The role of the single interchains disulfide bond in tetanus and botulinum neurotoxins and the development of antitetanus and antibotulism drugs (original) (raw)
Related papers
FEBS Letters, 2012
Tetanus and botulinum neurotoxins cause paralysis by cleaving SNARE proteins within the cytosol of nerve terminals. They are endocytosed inside acidic vesicles and the pH gradient across the membrane drives the translocation of their metalloprotease L domain in the cytosol. This domain is linked to the rest of the molecule by a single interchain disulfide bridge that has to be reduced on the cytosolic side of the membrane to free its enzymatic activity. By using specific inhibitors of the various cytosolic protein disulfides reducing systems, we show here that the NADPH-thioredoxin reductase-thioredoxin redox system is the main responsible for this disulfide reduction. In addition, we indicate auranofin, as a possible basis for the design of novel inhibitors of these neurotoxins.
Journal of Pharmacology and Experimental Therapeutics, 2003
All serotypes of botulinum toxin possess a disulfide bond that links the heavy chain and light chain components of the holotoxin. Experiments were done to assess the functional significance of this covalent bond, and the work was facilitated by use of mercurial compounds that modify residues in the vicinity of the catalytic site. The data indicated that reduction of the interchain disulfide bond had two major effects: 1.) changing conformation or orientation of the two chains, which diminished toxicity against intact cells, and 2.) loosening or relocating a heavy chain belt segment that encircles the light chain and occludes the catalytic site. Interestingly, disulfide bond reduction of all serotypes produced conformational changes that diminished toxicity against intact cells, but it produced conformational changes that led to exposure of the catalytic site in only three serotypes. For the other serotypes, the catalytic site was accessible even before disulfide bond reduction. Neither of the major structural effects was dependent upon separation of the heavy chain and light chain components of the toxin, nor were they dependent on toxin substrate. Depending on the initial state of the toxin molecule, the combination of disulfide bond reduction and treatment with a mercurial compound could abolish toxicity. Therefore, this combination of treatments was used to convert active toxin into a parenteral vaccine. Administration of the modified toxin evoked a substantial IgG response, and it produced complete protection against a large dose of native toxin.
Toxicon : official journal of the International Society on Toxinology, 2015
Botulinum neurotoxins (BoNTs) are Janus toxins, as they are at the same time the most deadly substances known and one of the safest drugs used in human therapy. They specifically block neurotransmission at peripheral nerves through the proteolysis of SNARE proteins, i.e. the essential proteins, which are the core of the neuroexocytosis machinery. Even if BoNTs are traditionally known as seven main serotypes, their actual number is much higher as each serotype exists in many different subtypes, with individual biological properties and little antigenic relations. Since BoNTs can be used as biological weapons, and the only currently available therapy is based on immunological approaches, the existence of so many different subtypes is a major safety problem. Nevertheless, all BoNT isoforms are structurally similar and intoxicate peripheral nerve endings via a conserved mechanism. They consist of two chains linked by a unique disulphide bond which must be reduced to enable their toxicit...
Cellular Microbiology, 2011
Tetanus and botulinum neurotoxins are di-chain proteins that cause paralysis by inhibiting neuroexocytosis. These neurotoxins enter into nerve terminals via endocytosis inside synaptic vesicles, whose acidic pH induces a structural change of the neurotoxin molecule that becomes capable of translocating its L chain into the cytosol, via a transmembrane protein-conducting channel made by the H chain. This is the least understood step of the intoxication process primarily because it takes place inside vesicles within the cytosol. In the present study, we describe how this passage was made accessible to investigation by making it to occur at the surface of neurons. The neurotoxin, bound to the plasma membrane in the cold, was exposed to a warm low pH extracellular medium and the entry of the L chain was monitored by measuring its specific metalloprotease activity with a ratiometric method. We found that the neurotoxin has to be bound to the membrane via at least two anchorage sites in order for a productive low-pH induced structural change to take place. In addition, this process can only occur if the single inter-chain disulfide bond is intact. The pH dependence of the conformational change of tetanus neurotoxin and botulinum neurotoxin B, C and D is similar and take places in the same slightly acidic range, which comprises that present inside synaptic vesicles. Based on these and previous findings, we propose a stepwise sequence of molecular events that lead from toxin binding to membrane insertion.
Bioorganic & Medicinal Chemistry, 2003
Botulinum neurotoxin type B causes the inhibition of acetylcholine release at the neuromuscular junction resulting in a flaccid paralysis designated botulism. This occurs through the cleavage of synaptobrevin, an intracellular critical component of neurotransmitter exocytosis, by the zinc-metallopeptidase activity of the smallest subunit of the toxin. Blocking the proteolytic activity may present an attractive approach to treat botulism as to date there is no efficient specific drug therapy avalaible. We have therefore recently described a series of b-amino-thiol derived pseudotripeptides able of inhibiting the toxin at low (10 À8 M) concentration. In this study, binding characteristics of the protein's active site are explored through various structural modifications of the thiol functionality which was supposed to be a key structural constituent for effective zinc-ion chelation. Surprisingly, sulfanylderivatives such as symmetric disulfides were shown to be better inhibitors than their thiol-counterparts, the most potent compound displaying a K i value of 3.4 nM.
Journal of Protein Chemistry, 1998
Botulinum neurotoxin (NT) serotype B, produced by Clostridium botulinum (proteolytic strain), is a ~150-kDa single-chain polypeptide of 1291 amino acids, of which 10 are Cys residues , Appl. Environ. Microbiol. 58, 2345-2354 The posttranslational modifications of the gene product were found to consist of excision of only the initiating Met residue, limited proteolysis (nicking) of the 1290-residue-long protein between Lys 440 and Ala 441, and formation of at least one disulflde bridge. The dichain (nicked) protein, in a mixture with the precursor single-chain (unnicked) molecules, was found to have a ~50-kDa light chain (Pro 1 through Lys 440) and ã 100-kDa heavy chain (Ala 441 through Glu 1290). The limited in vivo nicking of the singlechain NT to the dichain form, by protease endogenous to the bacteria, and the nonfacile in vitro cleavage by trypsin of the Lys 440-Ala 441 bond appear to be due to the adjacent Ala 441-Pro 442 imide bond's probable cis configuration in a mixed population of molecules with cis and trans configurations. The two chains were found connected by an interchain disulfide formed by Cys 436 and Cys 445. Six other Cys residues, at positions 70, 195, 308, 777, 954, and 1277, were found in sulfhydryl form. In addition, a Cys at position 1220 or 1257 appeared to be in sulfhydryl form, hence our experimental results could not unambiguously identify presence of an intrachain disulfide bridge near the C-terminus of the NT, A total of 384 amino acid residues, including the 6 Cys residues at positions 70, 195, 308, 436, 445, and 1277, were identified by direct proteinchemical analysis; thus 29.7% of the protein's entire amino acid sequence predicted from the nucleotide sequence was confirmed. The 6 amino acids, residues 945-950, did not match with the sequence predicted in 1992, but did match with a later report of 1995. The above determinations were made by a combination of chemical (CNBr and acidic cleavage at Asp-Pro) and enzymatic (trypsin, clostripain, and pepsin) cleavages of the NT, and NT carboxymethylated with iodoacetamide (with or without 14C label), separation and isolation of the fragments by SDS-PAGE (followed by electroblotting onto PVDF membrane), and/or reversed-phase HPLC, and analyses of the fragments for the N-terminal amino acid sequences by Edman degradation and amino acid compositions.