Two Antigenically Active, Complementary Polypeptide Fragments of Tetanus Neurotoxin

Matsuda, Morihiro; Yoneda, Masahiko

doi:10.1007/978-1-4684-0886-7_35

Cited by 16 publications

(17 citation statements)

References 13 publications

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“…Its molecular weight is about 150 000. Upon reduction, a heavy (MW about 95 000) and a light chain (MW about 52 000) can be separated by disk gel electrophoresis or, under slightly denaturing conditions, by gel chromatography (Matsuda and Yoneda 1975) and recombined to the active toxin (Matsuda and Yoneda 1976). Subfragments of the light chain have not been described so far, whereas hydrolysis of the heavy chain occurs upon treatment of the native toxin with papain.…”

Section: Introductionmentioning

confidence: 97%

Enzymatic hydrolysis of tetanus toxin by intrinsic and extrinsic proteases. Characterization of the fragments by monoclonal antibodies

Goretzki

Habermann

1985

Med Microbiol Immunol

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Enzymatic fragments of tetanus toxin were characterized by immunoblotting using a set of previously characterized antibodies (Ahnert-Hilger et al. (1983) and a set of novel antibodies. The selected antibodies recognized the light chain, fragment C (beta 1), and the complementary piece (beta 2) of the heavy chain when blotted on nitrocellulose. All toxin preparations contained intrinsic esteroprotease activity which became manifest in the presence of urea. The main split product was a fragment (MW 100 000) reacting with anti-fragment C and anti-beta 2 antibodies. Toxicity does not depend on this protease activity. Some crude preparations of tetanus toxin contain another split product (MW 47 000) which resembles fragment C. The main product of papain hydrolysis is fragment C, which appears as a double band under nonreducing conditions but is homogeneous when reduced. Chymotryptic digestion hydrolyses the heavy chain well but leaves the light chain largely intact. Tetanus toxin is very resistant against trypsin as compared with other proteases, although this enzyme splits numerous different links. Our data show the usefulness of immunoblotting with monoclonal antibodies in analytical work with tetanus toxin, and the relevance of intrinsic proteases.

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Section: Introductionmentioning

confidence: 97%

Enzymatic hydrolysis of tetanus toxin by intrinsic and extrinsic proteases. Characterization of the fragments by monoclonal antibodies

Goretzki

Habermann

1985

Med Microbiol Immunol

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“…Upon lysis of the bacterium, the toxin is released from the cells, concomitantly with proteolytic cleavage of the molecule by an endogenous protease. The resulting molecule, termed "extracellular toxin," is composed of two fragments designated the light and heavy chains (13). These chains are held together by one or more disulfide bonds.…”

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confidence: 99%

Cloning, nucleotide sequencing, and expression of tetanus toxin fragment C in Escherichia coli

Fairweather¹,

Lyness²,

Pickard³

et al. 1986

J Bacteriol

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The amino acid sequence of the first 30 residues of fragment C of tetanus toxin was determined, and a mixture of 32 complementary oligonucleotides, each 17 bases long, was synthesized. A 2-kilobase (kb) EcoI fragment of Clostridium tetani DNA was identified by Southern blotting and was cloned into the Escherichia coli plasmid vector pAT153 with the 32P-labeled oligonucleotide mixture as a probe. A second 3.2-kb BglII fragment was identified and cloned with the 2-kb EcoRI fragment as a probe. The nucleotide sequence of 1.8 kb of this DNA was determined and was shown to encode the entire fragment C and a portion of fragment B of tetanus toxin. The tetanus DNA was expressed in E. coli with pWRL507, a plasmid vector containing the trp promoter and a portion of the trpE gene. The trpE-tetanus fusion proteins were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were shown to react with anti-fragment C antibody.Tetanus toxin is a potent inhibitor of the central nervous system. It causes spastic paralysis by blocking the release of inhibitory transmitters from inhibitory synapses (for a review, see reference 1). The exact mode of action of tetanus toxin at the molecular level remains unknown. The toxin, produced by Clostridium tetani, is synthesized as a 150,000-dalton polypeptide (Fig. 1). Upon lysis of the bacterium, the toxin is released from the cells, concomitantly with proteolytic cleavage of the molecule by an endogenous protease. The resulting molecule, termed "extracellular toxin," is composed of two fragments designated the light and heavy chains (13). These chains are held together by one or more disulfide bonds. Purified heavy and light chains are by themselves virtually nontoxic, but when they are reassociated, toxicity is restored.Papain digesion of tetanus toxin results in cleavage of the heavy chain to give two fragments, B and C (8) (Fig. 1). Purified fragment C is completely nontoxic in animals, whereas fragment B retains some residual toxicity at high doses, although this activity is manifest as a flaccid paralysis in mice rather than the spastic paralysis characteristic of tetanus toxin (7,8).Immunity to tetanus toxin is provided by the administration of formaldehyde-treated toxin (tetanus toxoid). Fragments B and C have also been used to successfully immunize animals against tetanus, indicating that the entire molecule is not essential for protection (6). However, the preparation of these fragments is time-consuming and not commercially feasible. Studies with monoclonal antibodies have also shown that all three domains of the tetanus toxin molecule (the light chain, the amino-terminal half of the heavy chain, and the C fragment) may induce neutralizing antibodies (10, 25).To characterize tetanus toxin further at the molecular level, we undertook the cloning, nucleotide sequencing, and expression in Escherichia coli of DNA encoding fragment C of tetanus toxin. We used synthetic oligonucleotides com-* Corresponding author.plementary to the amino acid sequence of fragment C ...

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“…Tetanus toxin was prepared and purified from culture filtrates (21) and fragments [A-B] and [C] were separated and purified from mildly papain-treated toxin as described previously (22,23 (16). The toxin fragments on the gel were then transferred to a nitrocellulose membrane and treated with individual MAbs as described in the legend for figure 1.…”

Section: Tetanus Toxin and Fragments Of Tetanus Toxin -mentioning

confidence: 99%

Characteristics of toxin-neutralization by anti-tetanus human monoclonal antibodies directed against the three functional domains [A], [B], and [C] of the tetanus toxin molecule and a reliable method for evaluating the protective effects of monoclonal antibodies

et al. 1992

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Five anti-tetanus human monoclonal antibodies (MAbs) produced by hybrid cell lines we established previously were characterized. Their abilities to neutralize tetanus toxin in vitro and to protect mice against challenge with toxin were studied by observing the changes in the progress of symptoms in mice. Immunostaining showed that MAbs MAb-G4 and G2 recognized the N-terminal domain, [A] and the C-terminal domain, [C] of the tetanus toxin molecule, respectively, while MAbs MAb-G1, G3 and G6 recognized its middle domain, [B]. Enzyme-linked immunosorbent assay showed that the binding affinity of MAb-G3 was 2.9 x 10(10) M-1 and those of the other MAbs were as high as approximately 10(11) M-1. In in vitro neutralization experiments, at sufficient doses all the MAbs as single reagents protected mice completely against the effect of tetanus toxin. However, at lower doses than those sufficient to rescue mice, the kinetic patterns of progress of symptoms with the individual MAbs differed with each other and, except for MAb-G4, were different from that of anti-tetanus human polyclonal antibody. They suppressed the development and/or slowed the rate of progress of symptoms for over 96 h and delayed death of the mice. We propose that the comparison of the minimum survival dose with that of human polyclonal antibody of known international units is a reliable method for estimating the actual protective activity of a MAb. Intravenous (IV) injection of doses of individual MAbs or their mixtures at over 0.03 IU per mouse protected mice from subsequent challenge with 20 MLD of tetanus toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

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Two Antigenically Active, Complementary Polypeptide Fragments of Tetanus Neurotoxin

Cited by 16 publications

References 13 publications

Enzymatic hydrolysis of tetanus toxin by intrinsic and extrinsic proteases. Characterization of the fragments by monoclonal antibodies

Enzymatic hydrolysis of tetanus toxin by intrinsic and extrinsic proteases. Characterization of the fragments by monoclonal antibodies

Cloning, nucleotide sequencing, and expression of tetanus toxin fragment C in Escherichia coli

Characteristics of toxin-neutralization by anti-tetanus human monoclonal antibodies directed against the three functional domains [A], [B], and [C] of the tetanus toxin molecule and a reliable method for evaluating the protective effects of monoclonal antibodies

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