Covalent structure of botulinum neurotoxin type B; location of sulfhydryl groups and disulfide bridge and identification of C-termini of light and heavy chains.

Botulinum neurotoxin (NT) serotype B, produced by Clostridium botulinum (proteolytic strain), is a approximately 150-kDa single-chain polypeptide of 1291 amino acids, of which 10 are Cys residues [Whelan et al. (1992), 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 disulfide bridge. The dichain (nicked) protein, in a mixture with the precursor single-chain (unnicked) molecules, was found to have a approximately 50-kDa light chain (Pro 1 through Lys 440) and a approximately 100-kDa heavy chain (Ala 441 through Glu 1290). The limited in vivo nicking of the single-chain 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 protein-chemical 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.

Status of Cys residues in the covalent structure of botulinum neurotoxin types A, B, and E.

Clostridium botulinum neurotoxin (NT) serotypes A, B, and E have 9, 10, and 8 Cys residues, respectively, as deduced from nucleotide sequences [Whelan et al. (1992), Appl. Environ. Microbiol. 48, 2345-2354]. Each of the 150-kDa NTs has at least one disulfide; but type B, like types A and E, may have two disulfides. Using two different chemical reagents, we studied the status of the Cys residues in these three proteins after (i) the final anion exchange chromatographic step in their purification (fresh NT), (ii) 24 hr storage at 8 degrees C, (iii) precipitation with ammonium sulfate (precipitated NT), and (iv) dissolving the precipitated NT in 6 M guanidine HCl. In all three NT serotypes the number of Cys residues titrated with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) as free -SH groups varied, depending upon the absence or presence of EDTA added to the chromatography buffer, storage condition, age, and presence of the denaturant. Titration of 9.5-10 and 5.4-6.0 -SH groups in fresh NTs type B and E, respectively, indicated total and partial absence of disulfide bonds. Fewer titratable -SH groups in the precipitated NT than in the fresh NT suggested formation of disulfide and/or inaccessibility of the -SH groups due to protein's conformational change(s). When the precipitated NTs were dissolved in 6 M guanidine HCl, in the absence of any added reducing agent, all Cys residues of types B and E, and 6.4-8.3 Cys in type A NT were titratable with DTNB. Iodoacetamide modification of precipitated NT types A, B, and E carboxymethylated 4, 2, and 2 Cys residues, respectively; these numbers rose to 6, 9.4, and 8 when these proteins were carboxymethylated after dissolving in 6 M guanidine HCl in the absence of any added reducing agent. We propose that S-S- cleavage mediated by the -SH/-S-S- exchange observed in vitro after unfolding the NTs (also unfolded by 2 M guanidine HCl or urea) possibly mimicks a similar exchange process inside the endosomes, where the NTs are thought to undergo conformational changes, resulting in the reductive cleavage of the interchain disulfide between the 50-kDa light and 100-kDa heavy chain, which in turn releases the light chain and allows its egress out of the endosomes into the cytosol.

Covalent structure of botulinum neurotoxin type E: location of sulfhydryl groups, and disulfide bridges and identification of C-termini of light and heavy chains.

Botulinum neurotoxin (NT) serotype E is synthesized by Clostridium botulinum as an approximately 150-kDa single-chain polypeptide of 1252 amino acid residues of which 8 are Cys residues [Puolet et al. (1992); Biochem. Biophys. Res. Commun. 183, 107-113]. The posttranslational processing of the gene product removes only the initiating methionine. A very narrow segment of this 1251-residue-long mature protein--at one-third the distance from the N-terminus (between residues Lys 418 and Arg 421)--is highly sensitive to proteases, such as trypsin. The single-chain NT easily undergoes an exogenous posttranslational modification by trypsin; residues 419-421 (Gly-Ile-Arg) are excised. The proteolytically processed NT is a dichain protein in which Pro 1-Lys 418 constitute the approximately 50-kDa light chain, Lys 422-Lys 1251 constitute the approximately 100-kDa heavy chain; Cys 411-Cys 425 and Cys 1196-Cys 1237 form the interchain and intrachain disulfide bonds, respectively; the other four Cys residues at positions 25, 346, 941, and 1035 remain as free sulfhydryl groups. The approximately 150-kDa dichain NT, and separated light and heavy chains, were fragmented with CNBr and endoproteases (pepsin and clostripain); some of these fragments were carboxymethylated with iodoacetamide (with or without 14C label) before and after fragmentation. The fragments were separated and analyzed for amino acid compositions and sequences by Edman degradation to determine the complete covalent structure of the dichain type E NT. A total of 208 amino acid residues, i.e., 16.5% of the entire protein's sequence deduced from nucleotide sequence, was identified. Direct chemical identification of these amino acids was in complete agreement with that deduced from nucleotide sequence.

Tyrosine phosphorylation modulates the activity of clostridial neurotoxins.

Clostridial neurotoxins' metalloprotease domain selectively cleaves proteins implicated in the process of synaptic vesicle fusion with the plasma membrane and, accordingly, blocks neurotransmitter release into the synaptic cleft. Here we investigate the potential modulation of these neurotoxins by intracellular cascades triggered by environmental signals, which in turn may alter its activity on target substrates. We report that the nonreceptor tyrosine kinase Src phosphorylates botulinum neurotoxins A, B, and E and tetanus neurotoxin. Protein tyrosine phosphorylation of serotypes A and E dramatically increases both their catalytic activity and thermal stability, while dephosphorylation reverses the effect. This suggests that the biologically significant form of the neurotoxins inside neurons is phosphorylated. Indeed, in PC12 cells in which tyrosine kinases such as Src and PYK2 are highly abundant, stimulation by membrane depolarization in presence of extracellular calcium induces rapid and selective tyrosine phosphorylation of internalized light chain, the metalloprotease domain, of botulinum toxin A. These findings provide a conceptual framework to connect intracellular signaling pathways involving tyrosine kinases, G-proteins, phosphoinositides, and calcium with the action of botulinum neurotoxins in abrogating vesicle fusion and neurosecretion.

N-Ethylmaleimide-sensitive factor acts at a prefusion ATP-dependent step in Ca2+-activated exocytosis.

An ATP-dependent activity of NSF (N-ethylmaleimide-sensitive factor) that rearranges soluble NSF attachment protein (SNAP) receptor (SNARE) protein complexes was proposed to be the driving force for membrane fusion. The Ca2+-activated fusion of secretory vesicles with the plasma membrane in permeable PC12 cells requires ATP; however, the ATP requirement is for a priming step that precedes the Ca2+-triggered fusion reaction. While phosphoinositide phosphorylation is a key reaction required for priming, additional ATP-dependent reactions are also necessary. Here we report that the NSF-catalyzed rearrangement of SNARE protein complexes occurs during ATP-dependent priming. NSF with alpha-SNAP (soluble NSF attachment protein) were required for ATP-dependent priming but not Ca2+-triggered fusion, indicating that NSF acts at an ATP-dependent prefusion step rather than at fusion itself. NSF-catalyzed activation of SNARE proteins may reorganize membranes to generate a vesicle-plasma membrane prefusion intermediate that is poised for conversion to full fusion by Ca2+-dependent mechanisms.

SNAP-25 is required for a late postdocking step in Ca2+-dependent exocytosis.

The Ca2+-activated fusion of large dense core vesicles (LDCVs) with the plasma membrane is reconstituted in mechanically permeabilized PC12 cells by provision of millimolar MgATP and cytosolic proteins. Ca2+-activated LDCV exocytosis was inhibited completely by the type E but not the type A botulinum neurotoxin (BoNT) even though both BoNTs were equally effective in proteolytically cleaving the synaptosome-associated protein of 25 kDa (SNAP-25). The greater inhibition of exocytosis by BoNT E correlated with a greater destabilization of detergent-extracted complexes consisting of SNAP-25, synaptobrevin, and syntaxin. LDCVs in permeable PC12 cells can be poised at a late postdocking, prefusion state by MgATP-dependent priming processes catalyzed by N-ethylmaleimide sensitive factor and priming in exocytosis proteins. BoNT E completely blocked Ca2+-activated LDCV exocytosis in ATP-primed cells, whereas BoNT A was only slightly inhibitory, implying that the C-terminal region of SNAP-25 (Ile181-Gln197) between the cleavage sites for BoNT E and BoNT A is essential for late postdocking steps. A required role for SNAP-25 at this stage was also indicated by inhibition of Ca2+-activated LDCV fusion in ATP-primed cells by a C-terminal peptide antibody. We conclude that plasma membrane SNAP-25, particularly residues 181-197, is required for Ca2+-regulated membrane fusion at a step beyond LDCV docking and ATP utilization.

Reconstitution of transcytosis in SLO-permeabilized MDCK cells: existence of an NSF-dependent fusion mechanism with the apical surface of MDCK cells.

Recently, it was demonstrated that delivery from the trans-Golgi network (TGN) to the basolateral surface of Madin-Darby canine kidney (MDCK) cells required N-ethylmaleimide-sensitive factor (NSF)-alpha soluble NSF attachment protein (SNAP)-SNAP receptor (SNARE) complexes, while delivery from the TGN to the apical surface was independent of NSF-alpha SNAP-SNARE. To determine if all traffic to the apical surface of this cell line was NSF independent, we reconstituted the transcytosis of pre-internalized IgA to the apical surface and recycling to the basolateral surface. Transcytosis and the recycling of IgA required ATP and cytosol, and both were inhibited by treatment with N-ethylmaleimide. This inhibition was reversed by the addition of recombinant NSF. Botulinum neurotoxin serotype E, which is known to cleave the 25,000 Da synaptosomal associated protein, inhibited both transcytosis and recycling, although incompletely. We conclude that membrane traffic to a target membrane is not determined by utilizing a single molecular mechanism for fusion. Rather, a target membrane, e.g. the apical plasma membrane of MDCK cells, may use multiple molecular mechanisms to fuse with incoming vesicle.

Clostridium butyricum neurotoxin: partial amino acid sequence of major proteolytic fragments and intracellular activity in PC12 cells.

The approximately 150 kDa single-chain neurotoxin produced by Clostridium butyricum, reported to be similar to C. botulinum neurotoxin serotype E (Giménez and Sugiyama, Infect. Immun. 54, 926-929, 1988), was probed with trypsin and endoproteinase Lys-C. The two proteases cleaved the butyricum neurotoxin between residues Arg 421-Lys 422 and Lys 418-Gly 419, respectively. Cleavage at this region, highly susceptible to proteolysis, generated approximately 50 kDa light and approximately 100 kDa heavy chains, whose identities were established by amino acid sequence determination. In the approximately 150 kDa dichain neurotoxin these two chains remained linked by an interchain disulfide bond. The endoproteinase Lys-C also cleaved the heavy chain between residues Lys 594-Ile 595, yielding a approximately 73 kDa fragment. A total of 77 amino acid residues was identified by Edman degradation of the major fragments generated by proteolysis. By analogy with other botulinum neurotoxin serotypes, the light chain contains the intracellular inhibitory domain and the heavy chain the receptor-binding and channel-forming domains. Butyricum neurotoxin, whether in single-chain or dichain form, inhibited approximately 70% of the Ca2+ stimulated [3H]norepinephrine release from permeabilized PC12 cells. Reduction with 5 mM dithiothreitol enhanced the intracellular inhibitory activity of dichain neurotoxin about 30-fold and increased the extent of inhibition to about 80%, while the activity of single-chain neurotoxin was slightly affected. Increase in the intracellular inhibitory activity of butyricum neurotoxin following mild treatment with trypsin (i.e. conversion of the single-chain protein to the dichain form) was virtually complete within 6 min.

Covalent structure of botulinum neurotoxin type A: location of sulfhydryl groups, and disulfide bridges and identification of C-termini of light and heavy chains.

Botulinum neurotoxin Type A is synthesized by Clostridium botulinum as a approximately 150 kD single chain polypeptide. The posttranslational processing of the 1296 amino acid residue long gene product involves removal of the initiating methionine, formation of disulfide bridges, and limited proteolysis (nicking) by the bacterial protease(s). The mature dichain neurotoxin is made of a approximately 50-kD light chain and a approximately 100-kD heavy chain connected by a disulfide bridge. DNA derived amino acid sequence predicted a total of 9 Cys residues (Binz et al., 1990, J. Biol. Chem. 265, 9153-9158; Thompson et al., 1990, Eur. J. Biochem. 189, 73-81). Treatment of the dichain neurotoxin, dissolved in 6 M guanidine. HCl, with 4-vinylpyridine converted 5 Cys residues into S-pyridylethyl cysteine residues; but alkylation after mercaptolysis converted all 9 Cys residues in the S-pyridylethylated form. After confirming the predicted number of Cys residues by amino acid analysis, the positions of the 5 Cys residues carrying sulfhydryl groups and the 4 involved in disulfide bridges were determined by comparing the elution patterns in reversed-phase HPLC of the cyanogen bromide mixtures of the exclusively alkylated and the mercaptolyzed-alkylated neurotoxin. The chromatographically isolated components were identified by N-terminal amino acid sequence analysis. The HPLC patterns showed characteristic differences. The Cys residues predicted in positions 133, 164, 790, 966, and 1059 were found in the sulfhydryl form; Cys 429 and 453 were found disulfide-bridge connecting the light and heavy chains, and Cys 1234 and 1279 were found in an intrachain disulfide-bridge near the C-terminus in the heavy chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Nerve growth factor induces sensitivity to botulinum neurotoxin type A in norepinephrine-secreting PC12 cells.

Inhibition of Ca(2+)-activated norepinephrine secretion by the botulinum neurotoxin (NT) serotypes A and E was examined in permeabilized PC12 cells. The dichain type E NT reduced with dithiothreitol (DTT) completely inhibited secretion whereas the dichain type A NT reduced with DTT exhibited incomplete inhibitory activity. In contrast, Ca(2+)-activated secretion in PC12 cells treated with nerve growth factor (NGF) was completely inhibited by reduced type A NT. The NGF-treated PC12 cells retained a sensitivity to the type E NT similar to that of untreated PC12 cells. These results indicate that the intracellular mechanisms of inhibition of the types E and A NTs are distinct. NGF appears to either induce the expression of a component selectively required for type A NT sensitivity, or otherwise modifies the secretory apparatus to acquire type A NT sensitivity.

Calcium-dependent release of norepinephrine from permeabilized PC12 cells is inhibited by approximately 48 and approximately 112 kDa fragments of botulinum neurotoxin type E.

Permeabilized PC12 cells exhibit a Ca(2+)-stimulated norepinephrine secretory pathway which is sensitive to botulinum neurotoxin serotypes A, B and E [Lomneth R., Martin T.F.J. and DasGupta B. R. (1991) J. Neurochem. 57: 1413-1421]. Two novel amino terminal fragments of the 150 kDa neurotoxin serotype E (approximately 112 and 48 kDa), produced by digestion with pepsin, were tested in permeabilized PC12 cells. The intracellular inhibitory activity of the approximately 112 kDa amino terminal fragment, like that of the 150 kDa neurotoxin, was progressively enhanced after trypsinization and dithiothreitol reduction. The approximately 50 kDa C-terminal half of the heavy chain therefore does not contribute to the enhancement of inhibitory activity. The approximately 48 kDa amino terminal light chain-like fragment completely inhibited release of norepinephrine, with an IC50 = 500 pM (more potent than the light chain isolated after digestion with trypsin) not requiring reduction with dithiothreitol. These results clarify the molecular basis of activation of neurotoxin by trypsin and dithiothreitol.

Botulinum neurotoxins are zinc proteins.

The available amino acid sequences of 150-kDa botulinum and tetanus neurotoxins show the presence of a closely homologous segment in the middle of the light chain (NH2-terminal 50 kDa), which is the intracellularly active portion of the toxin. This segment contains the zinc binding motif of metalloendopeptidases, HEXXH. Atomic adsorption analysis of botulinum neurotoxins (serotypes A, B, and E) made on the basis of this observation demonstrated the presence of one zinc atom/molecule of 150-kDa neurotoxin. Conditions were found for the removal of the zinc ion with chelating agents and for the restoration of the normal metal content. The conserved segment, which includes the zinc binding motif, was synthesized and shown to bind [65Zn]2+. Chemical modification experiments indicated that two histidines and no cysteines are involved in Zn2+ coordination in agreement with a probable catalytic role for the zinc ion. The present findings suggest the possibility that botulinum neurotoxins are zinc proteases.

Botulinum neurotoxin type A: structure and interaction with the micellar concentration of SDS determined by FT-IR spectroscopy.

Secondary structures of botulinum neurotoxin type A have been determined using Fourier transform infrared spectroscopy in the amide I and amide III frequency regions. Using Fourier self-deconvolution, second derivatization, and curve-fit analysis, the amide I frequency contour was resolved into Gaussian bands at 1678, 1654, 1644, and 1634 cm-1. In the amide III frequency region, several small bands were resolved between 1320 and 1225 cm-1. Assignments of the bands in both amide I and amide III frequency regions to various types of secondary structures and the estimation of spectral band strengths by integrating areas under each band suggested that the neurotoxin contains 29% alpha-helix, 45-49% beta-sheets and 22-26% random coils. These values agreed very well with those determined earlier from CD spectra. The neurotoxin was treated with a micellar concentration of sodium dodecyl sulfate to simulate interaction between the protein and the amphipathic molecules. Sodium dodecyl sulfate micelles induced significant alterations both in the spectral band positions, and their strengths suggest refolding of the neurotoxin polypeptides. However, these changes were not entirely reversible, which could implicate the role of the altered structures in the function of the neurotoxin.

Botulinum neurotoxin light chain inhibits norepinephrine secretion in PC12 cells at an intracellular membranous or cytoskeletal site.

Botulinum neurotoxin (NT) is a potent inhibitor of neurotransmitter secretion, but its intracellular mechanism and site of action are unknown. In this study, the intracellular action of NT was investigated by rendering the secretory apparatus of PC12 cells accessible to macromolecules by a recently described "cell cracking" procedure. Soluble cytoplasmic factors were depleted from permeabilized cells by washing to generate cell "ghosts" which retained cellular structural components and intracellular organelles (including secretory granules). The PC12 cell ghosts exhibited Ca(2+)-activated [3H]norepinephrine release which was enhanced by cytosolic proteins and MgATP. PC12 cell ghosts provide the opportunity to distinguish the intracellular action of NT on soluble cytoplasmic components versus structural cellular components. The 150-kDa NT and the 50-kDa light chain of serotypes E and B, and to a lesser extent type A, inhibited Ca(2+)-activated [3H]norepinephrine release in PC12 ghosts, but not in intact PC12 cells. The 100-kDa heavy chain had no effect. This indicates that NT acts at an intracellular site in these cells permeabilized by "cell cracking." The inhibition of secretion by NT was rapid and irreversible under the incubation conditions used. NT inhibition of [3H]-norepinephrine release from PC12 ghosts occurred in the absence of cytosolic proteins and MgATP and was not reversed by the addition of cytosolic proteins and MgATP, indicating that NT acts at an intracellular membranous or cytoskeletal site.

Comparative molecular topography of botulinum neurotoxins from Clostridium butyricum and Clostridium botulinum type E.

Production of botulinum-like neurotoxin by a non-Clostridium botulinum organism has profound implications in the epidemiology of the disease botulism. Molecular topography of the approximately 150 kDa neurotoxic protein produced by Clostridium butyricum (strain 5839) and its activation kinetics were examined and compared with a serologically related botulinum neurotoxin produced by C. botulinum type E to further characterize the butyricum neurotoxin. Botulinum neurotoxin was fully activated within 30 min of incubation with trypsin, whereas butyricum neurotoxin achieved maximum activation within 5 min of incubation. Molecular topography of the two neurotoxins was analyzed in terms of secondary structures and the surface accessibilities of the polypeptide domains containing aromatic amino acids. The secondary structure parameters of the butyricum neurotoxin (alpha-helix 22%, beta-sheet 41% and random coil 37%), as estimated from the far ultraviolet circular dichroic spectra, appeared similar to that of botulinum neurotoxin. (Singh, B.R. and DasGupta, B.R., (1989) Mol. Cell. Biochem. 86, 87). Second derivative ultraviolet spectral analysis revealed 37 and 41 Tyr residues exposed on the surface of butyricum and botulinum neurotoxins, respectively, suggesting a differential surface accessibility of polypeptide segments containing Tyr residues. Fluorescent Trp residues in both the botulinum type E and butyricum neurotoxins were in a relatively hydrophobic environment as indicated by the blue-shifted emission maxima (334 nm). About half of the fluorescent Trp residues of both proteins were accessible to acrylamide, a neutral fluorescence quencher, and appeared to be in a similar molecular environment. The ionic surface probe, I-, quenched the Trp fluorescence of botulinum significantly, but not that of butyricum neurotoxin. Thus, a considerable number of fluorescent Trp residues were apparently located on the surface of the botulinum, but not on that of the butyricum neurotoxin. Botulinum and butyricum neurotoxins, indistinguishable by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, migrated differently in the absence of sodium dodecyl sulfate suggesting difference(s) in their surface charge distribution. These results provide the first report of the secondary and tertiary structure parameters of the neurotoxin produced by a non-botulinum species and comparison of the molecular topography of the neurotoxin with the antigenically related botulinum neurotoxin type E.

Structural analysis of botulinum neurotoxin types A and E in aqueous and nonpolar solvents by Fourier transform infrared, second derivative UV absorption, and circular dichroic spectroscopies.

Two pharmacologically similar but antigenetically distinct botulinum neurotoxins, types A and E with a 1000-fold difference in their toxicity, were examined for nonpolar solvent-induced changes in secondary structures and polypeptide foldings to understand their structural differences and their comparative responsiveness/susceptibility to solvent perturbation. Analysis of far UV circular dichroic spectra in aqueous buffer for types A and E neurotoxins yielded the following: the alpha-helix contents were 27 and 20%; the beta-sheets were 36 and 44%, the beta-turns were 6.0 and 0%, and the random coils were 31 and 36%, respectively. Fourier transform infrared spectra, obtained by using attenuated total reflection technique, indicated high content of alpha-helix and beta-pleated sheet structures for both neurotoxins as judged by strong bands at 1651 and 1633 cm-1 in the amide I frequency region and bands at 1314 and 1245 cm-1 in the amide III frequency region. The peak height ratio of 1314 and 1245 cm-1 bands, suggests that the type A neurotoxin has slightly higher alpha-helical content than the type E neurotoxin. These observations are consistent with the secondary structures estimated from far UV circular dichroic spectra. Fourier transform infrared spectra of the neurotoxins, exposed to methanol, showed sharp increases of the 1651 cm-1 band and a significant increase in the height of the 1314 cm-1 band, suggesting increases in the alpha-helical contents of the proteins. The changes were more in the type A than in the type E neurotoxin. The changes were reversible upon reexposure of the proteins to the aqueous buffer. Second derivative absorption spectroscopy demonstrated that methanol also induced changes in the degree of Tyr exposure to solvent. The results are discussed in terms of structural differences between the single and dichain neurotoxins and in terms of their mode of action.

Purification and characterization of a protease from Clostridium botulinum type A that nicks single-chain type A botulinum neurotoxin into the di-chain form.

A protease that nicks the approximately 150-kilodalton (kDa) single-chain type A botulinum neurotoxin into the approximately 150-kDa di-chain form in vitro was isolated from Clostridium botulinum type A (Hall strain) cultures. The di-chain neurotoxin generated in vitro is composed of an approximately 50-kDa light chain and an approximately 100-kDa heavy chain which are disulfide linked and is indistinguishable from the di-chain neurotoxin that forms in vivo and is routinely isolated (M.L. Dekleva and B.R. DasGupta, Biochem. Biophys. Res. Commun. 162:767-772, 1989). This enzyme was purified greater than 1,000-fold by ammonium sulfate precipitation, QAE-Sephadex Q-50, Sephadex G-100, and CM-Sephadex C-50 chromatography steps with the synthetic substrate N-benzoyl-DL-arginine-p-nitroanilide. The approximately 62-kDa amidase (protease) is a complex of 15.5- and 48-kDa polypeptides (determined by polyacrylamide gel electrophoresis) that could not be separated without sodium dodecyl sulfate. The enzyme has an isoelectric point of pH 5.73, a pH optimum of 6.2 to 6.4, an absolute requirement for a thiol-reducing agent as well as a divalent metallic cation (probably Ca2+) for activity, and a temperature optimum of 70 degrees C. Tests with several synthetic substrates indicated the high specificity of the enzyme for arginyl amide bonds.

Structure of heavy and light chain subunits of type A botulinum neurotoxin analyzed by circular dichroism and fluorescence measurements.

The secondary and tertiary structural features of botulinum neurotoxin (NT) serotype A, a dichain protein (Mr 145,000), and its two subunits, the heavy (H) and light (L) chains (Mr 97,000 and 53,000, respectively) were examined using circular dichroism and fluorescence spectorscopy. Nearly 70% of the amino acid residues in each of the three polypeptide preparations were found in ordered structure (sum of alpha helix, beta sheet and beta turns). Also, the alpha helix, beta sheet, beta turns and random coil contents of the dichain NT were nearly equal to the weighted mean of each of these secondary structure parameters of the L and H chains; e.g., sum of alpha helix of L chain (22%) and H chain (18.7%), as weighted mean, 19.8% was similar to that of NT (20%). These agreements suggested that the secondary structures of the subunits of the dichain NT do not significantly change when they are separated as isolated L and H chains. Fluorescence emission maximum of L chain, 4 nm less (blue shift) than that of H chain, suggested relatively more hydrophobic environment of fluorescent tryptophan residue(s) of L chain. Tryptophan fluorescence quantum yields of L chain, H chain and the NT, 0.072, 0.174 and 0.197, respectively, suggested that a) an alteration in the micro-environment of the tryptophan residues was possibly caused by interactions of L and H chain subunits of the NT and b) quantum yields for L and H chains were altered when they are together as subunits of the NT. Possible implications of structural features of the L and H chains, their interactions and the molecular mechanism of action of botulinum NT are assessed.

Botulinum neurotoxin type A: cleavage of the heavy chain into two halves and their partial sequences.

The 145-kDa type A botulinum neurotoxin (NT) is produced by the bacteria Clostridium botulinum (strain, Hall). The heavy (H) and light (L) chains (97- and 53-kDa, respectively) of this protein are linked by at least one disulfide bond. The N- and C-terminal halves of the H chain appear to have different functions in the mechanism of action of the NT [1987) FEBS Lett. 226, 115-120). Well-characterized and highly purified preparations of the two halves of the H chain are needed for such studies. Two different approaches were taken to cut the H chain with trypsin and isolate the fragments. In one method the cleavage products were: (i) 94-kDa fragment made of the L chain linked to the N-terminal half of the H chain (49 kDa) by a disulfide bond(s), and (ii) the C-terminal 44-kDa fragment. The N-terminal half of H chain was separated from the L chain by reducing the disulfide bond(s) linking them and then purified by ion-exchange chromatography. The 1-27 residues of 49-kDa N-terminal half of the H chain were Ala-Leu-Asn-Asp-Leu-Cys-Ile-Lys-Val-Asn-Asn-Trp-Asp-Leu-Phe-Phe-Ser-Pro- Ser-Glu - Asp-Asn-Phe-Thr-Asn-Asp-Leu-. The sequence of the other half of the H chain (44 kDa) was X-Ile-Ile-Asn-Leu-X-Ile-Leu-Asn-Leu-Arg-Tyr-Glu-X-Asn-His-Leu-Ile-Asp-Le u-Lys- X-Tyr-Ala-Ser-. In the second method, the H chain was first separated from the L chain, purified, and then cleaved. One product of cleavage, the 44-kDa fragment, was partially sequenced; the first 25 residues were identical to the sequence of the 44-kDa fragment generated by the first method. The present work also demonstrated that (i) The cysteine residue(s) located on the N-terminal half of the H chain form the -S-S- link(s) with the L chain. (ii) The other half of the H chain (44-kDa fragment, apparently the C-terminal half) is not linked via -S-S- to the L-chain or to the N-terminal half (49-kDa fragment) of the H chain.(ABSTRACT TRUNCATED AT 400 WORDS)

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)