that HmAb rCH5 had 4.55IU and HmAb rCH8 had 1.09IU/100 micro g of IgG, respectively. Mixing of the two HmAbs resulted in synergistic effects. On a weight basis (IU/100 micro g IgG), the highest potency values were obtained when the two HmAbs were combined in equal quantity. The neutralizing activity of rCH8 and rCH5 mixture was 6.94IU/100 micro g IgG.     

Micellar gangliosides mediate the lipid insertion of cholera toxin protomer A

The topology of the interaction of cholera toxin with ganglioside and detergent micelles was studied with the technique of hydrophobic photolabelling. Cholera toxin α and γ polypeptide chains appear to penetrate into the hydrophobic core of ganglioside micelles. Micelles of SDS cause the labelling also of the β polypeptide chains, while Triton X-100 micelles have little ability to mediate the labelling of the toxin. The specific reduction of the α-γ disulfide bond allows the penetration of the α polypeptide chain into Triton X-100 micelles, but does not affect the interaction of cholera toxin with either ganglioside or SDS micelles. Thus, ganglioside micelles appear to cause a conformational change of the native toxin, such as to induce the penetration of the α chain into the micelle hydrophobic core.     

Preparation and properties of monoclonal antibodies against lipopolysaccharide-sensitive serine protease zymogen, factor C, from horseshoe crab (Tachypleus tridentatus) hemocytes.

Seventeen murine monoclonal antibodies (mAbs) against horseshoe crab clotting factor, factor C, were prepared and characterized. When the binding sites of these mAbs were analyzed by immunoblotting, ten mAbs recognized nonreduced factor C, five mAbs were directed against the heavy chain, and two mAbs were directed against the B chain. Three mAbs, 1H4, 2C12, and 2A7, one selected from each group, were used for further study. The mAb 1H4, which recognized only nonreduced factor C molecule, inhibited the factor C activity in a dose-dependent manner. It also inhibited lipopolysaccharide (LPS)- and alpha-chymotrypsin-mediated activations of the zymogen factor C, suggesting that 1H4 binds close to the active site and/or the substrate-binding site located in the serine protease domain (B chain) of factor C. On the other hand, 2C12 and 2A7 recognized, respectively, an epitope located in the heavy and the B chains, and inhibited LPS-mediated activation of factor C, but not alpha-chymotrypsin-mediated activation of factor C or factor C activity. Both F(ab')2 and Fab' fragments derived from 2C12 inhibited LPS-mediated activation in the same manner. These three mAbs did not bind with LPS, although a factor C-mAb complex was able to bind LPS, suggesting that the LPS-mediated activation of the zymogen factor C was induced through intermolecular interaction between the LPS-bound factor C molecules. The dissociation constants (Kd) for 1H4, 2C12, and 2A7 binding to factor C were determined as 1.9 x 10(-9), 0.6 x 10(-10), and 1.8 x 10(-10) M, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation and structure of the C5b-7 complex of the lytic pathway of complement.

The formation and structure of the complement cytolytic intermediary complex, C5b-7, were studied with the aim of determining the interactive regions of C5, C6, and C7. The structure of human complement component C5 was elucidated by the application of limited proteolysis which generated well characterized major polypeptide fragments of this molecule. Plasmin, thrombin, and kallikrein cleave C5b with greater facility than C5. The most useful cleavage of C5b was effected by plasmin because the fragmentation pattern was similar to the processing of C3b by factors H, I, and kallikrein. Plasmin hydrolyzes peptide bonds within the alpha'-chain of C5b, resulting in a four-chain fragment, C5c (M(r) = 142,000), and a single chain fragment, C5d (M(r) = 43,000). Circular dichroism spectroscopic analyses indicated that C5d is substantially richer in alpha-helical content than is C5c (27 versus 9%). Polyclonal antibodies directed against C5c blocked the interaction of C5b-6 with C7, whereas antibodies directed against C5d inhibited the binding of C5 with C3b. Chemical cross-linking using a cleavable radioiodinated photoreactive reagent revealed that both C6 and C7 associate preferentially with the alpha'-chain of C5b. The reversible interactions of C5 with C6, C7, and major polypeptide fragments derived from these were investigated with solid phase binding assays. The results indicate that the carboxyl-terminal domains of C6 and C7, which have cysteine-rich modules homologous to those found in factors H and I, have the capacity to link specifically with C5.     

Structure-based sequence alignment for the beta-trefoil subdomain of the clostridial neurotoxin family provides residue level information about the putative ganglioside binding site

Clostridial neurotoxins embrace a family of extremely potent toxins comprised of tetanus toxin (TeNT) and seven different serotypes of botulinum toxin (BoNT/A-G). The beta-trefoil subdomain of the C-terminal part of the heavy chain (H(C)), responsible for ganglioside binding, is the most divergent region in clostridial neurotoxins with sequence identity as low as 15%. We re-examined the alignment between family sequences within this subdomain, since in this region all alignments published to date show obvious inconsistencies with the beta-trefoil fold. The final alignment was obtained by considering the general constraints imposed by this fold, and homology modeling studies based on the TeNT structure. Recently solved structures of BoNT/A confirm the validity of this structure-based approach. Taking into account biochemical data and crystal structures of TeNT and BoNT/A, we also re-examined the location of the putative ganglioside binding site and, using the new alignment, characterized this site in other BoNT serotypes.     

Dissociation of tetanus neurotoxin into two polypeptide fragments

Analyses of neurotoxin protein of $\text{Clostridium tetani}$ by polyacrylamide gel electrophoresis showed that the toxin as purified from culture filtrates (“extracellular” toxin, molecular weight 160,000) could be dissociated into two polypeptide chains of molecular weight 53,000 (Fragment α) and 107,000 (Fragment β) by treatment with dithiothreitol and sodium dodecyl sulfate. The toxin as purified from bacterial extracts (“intracellular” toxin) was found to consist of a single 160,000 dalton polypeptide chain, which is undissociable by such treatment but, when pretreated with trypsin, becomes dissociable into two fragments apparently identical with α and β.     

GANGLIOSIDES IN NERVOUS TISSUE CULTURES AND BINDING OF 125I-LABELLED TETANUS TOXIN, A NEURONAL MARKER

Abstract— —Continuous cell lines, primary cell cultures derived from embryonic CNS, and homogenates made from adult and embryonic CNS were compared with respect to their lipid pattern and their ability to bind ¹²⁵I-labelled tetanus toxin. In parallel experiments de novo synthesis of gangliosides in the cell lines was studied, using [¹⁴C]glucosamine as precursor. Of the total lipid only gangliosides were specifically labelled by [¹⁴C]glucosamine. The patterns of the de novo synthesized gangliosides corresponded to those present in the respective cells.
Pronounced binding of ¹²⁵I-labelled toxin was only detectable in tissues containing long-chain gangliosides (ganglioside C which represents GDIb and GTI).
Accordingly, hybrid (neuroblastoma x glioma) cells, due to their lack of long-chain gangliosides, bound just-discernible amounts of labelled toxin. When previously exposed to gangliosides, their binding of tetanus toxin tremendously increased.
It was concluded that only the long-chain gangliosides in the neuronal cells are functionally involved in the binding of the tetanus toxin and that these acceptors of tetanus toxin can be transplanted.