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B R DasGupta

Publications and source records attributed to B R DasGupta.

At least 55 records · Page 3Linked to original sources
PubMed1990

Response of the chick ciliary ganglion-iris neuromuscular preparation to botulinum neurotoxin.

Response of the chick ciliary ganglion-iris muscle neuromuscular junction (NMJ) preparation to the botulinum neurotoxin (NT) was investigated. The 150 kDa serotypes A and E NTs inhibited muscle contraction in a dose dependent fashion. Neurotoxicity of type E NT increased 20-40 fold after mild digestion with trypsin. The 50 kDA light and 100 kDa heavy chains of type A NT, following separation, applied individually, did not paralyze the tissues. Preincubation of the NMJ preparations with the isolated type A heavy chain delayed (antagonized) the paralytic action of the 150 kDa dichain type A NT. Sequential administration of type A heavy chain, followed by type A light chain mimicked the action of the parent NT. The chick ciliary preparation therefore is a useful NMJ preparation to study neurotoxicity of botulinum neurotoxins.

Animals↗
PubMed1990

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.

Botulinum Toxins↗
PubMed1990

Botulinum neurotoxin type A: sequence of amino acids at the N-terminus and around the nicking site.

Clostridium botulinum synthesizes the type A botulinum neurotoxin (NT) as a approximately 150 kDa single chain protein. Post-translational proteolytic processing yields a approximately 150 kDa dichain protein composed of a approximately 50 kDa light and approximately 100 kDa heavy chain, which has higher toxicity. Trypsin's action mimics the endogenous proteolytic processing. The proteolytic cleavages could occur at 4 sites. We have examined 2 such sites and defined the peptide sequences before and after proteolytic processing. The N-terminal residues of the newly synthesized approximately 150 kDa single chain NT, Pro-Phe-Val-Asn-Lys-, remain intact at the N-terminus of the approximately 50 kDa light chain generated either in the clostridial culture or in vitro with trypsin or with a protease purified from the homologous bacterial culture. The clostridial protease cleaves the single chain NT in vitro, at 1/3 the distance from its N-terminus, on the amino side of Gly of the sequence -Gly-Tyr-Asn-Lys-Ala-Leu-Asn-Asp-Leu- before cleaving the bond Lys-Ala at a slower rate. The data indicate that the dichain NT is formed in the bacterial culture in at least 2 steps. Cleavage at X-Gly produces a approximately 100 kDa heavy chain-like fragment which is then truncated; cleavage 4 residues downstream at Lys-Ala, and excision of the tetrapeptide Gly-Tyr-Asn-Lys, generates the mature heavy chain with Ala as its N-terminal residue. The approximately 100 kDa heavy chain generated in vitro, by nicking the single chain NT with trypsin, also has Ala-Leu-Asn- as the N-terminal residues.

Amino Acid Sequence↗
PubMed1990

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.

Amino Acid Sequence↗
PubMed1990

Conformational changes associated with the nicking and activation of botulinum neurotoxin type E.

Secondary and tertiary structural parameters of type E botulinum neurotoxin in the unactivated single-chain and activated two-chain (i.e., after proteolytic cleavage) forms were analyzed using circular dichroism, derivative absorption and fluorescence spectroscopy. The estimated secondary structures (22 and 20% alpha-helix, 44 and 44% beta-pleated sheets, and 34 and 36% random coils for the single- and two-chain neurotoxins, respectively) indicated that virtually no change occurred upon nicking of the single-chain neurotoxin. About 57% of the 70 Tyr residues were exposed in the single-chain form, which increased to 62% in the two-chain form. Fluorescence quenching experiments with neutral, anionic and cationic quenchers indicated that about 40% of the maximum accessible fluorescent Trp residues were exposed on the surface of the single-chain neurotoxin as compared to only 20% in the case of the two-chain neurotoxin. Acrylamide was the most effective quencher with a fraction accessibility of 0.56 and 0.48 of maximum accessible Trp fluorescence residues in the single and two-chain forms of the neurotoxin, respectively. Native polyacrylamide gel electrophoresis of the two forms of the neurotoxin revealed greater mobility for the two chain form. This indicates that the surface charges in the single-chain neurotoxin were altered upon nicking. These observations suggest that nicking of the single-chain type E neurotoxin results in refolding and redistribution of the surface charges of the neurotoxin.

Botulinum Toxins↗
PubMed1990

Structure and biological activity of botulinum neurotoxin.

Botulinum neurotoxin appears to undergo structural alterations after synthesis and also before it inhibits neurotransmitter release. Discussions and conjectures are presented in this context: 1. At what sites on the 150 kDa neurotoxin does posttranslational proteolytic processing occur? 2. Does neurotransmitter inhibition depend on separation of a segment of the neurotoxin from the rest of the molecule? 3. At what step in the intoxication pathway does activation of neurotoxin (enhanced lethality following limited proteolysis) manifest? 4. Can the receptor binding parameters (based on bovine brain synaptosome and lipid membrane), channel forming property (lipid bilayer membrane) and intracellular inhibitory activity (based on permeabilized chromaffin and PC 12 cells) provide clues to differences in the lethal potency between the neurotoxin serotypes? In addition, the following issues are considered: 5. The spontaneous fragmentation of isolated 50 kDa light chain, after its separation from 100 kDa heavy chain, 6. Effect of specific chemical modification of Arg, His, Lys, Trp, Tyr and Asp/Glu residues of types A, B and E neurotoxins on lethality and antigenicity, and 7. Development of second generation toxoids.

Binding Sites↗
PubMed1989

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.

Animals↗
PubMed1989

Isolated light chains of botulinum neurotoxins inhibit exocytosis. Studies in digitonin-permeabilized chromaffin cells.

The effects of botulinum neurotoxins or their light and heavy chain subunits were investigated in digitonin-permeabilized adrenal chromaffin cells. Because these cells are permeable to proteins, the toxin had direct access to the cell interior. Botulinum type A neurotoxin and its light chain subunit inhibited Ca2+-dependent catecholamine secretion in a dose-dependent manner. The heavy chain subunit had no effect. Inhibition required introduction of the neurotoxin or light chain into the cell and was not seen when intact cells were incubated with these proteins. The inhibition of secretion by type A neurotoxin and light chain was incomplete, the maximal response being 65%. The inhibition was not overcome by increasing Ca2+ concentrations. The action of the light chain was irreversible and rapid. Botulinum type E neurotoxin also inhibited secretion in a dose-dependent manner. Its potency was increased 30-fold following mild trypsinization, which nicked the single chain protein to the dichain form. In contrast to the results seen with types A and E, botulinum type B neurotoxin did not inhibit secretion, while its light chain totally abolished secretion. Trypsinization of the neurotoxin produced the dichain form, which did not inhibit secretion. Reduction of the trypsinized neurotoxin with dithiothreitol produced inhibition equivalent to that seen with the purified light chain subunit. Isolated type A heavy chain had no effect on the inhibitory action of type A or B light chains. The data demonstrate that the ability of botulinum neurotoxins to inhibit secretion is confined to the light chain region of these proteins. Furthermore, while the botulinum neurotoxin types A, B, and E have similar macrostructures, they are not identical with respect to their biological activities.

Adrenal Medulla↗
PubMed1989

Molecular topography and secondary structure comparisons of botulinum neurotoxin types A, B and E.

Botulinum neurotoxin (NT) serotypes A, B and E differ in microstructure and biological activities. The three NTs were examined for secondary structure parameters (alpha-helix, beta-sheet, beta-turn and random coil content) on the basis of circular dichroism; degree of exposed Tyr residues (second derivative spectroscopy) and state of the Trp residues (fluorescence and fluorescence quantum yield). The proteins are high in beta-pleated sheet content (41-44%) and low in alpha-helical content (21-28%). About 30-36% of the amino acids are in random coils. The beta-sheet contents in the NTs are similar irrespective of their structural forms (i.e. single or dichain forms) or level of toxicity. About 84%, 58% and 61% of Tyr residues of types A, B, and E NT, respectively, were exposed to the solvent (pH 7.2 phosphate buffer). Although the fluorescence emission maximum of Trp residues of type B NT was most blue shifted (331 nm compared to 334 for types A and E NT, and 346 nm for free tryptophan) the fluorescence quantum yields of types A and B were similar and higher than type E. In general the NTs have similar secondary (low alpha-helix and high beta-sheets) and tertiary (exposed tyrosine residues and tryptophan fluorescence quantum yield) structures. Within this generalized picture there are significant differences which might be related to the differences in their biological activities.

Botulinum Toxins↗
PubMed1989

Effect of tetranitromethane on the biological activities of botulinum neurotoxin types A, B and E.

Botulinum neurotoxin serotypes A, B and E were modified at pH 7.9 with tetranitromethane, a reagent highly specific for tyrosine residues. The type B and E neurotoxins were completely detoxified without significant damage to their serological activities. Under similar modification conditions, the type A neurotoxin was incompletely detoxified with some alteration in its serological reactivity. Modification of only tyrosine residues to nitrotyrosine was evident from amino acid analysis of the acid hydrolysates of the modified proteins. The completely detoxified type B and E neurotoxins, used as toxoid, elicited antibodies in rabbits. The antisera precipitated and neutralized the homologous neurotoxin. The two toxoids, type B and E, were prepared with greater than 99% pure neurotoxins as tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis whereas the traditional toxoids produced with formaldehyde are very crude preparations of the neurotoxin (approximately 90% impure). Chemical modification using tetranitromethane is more specific than products that form during approximately 7 days of reaction between a protein and formaldehyde. The toxoids produced with tetranitromethane may be considered second-generation toxoids, compared with the first-generation toxoids (crude preparation of neurotoxins detoxified with formaldehyde).

Amino Acids↗
PubMed1989

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 Toxins↗
PubMed1989

Botulinum neurotoxin type A radiolabeled at either the light or the heavy chain.

Botulinum neurotoxin (NT) has two distinct structural regions called L and H chains (approximately 50 and approximately 100 kDa, respectively). Although the H chain is responsible for binding of the NT to neuronal cells, it is not known which of the subunits is internalized and therefore responsible for causing the blockage of acetylcholine release in susceptible neuronal cells. In this report we describe for the first time the preparation of type A NT which is selectively radiolabeled at either the L or the H chain subunit. Such NT preparations will be useful as tools for determining the distribution of L and H chains in poisoned neuronal cells and the role that each subunit plays in inducing toxicity. The L and H chains of the NT (approximately 150 kDa) were separated, purified, and then individually radiolabeled by reductive methylation of the lysine residues using [3H]- or [14C]formaldehyde. The labeled L and H chains were reconjugated with the complementary unlabeled L and H chains. Formation of -S-S- and noncovalent bonds between the L and H chains regenerated the approximately 150 kDa NT. Autoradiographs of sodium dodecyl sulfate polyacrylamide gels confirmed that each reconstituted NT preparation was labeled at only one subunit chain. NT selectively labeled at either the L or the H chain had specific radioactivities of ca. 25-30 and 45-55 microCi/mumol, respectively, and toxicity (mouse LD50/mg protein) values of 2.2 +/- 1.1 X 10(7) and 3.0 +/- 1.0 X 10(7), respectively. A linear increase in the specific radioactivity of L and H chain subunits was observed with increasing concentrations of 3H- or 14C-labeled formaldehyde in the reaction mixture and with increasing concentrations of L or H chain in the reaction mixture.

Botulinum Toxins↗
PubMed1989

Modification of carboxyl groups in botulinum neurotoxin types A and E.

Effects of chemical modification of carboxyl groups of botulinum neurotoxin serotypes A and E were studied by using a water soluble carbodiimide-nucleophile reaction that is highly specific for modifying carboxyl groups of proteins. In both types A and E, increasing levels of the reagents, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and norleucine methyl ester or glycine methyl ester, at pH 4.8 caused increased loss of toxicity. More glycine could be incorporated than norleucine. Amino acid analysis did not reveal modification of any amino acid residue other than carboxyl groups (possible reaction of sulfhydryl groups was not studied). Loss of one carboxyl group did not severely affect toxicity, but modification of three carboxyl groups caused greater than 95% detoxification in both types. Complete detoxification could not be achieved with any amount of the reagents. Modification of three to five carboxyl groups did not affect serological activity.

Animals↗
PubMed1989

Molecular differences between type A botulinum neurotoxin and its toxoid.

The neurotoxins (seven serotypes, Mr approximately 150,000) produced by Clostridium botulinum cause the neuroparalytic disease botulism. Prophylaxis, definitive diagnosis and the only effective therapy for botulism depend, at present, on chemically detoxified form(s) of the neurotoxins, i.e. toxoids (immunogens), and the antisera raised with the immunogens. And yet, the toxoids currently used for immunization of humans and animals and for raising antibody are very crude preparations (approximately 90% impure) of the neurotoxins. Hence, the highly heterogenous toxoids were not suitable for physicochemical studies. We have detoxified a pure (greater than 99%) neurotoxin (serotype A) with formaldehyde. The native neurotoxin is composed of two subunit chains (Mr 53,000 and 97,000). The physicochemical properties of the toxoid (immunogenic in rabbits) were analyzed. The chemical modification produced inter- and intrasubunit covalent links at multiple sites and thus extensive aggregation of the neurotoxin. The secondary structure parameters (alpha-helix, beta-sheet, beta-turn and random coil) of the native protein were not significantly altered. Tertiary structure, as measured by exposure of tyrosine residues and fluorescence quantum yield of tryptophan residues, was considerably altered. The data imply that conformational (topographical) antigenic determinants may not contribute significantly to the serological property of the neurotoxin.

Botulinum Toxins↗
PubMed1989

Changes in the molecular topography of the light and heavy chains of type A botulinum neurotoxin following their separation.

Botulinum neurotoxin serotype A, an approx. 150 kDa protein, is composed of two subunits, the light and heavy chains (approximately 50 and approximately 100 kDa, respectively). The neurotoxin's mode of action is believed to depend on coordinated but independent actions of the two subunit chains. The molecular environments of the aromatic amino acid residues of the dichain neurotoxin and the two isolated subunit chains were analyzed using near-ultraviolet circular dichroism (CD) (between 250 and 320 nm) and second-derivative ultraviolet absorption spectroscopy (between 240 and 320 nm) to investigate the conformational variations of the subunit chains in separated and conjugated forms. The mean residue weight ellipticities showed virtually no change (i.e., 1.7%) in the vicinities of Phe (268 nm), and only a small change (11%) around Tyr (279 nm) residues following dissociation of the subunit chains. However, significant changes (23-26%) at 286 nm as well as at 292 nm were noted, suggesting considerable alteration in the conformation of the subunits. Second-derivative ultraviolet absorption spectra indicated the degree of Tyr exposure in the dichain neurotoxin, isolated heavy and light chains at 70.7, 81.5 and 46.4%, respectively. A weighted mean of the degree of exposed Tyr residues in the separated heavy and light chains was 69.6%, virtually same as the 70.7% exposed Tyr residues observed in the intact dichain neurotoxin, indicating no difference in their Tyr exposure upon separation of the two chains. This was corroborated by the CD data which revealed only small changes in the CD signals of Tyr residues, and no alteration in those of the Phe residues following separation of the subunit chains. However, a change in the CD signal at 292 nm suggested that the conformations of Trp-containing segments of the two chains were significantly influenced upon their separation. The heavy and light chains of the neurotoxin therefore appear to exist as two semi-independent domains, in spite of being linked by disulfide and noncovalent bonds, and at least part of their conformations depends on interactions between them.

Botulinum Toxins↗
PubMed1988

Fast protein liquid chromatography of botulinum neurotoxin types A, B and E.

Three antigenically different botulinum neurotoxins (NTs, relative molecular mass approximately 150,000), classically distinguished only by specific antisera, were for the first time chromatographically resolved. Mixed NTs eluted from a Mono-Q column in order of types E, A and B, and from Mono-S as B, E and A. Type A and B NTs were successfully chromatographed on the cation-exchange Mono-S column above their isoelectric points. Purification of type A and B NTs by automated liquid chromatography was also accomplished for the first time. Type A, B and E NTs were purified by application on an anion-exchange Mono-Q column, followed by use of a cation-exchange Mono-S column.

Botulinum Toxins↗
PubMed1988

Interaction of botulinum and tetanus toxins with the lipid bilayer surface.

The interaction of botulinum neurotoxins serotypes A, B and E (from Clostridium botulinum) and of tetanus neurotoxin (from Clostridium tetani) with the surface of liposomes made of different lipid compositions was studied by photolabelling with a radioiodinated photoactive phosphatidylethanolamine analogue [125I-dipalmitoyl (3,4-azidosalicylamido)phosphatidylethanolamine]. When the vesicles were made of negatively charged lipids (asolectin), each of these neurotoxic proteins was radioiodinated, thus providing evidence for their attachment to the membrane surface. The presence of gangliosides on liposome membranes enhanced fixation of the neurotoxic proteins to the lipid vesicle surface. Both the heavy and light chains of the clostridial neurotoxins were involved in the attachment to the lipid bilayer surface. Each of the toxins tested here attached poorly to liposomes made of zwitterionic lipids (egg phosphatidylcholine), even when polysialogangliosides were present. The data suggest that the binding of botulinum and tetanus neurotoxins to their target neuronal cells involves negatively charged lipids and polysialogangliosides on the cell membrane.

Botulinum Toxins↗