Partial amino acid sequences of the heavy and light chains of botulinum neurotoxin type E

The dichain type E botulinum neurotoxin, a product of nicking the single chain protein by trypsin, is composed of a heavy and light chains. Sequence of the first 13 and 20 N-terminal residues of these two chains were determined. Also, proof is provided here that (i) the light chain of the nicked (dichain) is derived from the N-terminal one-third of the parent single chain neurotoxin, and (ii) molecular events leading to the activation, of the single chain neurotoxin cannot involve tryptic cleavage at or very close to the N-terminal of the single chain protein. The partial amino acid sequence of the light chain of botulinum type E and tetanus neurotoxins show significant similarity between the two clostridial neurotoxins.     

Partial amino acid sequences of botulinum neurotoxins types B and E

Clostridium botulinum type E neurotoxin, a single-chain protein of Mr 147,000, was purified and subjected to amino acid sequencing. The same was done for single-chain botulinum type B neurotoxin (Mr 152,000), and for the heavy and light chains (Mr 104,000 and 51,000 respectively) derived from type B by limited trypsin digestion. Twelve to eighteen residues were identified and the following conclusions were drawn: The light chain of the nicked (dichain) type B is derived from the N-terminal one-third of the single-chain (unnicked) parent neurotoxin; sequence homologies are present between single-chain types B and E and the light chain of the nicked type A [J. J. Schmidt, V. Sathyamoorthy, and B. R. DasGupta (1984) Biochem. Biophys. Res. Commun. 119, 900-904]; the N-terminal regions of the heavy chains of types A and B have some structural similarity; and activation of type B neurotoxin cannot involve removal of amino acids or peptides from the N terminus.     

Nicking of single chain Clostridium botulinum type A neurotoxin by an endogenous protease

Botulinum neurotoxin (NT) serotype A isolated from cells from young cultures (approximately 8 h) of Clostridium botulinum type A is a approximately 150 kDa single chain protein. Supernatant from older cultures (96 h) yields approximately 150 kDa dichain NT composed approximately 50 and approximately 100 kDa subunits, that remain associated by disulfide and noncovalent bonds. This had led to the assumption that an endogenous protease cleaves a peptide bond at 1/3rd the distance from the N- or C-terminals of the single chain protein. An endogenous protease that causes such a cleavage (nicking) has now been purified greater than 1,000-fold from C. botulinum type A (Hall strain) culture; this culture also produces the single chain NT and eventually yields the dichain NT. The purified protease nicked the pure preparation of single chain type A NT, in vitro at pH 5.6, into a dichain form that was indistinguishable from the dichain NT normally isolated from 96 h cultures. The protease appears specific for nicking serotype A NT because it did not nick single chain serotype B and E NT nor did it enhance toxicity of serotype A, B and E NT.     

Evidence for a vesicle-mediated maintenance of store-operated calcium channels in a human embryonic kidney cell line

Direct microinjection of the clostridial neurotoxins botulinum neurotoxin A light chain or tetanus neurotoxin into cells of a human embryonic kidney cell line significantly reduced calcium entry after depletion of internal calcium stores by cyclopiazonic acid, a reversible inhibitor of the sarcoplasmic-endoplasmic reticular calcium-ATPases. Botulinum neurotoxin A light chain specifically hydrolyzes a synaptosomal-associated protein of 25 kilodaltons (SNAP-25), and tetanus neurotoxin specifically hydrolyzes synaptobrevin-2 (vesicle-associated membrane protein 2, VAMP-2) and cellubrevin (vesicle-associated membrane protein 3, VAMP-3). Since these substrate proteins are required for vesicle docking and fusion, inhibition of store-operated calcium entry by botulinum neurotoxin A light chain and tetanus neurotoxin supports a model in which vesicle fusion is a prerequisite for activation of store-operated calcium entry. Brefeldin A, a fungal metabolite that interferes with vesicle traffic, partially reduced calcium entry following store depletion. The size of the reserve pool of vesicles or parallel vesicle recycling pathways employing brefeldin A-sensitive and brefeldin A-insensitive ADP-ribosylation factors may explain the failure of brefeldin A to completely inhibit store-operated calcium entry.     

Botulinum neurotoxin type B (strain 657): partial sequence and similarity with tetanus toxin

The type B neurotoxin (NT) isolated from Clostridium botulinum (strain 657) behaved as a mixture of single (unnicked) and dichain (nicked) proteins, both of Mr approximately 150 kDa. When the dichain NT was reduced by mercaptoethanol, the two chains migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as separate polypeptides of Mr approximately 100 and 50 kDa that appeared similar to the heavy and light chains of other serotypes of botulinum NT. The N-terminal amino acid sequences of the two chains were determined. They were as follows: light chain: Pro-Val-Thr-Ile-Asn-Asn-Phe-Asn-Tyr-Asn-Asp-Pro-Ile-Asp-Asn-Asn-Asn-Ile- Ile-Met - Met-Glu-Pro-Pro-Phe-Ala-Arg-Gly-Met-Gly-Arg-Tyr-Tyr-Lys-Ala-Phe-Lys-Ile- Thr-Asp - Arg-Ile-Trp-Ile-; and heavy chain: Ala-Pro-Gly-Ile-X-Ile-Asp-Val-Asp-Asn-Glu-Asp-Leu-Phe-Phe-Ile-Ala-Asp-Ly s-Asn- Ser-Phe-Arg-Asp-Asp-Leu-. These two sequences matched exactly with those of the light and heavy chains of type B NT (strain Okra) of which only 16 and 18 residues were known (J. Biol. Chem. (1985) 260, 10461). The above sequences were different from those of type A NT. Immunoprecipitation reactions of type B NT isolated from strains 657 and Okra were indistinguishable against polyclonal anti-type B NT serum. These two preparations did not produce precipitin reactions with polyclonal anti-type A NT serum.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-throughput fluorogenic assay for determination of botulinum type B neurotoxin protease activity

Botulinum neurotoxins are responsible for botulism, a flaccid muscular paralysis caused by inhibition of acetylcholine release at the neuromuscular junction. This occurs by cleavage of conserved proteins involved in exocytosis such as synaptobrevin by the zinc metallopeptidase activity of the light chain of some botulinum neurotoxins. Botulism, for which there is presently no therapy available, is a relatively widespread disease that may result in death. Consequently, the development of drugs able to inhibit the hydrolytic activity of these neurotoxins is of great interest. Design and screening of such inhibitors could be largely facilitated by using high-throughput assays. With this aim, a novel in vitro test for quantifying the proteolytic activity of botulinum type B neurotoxin was developed. The substrate is the 60--94 fragment of human synaptobrevin-1 which was modified by introduction of the fluorescent amino acid l-pyrenylalanine in position 74 and a p-nitrophenylalanyl residue as quenching group in position 77. The cleavage of Syb 60-94 [Pya(74), Nop(77)] by the toxin active chain occurs selectively between residues 76 and 77 as in the case of the unmodified synaptobrevin and is directly quantified by measuring the strong fluorescence of the formed metabolite Syb 60-76 [Pya(74)]. This is the easiest, quickest, and cheapest assay described to date for measuring the proteolytic activity of botulinum type B neurotoxin. It can be easily automated for high-throughput screening. Moreover, amounts of about 3.5 pg/ml of botulinum type B neurotoxin could be detected by this method.     

Immuno-crossreactivity between botulinum neurotoxin type C1 or D and exoenzyme C3

Botulinum neurotoxin type D and exoenzyme C3 have been separately purified from Clostridium botulinum strain D-1873 to apparent homogeneity. Both ADP-ribosylated a rat liver cytosolic protein of 24 kDa. The N-terminal amino acid sequence of C3 was determined and showed a low degree of homology with those of the light and heavy chains of neurotoxins of various types which have been reported previously. However, a polyclonal antibody raised against C3 cross-reacted with the light chains, but not with the heavy chains, of type C1 and D neurotoxins. Furthermore, a monoclonal antibody recognizing the light chains of type C1 and D neurotoxins interacted with C3. These results suggest that the light chain of type C1 or D neurotoxin and exoenzyme C3 share at least one epitope in common with each other.     

Botulinum neurotoxin type E fragmented with endoproteinase Lys-C reveals the site trypsin nicks and homology with tetanus neurotoxin

Botulinum neurotoxin type E, a 150 kDa single chain protein, cleaved with endoproteinase Lys-C yielded 113, 73, and 50 kDa fragments. The N-terminal sequence of the 113 kDa fragment, Gly-Ile-Arg-Lys-Ser-Ile-Cys-Ile, overlaps the N-terminal sequence, Lys-Ser-Ile-Cys-Ile, of the 103 kDa heavy chain produced by nicking the neurotoxin with trypsin. The -Arg-Lys- bond is therefore the site on the single chain type E NT where trypsin nicks generating the 50 kDa light and 103 kDa heavy chains of the dichain NT. The sequence of the first 50 N-terminal residues of the 73 kDa fragment were determined. This fragment is a segment of the heavy chain; 50% of the 50 residues are present in identical positions in a similar segment of the heavy chain of tetanus neurotoxin.     

Ganglionic synapses of Aplysia as a model for the study of the mechanism of action of botulinum neurotoxins

The action of type A and type B botulinum neurotoxin on neurotransmitter release was studied on identified ganglionic synapses of Aplysia. Using this model, we have shown that botulinum neurotoxins at concentrations used in vertebrate preparations had the same specificity of action and that both heavy and light chains of these toxins are intracellularly required to inhibit neurotransmitter release.     

Role of the heavy and light chains of botulinum neurotoxin in neuromuscular paralysis

Botulinum neurotoxin (NT) is synthesized by Clostridium botulinum in any of seven antigenically distinct forms called types A-G. NT, when fully active, is a dichain protein, composed of two polypeptides, a heavy (H) and a light (L) chain (approximately 100,000 and approximately 50,000 Da, respectively) that are held together by noncovalent bonds and at least one disulfide bond. Two types of dichain NT, A and B, and their respective H and L chains were applied to nerve-muscle (NM) preparations (phrenic nerve-hemidiaphragm of the mouse), in order to develop a broader, comparative understanding of the neuroparalytic actions of NT types. It was found that the paralysis induced by dichain NT was delayed or antagonized if NM preparations were incubated with isolated and purified H chain prior to, or during, incubation with the parent, dichain NT. NM preparations preincubated with H chain and then washed free of unbound H chain became paralyzed after subsequent incubation with L chain. Paralysis did not occur if NM preparations were incubated first with L chain, washed, and then incubated with H chain. These observations suggest that the H chain binds with specific sites on the nerve terminal. This binding appears to permit the L chain, or some combination of the L and H chain, to bring about neuroparalysis through a mechanism very similar to that of the parent, dichain NT.     

Partial amino acid sequence of the heavy and light chains of botulinum neurotoxin type A

The dichain (nicked) type A botulinum neurotoxin is a protein (mol. wt. 145,000) composed of a heavy and a light chain (mol. wt. 97,000 and 53,000, respectively) that are held together by disulfide bond(s). We report here the sequence of the first 17 amino acid residues of the light chain, and the first 10 residues of the heavy chain. The heavy chain was isolated from the neurotoxin by two different methods, while the light chain was isolated by the only available method. The identical amino acid sequence was found in both preparations of heavy chain. Two samples of the light chain isolated from two separately prepared batches of the neurotoxin also had identical sequences.     

Dichain structure of botulinum neurotoxin: identification of cleavage sites in types C, D, and F neurotoxin molecules

Botulinum neurotoxin (NT) is synthesized by Clostridium botulinum as about a 150-kDa single-chain polypeptide. Posttranslational modification by bacterial or exogenous proteases yielded dichain structure which formed a disulfide loop connecting a 50-kDa light chain (Lc) and 100-kDa heavy chain (Hc). We determined amino acid sequences around cleavage sites in the loop region of botulinum NTs produced by type C strain Stockholm, type D strain CB16, and type F strain Oslo by analysis of the C-terminal sequence of Lc and the N-terminal sequence of Hc. Cleavage was found at one or two sites at Arg444/Ser445 and Lys449/Thr450 for type C, and Lys442/Asn443 and Arg445/Asp446 for type D, respectively. In culture fluid of mildly proteolytic strains of type C and D, therefore, NT exists as a mixture of at least three forms of nicked dichain molecules. The NT of type F proteolytic strain Oslo showed the Arg435 as a C-terminal residue of Lc and Ala440 as an N-terminal residue of Hc, indicating that the bacterial protease cuts twice (Arg435/Lys436 and Lys439/Ala440), with excision of four amino acid residues. The location of cleavage and number of amino acid residue excisions in the loop region could be explained by the degree of exposure of amino acid residues on the surface of the molecule, which was predicted as surface probability from the amino acid sequence. In addition, the observed correlation may also be adapted to the cleavage sites of the other botulinum toxin types, A, B, E, and G.     

Cloning, high-level expression, single-step purification, and binding activity of His6-tagged recombinant type B botulinum neurotoxin heavy chain transmembrane and binding domain

Botulinum neurotoxins (BoNTs) are highly potent toxins that inhibit neurotransmitter release from peripheral cholinergic synapses and associate with infant botulism. BoNT is a approximately 150kDa protein, consisting of a binding/translocating heavy chain (HC; 100kDa) and a toxifying light chain (LC; 50kDa) linked through a disulfide bond. C-terminal half of the heavy chain is binding domain, and N-terminal half of the heavy chain is translocation domain that includes transmembrane domain. A functional botulinum neurotoxin type B heavy chain transmembrane and binding domain (Ile 624-Glu 1291) has been cloned into a bacterial expression vector pET 15b and produced as an N-terminally six-histidine-tagged fusion protein (BoNT/B HC TBD). (His(6))-BoNT/B HC TBD was highly expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL and isolated from the E. coli inclusion bodies. After solubilizing the purified inclusion bodies with 6M guanidine-HCl in the presence of 10mM beta-mercaptoethanol, the protein was purified and refolded in a single step on Ni(2+) affinity column by removing beta-mercaptoethanol first, followed by the removal of urea. The purified protein was determined to be 98% pure as assessed by SDS-polyacrylamide gel. (His(6))-BoNT/B HC TBD retained binding to synaptotagmin II, the receptor of BoNT/B, which was confirmed by immunological dot blot assay, also to ganglioside, which was investigated using enzyme-linked immunosorbent assay.     

Is formation of visible channels in a phospholipid bilayer by botulinum neurotoxin type B sensitive to its disulfide?

Botulinum neurotoxin, produced by Clostridium botulinum as a approximately 150-kDa single-chain protein, is nicked proteolytically either endogenously or exogenously. The approximately 50- and approximately 100-kDa chains of the dichain molecule remain held together by an interchain disulfide bridge and noncovalent interactions. The neurotoxin binds to receptors of the target cell and is internalized by endocytosis. Thereafter, a portion of the neurotoxin, the approximately 50-kDa chain, escapes to the cytosol, where it blocks neurotransmitter release. Botulinum neurotoxin serotype B is released by the bacteria primarily as an unnicked single chain. We reduced this unnicked protein and used its binding to ganglioside in a lipid layer to produce helical tubular crystals of unnicked botulinum neurotoxin type B in its disulfide-reduced state. The helical arrangement of the neurotoxin allowed determination of the structure of the molecule using cryo-electron microscopy and image processing. The resulting model reveals that neurotoxin molecules formed loops extending out from the surface of the bilayer and bending toward a neighboring loop. Although channels have been seen with disulfide-linked neurotoxin (Schmid, Robinson, and DasGupta (1993) Direct visualization of botulinum neurotoxin-induced channels in phospholipid vesicles, Nature 364, 827-830), no channels were seen here, a finding which suggests that the reduced, unnicked neurotoxin is incapable of forming a visible channel.     

Effect of pH on the interaction of botulinum neurotoxins A, B and E with liposomes.

The interaction of botulinum neurotoxin serotypes A, B and E with membranes of different lipid compositions was examined by photolabelling with two photoreactive phosphatidylcholine analogues that monitor the polar region and the hydrophobic core of the lipid bilayer. At neutral pH the neurotoxins interacted both with the polar head groups and with fatty acid chains of phospholipids. At acidic pHs the neurotoxins underwent structural changes characterized by a more extensive interaction with lipids. Both the heavy and light chain subunits of the neurotoxins were involved in the process. The change in the nature and extent of toxin-lipid interaction occurred in the pH range 4-6 and was not influenced by the presence of polysialogangliosides. The present data are in agreement with the idea that botulinum neurotoxins enter into nerve cells from a low pH intracellular compartment.     

Role of the disulfide cleavage induced molten globule state of type a botulinum neurotoxin in its endopeptidase activity.

Botulinum neurotoxins are produced by anaerobic Clostridium botulinum in an inactive form. The endopeptidase activity of type A botulinum neurotoxin (BoNT/A) is triggered by reduction of its disulfide bond between its heavy chain and light chain. By using circular dichroism spectroscopy, we show that, upon reduction of BoNT/A and under physiological temperature (37 degrees C), the BoNT/A loses most of its native tertiary structure, while retaining most of its secondary structure. This type of structure is characterized as a molten globule type conformation, which was further confirmed for BoNT/A by the characteristic binding of 1-anilinonaphthalene-8-sulfonic acid. Under nonreducing conditions where the interchain disulfide bond is intact, the enzymatically inactive BoNT/A did not show a molten globule type of structure. A temperature profile of the structure and enzyme activity of BoNT/A revealed that, under reducing conditions, there was a strong correlation in the existence of the molten globule structure and optimum endopeptidase activity at about 37 degrees C.     

Circular dichroic and fluroescence spectroscopic study of the conformation of botulinum neurotoxin types A and E

Summary Botulinum neurotoxin (NT) is synthesized byClostridium botulinum in any of seven antigenically distinct forms, called types A through G. Protease(s) endogenous to the bacteria, or trypsin, nicks the single chain protein to a dichain molecule which generally is more toxic. The conformation of dichain type A (nicked by endogenous protease), single chain type E, and dichain type E NT (nicked by trypsin) have been determined using circular dichroism (CD) and fluorescence spectroscopy. The high degree of ordered secondary structure (α helix 28%, β sheet 42%, total 70%) found in type A NT at pH 6.0 was similar to that found at pH 9.0 (α 22%, β 47%, total 69%). The secondary structure of the single chain type E NT at pH 6.0 (α 18%, β 37%, total 55%) differed somewhat from these values at pH 9.0 (α 22%, β 43%, total 65%). The dichain type E NT at pH 6.0 assumed a secondary structure (α 20%, β 47%, total 67%) more similar to that of dichain type A than the single chain type E NT. Examination with the fluorogenic probe toluidine napthalene sulfonate revealed that the hydrophobicity of the type A and E NTs were higher at pH 9.0 than at pH 6.0. Also, the hydrophobicity of the dichain type E NT was higher than its precursor the single chain protein and appeared similar to that of the dichain type A NT. The CD and fluorescence studies indicate that conversion of the single chain type E NT to the dichain form (i.e. nicking by trypsin) induced changes in conformation. The ordered secondary structure (a + β contents) of botulinum NT, 70% for type A and 67% for dichain type E, agree well with 65% of α + β contents of tetanus toxin [21] that is produced byClostridium tetani.     

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.     

DNA vaccination using the fragment C of botulinum neurotoxin type A provided protective immunity in mice

Botulinum neurotoxin (BoNT) is one of the most toxic substances known to produce severe neuromuscular paralysis. The currently used vaccine is prepared mainly from biohazardous toxins. Thus, we studied an alternative method and demonstrated that DNA immunization provided sufficient protection against botulism in a murine model. A plasmid of pBoNT/A-Hc, which encodes the fragment C gene of type A botulinum neurotoxin, was constructed and fused with an Igkappa leader sequence under the control of a human cytomegalovirus promoter. After 10 cycles of DNA inoculation with this plasmid, mice survived lethal doses of type A botulinum neurotoxin challenges. Immunized mice also elicited cross-protection to the challenges of type E botulinum neurotoxin. This is the first study demonstrating the potential use of DNA vaccination for botulinum neurotoxins.     

Effect of diethylpyrocarbonate on the biological activities of botulinum neurotoxin types A and E

The single chain (unnicked) type-E and the dichain (nicked) type-A botulinum neurotoxins were modified with diethylpyrocarbonate (ethoxyformic anhydride), a reagent highly specific for histidine residues. The type-E neurotoxin could be completely detoxified without causing detectable damage to its serological reactivity. Under identical modification reaction conditions, the type A was incompletely detoxified with some alteration in its serological reactivity. Modification of histidine residues was evident from the increase in absorbance at 240 nm, and reactivation of the detoxified proteins by reversing the modification with hydroxylamine. The completely detoxified type-E neurotoxin, used as toxoid, elicited antibodies in rabbits. The antiserum precipitated and neutralized the neurotoxin. This toxoid is homogeneous as tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas the traditional toxoid produced with formaldehyde is heterogeneous.