Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.11 (CD10)
 9,792 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A novel assay method based on the endopeptidase activities of the botulinum neurotoxins has been developed and applied to the detection of botulinum type A and B toxins. An assay system developed for the detection of botulinum type B neurotoxin (BoNT/B) is based on the cleavage of a synthetic peptide substrate representing amino acid residues 60 to 94 of the intracellular target protein for the toxin, VAMP (vesicle-associated membrane protein, or synaptobrevin). In this assay system, immobilized VAMP (60-94) peptide substrate is cleaved by BoNT/B at the Gln-76-Phe-77 bond, leaving the C-terminal cleavage fragment on the solid phase. This fragment is then detected by the addition of an antibody-enzyme reagent which specifically recognizes the newly exposed N terminus of the cleavage product. The developed assay was specific to BoNT/B, showing no cross-reactivity with other clostridial neurotoxins, and had a sensitivity for BoNT/B of 0.6 to 4.5 ng/ml, which could be increased to 0.1 to 0.2 ng/ml by using an assay amplification system based on catalyzed reporter deposition. Trypsin treatment of BoNT/B samples, which converts the single-chain toxin to the active di-chain form, was found to increase the sensitivity of the endopeptidase assay from 5- to 10-fold. An endopeptidase assay for BoNT/A, based on the cleavage of a peptide substrate derived from the protein SNAP-25 (synaptosome-associated protein), was also developed and characterized.
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PMID:Development of novel assays for botulinum type A and B neurotoxins based on their endopeptidase activities. 881 85

Bacterial neurotoxins are now being used routinely for the treatment of neuromuscular conditions. Alternative assays to replace or to complement in vivo bioassay methods for assessment of the safety and potency of these botulinum neurotoxin-based therapeutic products are urgently needed. Advances made in understanding the mode of action of clostridial neurotoxins have provided the basis for the development of alternative mechanism-based assay methods. Thus, the identification of SNAP-25 (synaptosomal-associated protein of molecular mass 25 kDa) as the intracellular protein target which is selectively cleaved during poisoning by botulinum neurotoxin type A (BoNT/A) has enabled the development of a functional in vitro assay for this toxin. Using recombinant DNA methods, a segment of SNAP-25 (aa residues 134-206) spanning the toxin cleavage site was prepared as a fusion protein to the maltose-binding protein in Escherichia coli. The fusion protein was purified by affinity chromatography and the fragment isolated after cleavage with Factor Xa. Targeted antibodies specific for the N and C termini of SNAP-25, as well as the toxin cleavage site, were prepared and used in an immunoassay to demonstrate BoNT/A endopeptidase activity towards recombinant SNAP-25 substrates. The reaction required low concentrations of reducing agents which were inhibitory at higher concentrations as were metal chelators and some inhibitors of metallopeptidases. The endopeptidase assay has proved to be more sensitive than the mouse bioassay for detection of toxin in therapeutic preparations. A good correlation with results obtained in the in vivo bioassay (r = 0.95, n = 23) was demonstrated. The endopeptidase assay described here may provide a suitable replacement assay for the estimation of the potency of type A toxin in therapeutic preparations.
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PMID:Recombinant SNAP-25 is an effective substrate for Clostridium botulinum type A toxin endopeptidase activity in vitro. 935 35

Botulinum neurotoxins type A (BoNT/A), the most toxic substance known to man, is produced by Clostridium botulinum type A as a complex with a group of neurotoxin-associated proteins (NAPs), possibly through a polycistronic expression of a clustered group of genes. The botulinum neurotoxin complex is the only known example of a protein complex where a group of proteins (NAPs) protect another protein (BoNT) against acidity and proteases of the GI tract. We now report that NAPs also potentiate the Zn2+ endopeptidase activity of BoNT/A in both in vitro and in vivo assays against its known intracellular target protein, 25 kDa synaptosomal associated protein (SNAP-25). While BoNT/A exhibited no protease activity prior to reduction with dithiothreitol (DTT), the BoNT/A complex exhibited a high protease activity even in its nonreduced form. Our results suggest that the bacterial production of NAPs along with BoNT is designed for the NAPs to play an accessory role in the neurotoxin function, in contrast to their previously known limited role in protecting the neurotoxin in the GI tract and in the external environment. Structural features of BoNT/A change considerably upon disulfide reduction, as revealed by near-UV circular dichroism spectroscopy. BoNT/A in the reduced form adopts a more flexible structure than in the unreduced form, as also indicated by large differences in DeltaH values (155 vs 248 kJ mol-1) of temperature-induced unfolding of BoNT/A.
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PMID:Enhancement of the endopeptidase activity of botulinum neurotoxin by its associated proteins and dithiothreitol. 1034 12

Botulinum neurotoxin light chain (BoNT LC, 50 kDa) is responsible for the zinc endopeptidase activity specific for proteins of neuroexocytosis apparatus. We describe the expression of recombinant type A BoNT LC in Escherichia coli as well as the purification and characterization of the recombinant protein. A high level of expression of BoNT/A LC was obtained by an extended postinduction time of 15 h at 30 degrees C. Recombinant BoNT/A LC was isolated from an Ni(2+) column. Due to its high pI ( approximately 8.7), purification was achieved by a single step of passing the protein through anion-exchange chromatography at pH 8.0 without the need of elution. The purified recombinant BoNT/A LC retained proteolytic activity and had a secondary structure similar to that of native LC determined by CD measurement.
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PMID:High-level expression, purification, and characterization of recombinant type A botulinum neurotoxin light chain. 1060 Apr 50

Type A botulinum neurotoxin (BoNT/A) is a zinc endopeptidase that contains the consensus sequence HEXXH (residues 223-227) in the toxic light chain (LC). The X-ray structure of the toxin has predicted that the two histidines of this motif are two of the three zinc-coordinating ligands and that the glutamate is a crucial amino acid involved in catalysis. The functional implication of E224 in the motif of LC was investigated by replacing the residue with glutamine and aspartate using site-directed mutagenesis. Substitution of Glu-224 with Gln (E224Q) resulted in a total loss of the endopeptidase activity, whereas substitution with Asp (E224D) retained about 1.4% of the enzymatic activity (k(cat) 140 vs 1.9 min(-1), respectively). However, K(m) values for wild-type and E224D BoNT/A LC were similar, 42 and 50 microM, respectively. Global structure, in terms of secondary structure content and topography of aromatic amino residues, Zn(2+) content, and substrate binding ability are retained in the enzymatically inactive mutants. Titration of Zn(2+) to EDTA-treated wild-type and mutant proteins indicated identical enthalpy for Zn(2+) binding. These results suggest an essential and direct role of the carboxyl group of Glu-224 in the hydrolysis of the substrate. The location of the carboxyl group at a precise position is critical for the enzymatic activity, as replacement of Glu-224 with Asp resulted in almost total loss of the activity.
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PMID:Probing the mechanistic role of glutamate residue in the zinc-binding motif of type A botulinum neurotoxin light chain. 1069 9

Clostridial botulinum neurotoxins (BoNTs) cause neuroparalysis by blocking neurotransmitter release at the neuromuscular junctions. While the toxin's heavy chain (HC) is involved in binding and internalization, the light chain (LC) acts as a unique Zn(2+)-endopeptidase against a target protein in the exocytotic docking/fusion machinery. During the translocation of the LC to the cytosol, it is exposed to the endosomal low pH. Low pH showed a dramatic change in the BoNT/A LC polypeptide folding as indicated by differential heat denaturation. Furthermore, binding of 1-anilinonaphthalenesulfonate (ANS) revealed exposure of hydrophobic domains of BoNT/A LC at low pH. Low-pH-induced structural (and by implication the endopeptidase activity) changes were completely reversible. Exposure of BoNT/A LC to low pH (4.7) did not, however, evoke the loss of Zn(2+) bound to its active site. Implications of these observations to the delivery of active BoNT/A LC to the nerve cell are discussed. We further analyzed the nature of low-pH-induced change in the polypeptide folding of BoNT/A LC by Trp fluorescence measurements. The Trp fluorescence peak was observed at 322 nm, and the two fluorescence lifetime components estimated at 2.1 ns (88%) and 0.6 ns (12%) did not change much at low pH. These observations suggested that the two Trp residues are buried and constrained in a hydrophobic environment, and it is likely that the core of the BoNT/A LC protein matrix does not participate in the low-pH-induced structural alteration. This conclusion was further supported by the near-UV circular dichroism spectra under two pH conditions.
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PMID:Spectroscopic analysis of pH-induced changes in the molecular features of type A botulinum neurotoxin light chain. 1082 61

Botulinum neurotoxin type A is one of the most toxic substances known to man (LD(50) for mouse 0.1 ng/kg). It is also an effective therapeutic drug against involuntary muscle disorders and for pain management. BoNT/A is a Zn(2+) endopeptidase which selectively cleaves SNAP-25 (synaptosomal-associated protein of 25 kDa), a critical component of the exocytotic machinery. Based on nucleotide sequence, BoNT/A is a 145 kDa protein, which appears as a 145 kDa protein band on sodium dodecyl sulfate--polyacrylamide gel electrophoresis. We have examined the structure of BoNT/A in aqueous solution, and found the structure in aqueous solution differs dramatically from that resolved by X-ray crystallography, both at secondary and at quaternary levels. In terms of secondary structure, BoNT/A in aqueous solution has about 47% beta-sheet structure as revealed by infrared spectroscopy, while X-ray crystallography revealed only 17% beta-sheet structure. In terms of quaternary structure, the estimated molecular mass of the native BoNT/A in aqueous solution ranged between 230 and 314 kDa, based on results from different chemical and biophysical techniques (native gel electrophoresis, chemical cross-linking, size exclusion chromatography, and fluorescence anisotropy). These results indicate that BoNT/A exists as a dimer in aqueous solution, which contrasts with the reported monomeric structure of BoNT/A based on X-ray crystallography. The dimeric form of BoNT/A can self-dissociate into the monomeric form at a concentration lower than 50 nM. This concentration-dependent structural change has a significant impact on the endopeptidase activity of BoNT/A: the catalytic efficiency of the monomeric BoNT/A is about 4-fold higher than that of its dimeric form. This difference implies a sterically restricted catalytic site of BoNT/A in the dimeric form of BoNT/A.
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PMID:A correlation between differential structural features and the degree of endopeptidase activity of type A botulinum neurotoxin in aqueous solution. 1129 37

Tetanus neurotoxin (TeNT) blocks neurotransmitter release by cleaving VAMP/synaptobrevin, a membrane associated protein involved in synaptic vesicle fusion. Such activity is exerted by the N-terminal 50kDa domain of TeNT which is a zinc-dependent endopeptidase (TeNT-L-chain). Based on the three-dimensional structure of botulinum neurotoxin serotype A (BoNT/A) and serotype B (BoNT/B), two proteins closely related to TeNT, and on X-ray scattering studies of TeNT, we have designed mutations at two active site residues to probe their involvement in activity. The active site of metalloproteases is composed of a primary sphere of residues co-ordinating the zinc atom, and a secondary sphere of residues that determines proteolytic specificity and activity. Glu-261 and Glu-267 directly co-ordinates the zinc atom in BoNT/A and BoNT/B respectively and the corresponding residue of TeNT was replaced by Asp or by the non conservative residue Ala. Tyr-365 is 4.3A away from zinc in BoNT/A, and the corresponding residue of TeNT was replaced by Phe or by Ala. The purified mutants had CD, fluorescence and UV spectra closely similar to those of the wild-type molecule. The proteolytic activity of TeNT-Asp-271 (E271D) is similar to that of the native molecule, whereas that of TeNT-Phe-375 (Y375F) is lower than the control. Interestingly, the two Ala mutants are completely devoid of enzymatic activity. These results demonstrate that both Glu-271 and Tyr-375 are essential for the proteolytic activity of TeNT.
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PMID:Active-site mutagenesis of tetanus neurotoxin implicates TYR-375 and GLU-271 in metalloproteolytic activity. 1130 25

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.
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PMID:Role of the disulfide cleavage induced molten globule state of type a botulinum neurotoxin in its endopeptidase activity. 1173 15

The botulinum neurotoxin type A (BoNT/A) light chain (LC) acts as zinc endopeptidase. The X-ray structure of the toxin demonstrated that Zn(2+) is coordinated by His(222) and His(226) of the Zn(2+) binding motif HisGluXXHis and Glu(261), whereas Glu(223) coordinates the water molecule required for hydrolysis as the fourth ligand. Recent analysis of a cocrystal of the BoNT/B LC and its substrate synaptobrevin 2 suggested that Arg(362) and Tyr(365) of the homologous BoNT/A may be directly involved in catalysis. Their role and that of Glu(350) which is also found in the vicinity to the active site were analyzed by site-directed mutagenesis. Various replacements of Arg(362) and substitution of Tyr(365) with Phe resulted in 79- and 34-fold lower k(cat)/K(m) values, respectively. These changes were provoked by decreased catalytic rates (k(cat)) and not by alterations of ground state substrate binding as evidenced by largely unchanged K(d) and K(m) values. None of these mutations affected the overall secondary structure or zinc content of the LC. These findings suggest that the guanidino group of Arg(362) and the hydroxyl group of Tyr(365) together accomplish transition state stabilization as was proposed for thermolysin, being the prototypical member of the gluzincin superfamily of metalloproteases. Mutation of Glu(350) dramatically diminished the hydrolytic activity which must partly be attributed to an altered active site fine structure as demonstrated by an increased sensitivity toward heat-induced denaturing and a lower Zn(2+) binding affinity. Glu(350) apparently occupies a central position in the active site and presumably positions His(222) and Arg(362).
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PMID:Arg(362) and Tyr(365) of the botulinum neurotoxin type a light chain are involved in transition state stabilization. 1182 15


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