[Show abstract][Hide abstract] ABSTRACT: The large toxin complex (L-TC) produced by Clostridium botulinum is formed from the M-TC (BoNT/NTNHA complex) by conjugation of M-TC with HA-33/HA-17 trimer consists of two HA-33 proteins and a single HA-17 protein. This association is mediated by HA-70, which interacts with HA-17. The current study aims to identify the regions of the HA-70 molecule that adhere to the HA-33/HA-17 complex. Products from limited proteolysis of HA-70 were resolved by SDS-PAGE and transferred onto PVDF membranes, where they were probed with HA-33/HA-17 in a far-western blot. Among the HA-70 fragments, HA-33/HA-17 bound to those containing at least the C-terminal half of the HA-70 molecule, but not those carrying the N-terminal half. Additional docking simulation analysis indicated that the HA-70 region Gln420-Tyr575 is responsible for binding to HA-17, which is consistent with the far-western blot data. The findings here reveal additional details concerning the three-dimensional structure of the functional HA sub-complex in the botulinum toxin complex.
International journal of biological macromolecules 01/2014; · 2.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In Clostridium botulinum, the characteristics of type C and D strains are quite different from other types, and they are classified as group III. They produce C2 binary toxin and C3 exoenzyme in addition to type C and D neurotoxins. Two different phages and many plasmids are identified in the organisms. The genes of neurotoxin and C3 exoenzyme are converted from toxigenic strains to non-toxigenic strains by the specific bacteriophages (phages), whereas, the C2 toxin gene is carried by large or small plasmids. Classification of type C and D strains has been in confusion because 1) antigenicity of type C and D neurotoxins is complex, 2) the cells produce two types of toxins, neurotoxin and C2 toxin, and 3) some non-toxigenic strains can be converted to produce C or D neurotoxin by the infection with phages. Until now, entire nucleotide sequences of cell chromosomes, phages, and plasmids have been determined. Since both genetic and protein-chemical analyses have been clarifying the above confusions, these data are reviewed historically.
[Show abstract][Hide abstract] ABSTRACT: Large-sized botulinum toxin complex (L-TC) is formed by conjugation of neurotoxin, nontoxic nonhemagglutinin and hemagglutinin (HA) complex. The HA complex is formed by association of three HA-70 molecules and three HA-33/HA-17 trimers, comprised of a single HA-17 and two HA-33 proteins. The HA-33/HA-17 trimer isolated from serotype D L-TC has the ability to bind to and penetrate through the intestinal epithelial cell monolayer in a sialic acid-dependent manner, and thus it plays an important role in toxin delivery through the intestinal cell wall. In this study, we determined the solution structure of the HA-33/HA-17 trimer by using small-angle X-ray scattering (SAXS). The SAXS image of HA-33/HA-17 exhibited broadly similar appearance to the crystal image of the complex. On the other hand, in the presence of N-acetylneuraminic acid, glucose and galactose, the solution structure of the HA-33/HA-17 trimer was drastically altered compared to the structure in the absence of the sugars. Sugar-induced structural change of the HA-33/HA-17 trimer may contribute to cell binding and subsequent transport across the intestinal cell layer.
Biochemical and Biophysical Research Communications 08/2013; · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Clostridium botulinum produces a large toxin complex (L-TC) that increases paracellular permeability in intestinal epithelial cells by a mechanism that remains unclear. Here, we show that mitogen-activated protein kinases (MAPKs) are involved in this permeability increase. Paracellular permeability was measured by FITC-dextran flux through a monolayer of rat intestinal epithelial IEC-6 cells, and MAPK activation was estimated from western blots. L-TC of C. botulinum serotype D strain 4947 increased paracellular dextran flux and activated extracellular signal-regulated kinase (ERK), p38, but not c-Jun N-terminal kinase (JNK) in IEC-6 cells. The permeability increase induced by L-TC was abrogated by the p38 inhibitor SB203580. These results indicate that L-TC increases paracellular permeability by activating p38, but not JNK and ERK.
Journal of Veterinary Medical Science 07/2013; · 0.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Botulinum neurotoxin (BoNT) binds to nontoxic nonhemagglutinin (NTNHA) protein in a pH-dependent manner, and yields the protease-resistant BoNT/NTNHA complex. Here, we screened short peptides that bind to the serotype D NTNHA (NTNHA-D) using random phage display technique. NTNHA was fixed onto electrode of quartz crystal microbalance (QCM) apparatus, and then the phages displaying random heptapeptides were exposed to the NTNHA-D under the acidic condition. After rinsing with acidic buffer, the released phages under the alkaline condition were collected. The binding and release of the phage were monitored by the frequency shift on the QCM. As a result of the screening, 16 were selected as peptides that bind to NTNHA-D. The selected peptides do not share any conserved sequence, but tend to be rich in basic and/or hydrophobic amino acid. This would explain the binding manner of the BoNT to the NTNHA protein.
Current Microbiology 03/2013; · 1.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Clostridium botulinum type C and D strains recently have been found to produce PLC on egg yolk agar plates. To characterize the gene, enzymatic and biological activities of C. botulinum PLCs (Cb-PLCs), the cb-plc genes from 8 strains were sequenced, and 1 representative gene was cloned and expressed as a recombinant protein. The enzymatic and hemolytic activities of the recombinant Cb-PLC were measured and compared with those of the Clostridium perfringens alpha-toxin. Each of the eight cb-plc genes encoded a 399 amino acid residue protein preceded by a 27 residue signal peptide. The protein consists of 2 domains, the N- and C-domains, and the overall amino acid sequence identity between Cb-PLC and alpha-toxin was greater than 50%, suggesting that Cb-PLC is homologous to the alpha-toxin. The key residues in the N-domain were conserved, whereas those in the C-domain which are important in membrane interaction were different than in the alpha-toxin. As expected, Cb-PLC could hydrolyze egg yolk phospholipid, p-nitrophenylphosphorylcholine, and sphingomyelin, and also exhibited hemolytic activity;however, its activities were about 4- to over 200-fold lower than those of alpha-toxin. Although Cb-PLC showed weak enzymatic and biological activities, it is speculated that Cb-PLC might play a role in the pathogenicity of botulism or for bacterial survival.
[Show abstract][Hide abstract] ABSTRACT: Clostridium botulinum produces seven immunological distinct poisonous neurotoxins, A to G, with molecular masses of approximately 150kDa. In acidic foods and culture fluid, the neurotoxins associate with non-toxic components, and form large complexes designated progenitor toxins. The progenitor toxins are found in three forms named LL, L, and M. These neurotoxins and progenitor toxins were purified, and whole nucleotide sequences of their structure genes were determined. In this manuscript, the structure and function of these toxins, and the application of these toxins to clinical usage have been described.
Nippon rinsho. Japanese journal of clinical medicine 08/2012; 70(8):1329-37.
[Show abstract][Hide abstract] ABSTRACT: Several studies have demonstrated the efficacy of intraprostatic injection of botulinum neurotoxin type A (BoNT/A) against symptomatic benign prostatic hyperplasia (BPH). The most commonly used BoNT/A product, Botox(®), forms large complexes and composed of neurotoxin (NTX) as well as non-toxic components. We purified NTX lacking non-toxic components. We investigated the efficacy of this newly purified NTX for men with BPH. Ten male patients (mean age, 70.0 years) with BPH received 100 units (prostate volume [PV] ＜30 ml) or 200 units (PV >30 ml) of NTX injected into the prostate via a minimally invasive outpatient technique. Evaluation included uroflowmetry, postvoid residual urine volume (PVR), PV, and International Prostate Symptom Score (IPSS) measured at baseline and 1, 3, 6, and 12 months post-treatment. The status of 7 of the 10 patients examined was found to have improved within 1 month of treatment. The mean IPSS decreased from 23.8 ± 7.0 to 16.3 ± 10.3 (p＝0.0093) at 1 month, to 14.9 ± 8.2 (p＝0.0074) at 3 months, and to 16.9 ± 7.3 (p＝0.018) at 12 months. The mean PV decreased from 47.8 ± 21.2 to 39.2 ± 19.5 ml (p＝0.0076) at 3 months. The PVR improved at 3 and 6 months post-treatment. Intraprostatic NTX injection induces prostate shrinkage and is effective in men with BPH.
[Show abstract][Hide abstract] ABSTRACT: In cell culture supernatants, the botulinum neurotoxin (BoNT) exists as part of a toxin complex (TC) in which nontoxic nonhemagglutinin (NTNHA) and/or hemagglutinins (HAs) are assembled onto the BoNT. A series of investigations indicated that formation of the TC is vital for delivery of the toxin to nerve cells through the digestive tract. In the assembly process, BoNT binds to NTNHA yielding M-TC, and it then matures into L-TC by further association with the HAs via NTNHA in the M-TC. Here, we report a crystal structure of the NTNHA from Clostridium botulinum serotype D strain 4947. Additionally, we performed small-angle X-ray scattering (SAXS) analysis of the NTNHA and the M-TC to elucidate the solution structure. The crystal structure of D-4947 NTNHA revealed that BoNT and NTNHA share a closely related structure consisting of three domains. The SAXS image indicated that, even though the N-terminal two-thirds of the NTNHA molecule had an apparently similar conformation in both the crystal and solution structures, the C-terminal third of the molecule showed a more extended structure in the SAXS image than that seen in the crystallographic image. The discrepancy between the crystal and solution structures implies a high flexibility of the C-terminal third domain of NTNHA, which is involved in binding to BoNT. Structural dynamics of the NTNHA molecule revealed by SAXS may explain its binding to BoNT to form the BoNT/NTNHA complex.
Biochemical and Biophysical Research Communications 07/2012; 425(2):256-60. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cultured Clostridium botulinum strains produce progenitor toxins designated as 12S, 16S, and 19S toxins. The 12S toxin consists of a neurotoxin (NTX, 7S) and a non-toxic non-hemagglutinin (NTNH). The 16S and 19S toxins are formed by conjugation of the 12S toxin with hemagglutinin (HA), and the 19S toxin is a dimer of the 16S toxin. Type A cultures produce all 3 of these progenitor toxins, while type E produces only the 12S toxin. The 7S toxin is cleaved into heavy (H) and light (L) chains by a protease(s) in some strains, and the H chain has 2 domains, the N-terminus (Hn) and C-terminus (Hc). It has been reported that type A toxins bind to the intestinal cells or cultured cells via either HA or Hc. In this study, we investigated the binding of type A and E toxins to Caco-2 cells using Western blot analysis. Both the type E 7S and 12S toxins bound to the cells, with the 7S toxin binding more strongly, whereas, in the type A strain, only the 16S/19S toxins showed obvious binding. Pre-incubation of the type E 7S toxin with IgG against recombinant type E Hc significantly inhibited the 7S toxin binding, indicating that Hc might be a main binding domain of the type E toxin.
[Show abstract][Hide abstract] ABSTRACT: The large-sized botulinum toxin complex (L-TC) is composed of botulinum neurotoxin (BoNT) and nontoxic proteins, e.g. nontoxic nonhemagglutinin (NTNHA) and three types of hemagglutinins (HAs; HA-33, HA-17 and HA-70). The nontoxic proteins play a critical role in L-TC oral toxicity by protecting the BoNT in the digestive tract, and facilitating absorption of the L-TC across the intestinal wall. Under alkaline conditions, the L-TC separates into BoNT and the nontoxic protein complex (NC). In this study, we established a two-step procedure to yield highly pure NC from the L-TC produced by Clostridium botulinum serotype D strain 4947 in which the NC was isolated from the L-TC by gel filtration under alkaline conditions followed by immunoprecipitation with an anti-BoNT antibody to remove contaminating BoNT from the NC fraction. Western blotting and electrophoretic analysis showed that the highly purified NC fraction had only very slight or no BoNT contamination. In addition, the purified NC fraction showed no intraperitoneal (ip) toxicity to mice at a dose of 38 ng per animal whereas the L-TC exhibited an ip median lethal dose of 0.38 ng per mouse. The highly purified NC displayed the same hemagglutination titer as the L-TC. The NC, as well as the L-TC, demonstrated cell binding and monolayer transport in the rat intestinal epithelial cell line IEC-6. Consequently, the highly purified NC can function as a "delivery vehicle" even without the BoNT.
The Protein Journal 05/2012; 31(5):387-92. · 1.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Many papers report the clinical success of botulinum toxin A as a method of management of various bladder dysfunctions. The rationale was that botulinum toxin A was able to block the presynaptic release of acetylcholine from the parasympathetic efferent nerve. The efficacy might result not only from an inhibitory effect on detrusor muscle, but also some effects might be mediated by altering the afferent nerve input. This systematic literature review discusses the efficacy and safety of botulinum toxin A therapy for idiopathic detrusor overactivity, neurogenic detrusor overactivity, interstitial cystitis/painful bladder syndrome and benign prostatic hyperplasia. The information was gathered from a PubMed literature research for abstracts from recent urological meetings. Injection of botulinum toxin A appears to have a positive therapeutic effect in multiple urological conditions, such as refractory idiopathic detrusor overactivity, neurogenic detrusor overactivity, interstitial cystitis/painful bladder syndrome and benign prostatic hyperplasia. Because the United States Food and Drug Administration has approved botulinum toxin A (Botox) for injection for the treatment of urinary incontinence as a result of neurogenic detrusor overactivity (e.g. spinal cord injury, multiple sclerosis) in adults who have an inadequate response to or are intolerant of an ant cholinergic medication, the use of botulinum toxin A will spread and be a more familiar therapy in the urological arena. However, further robust evidence should be awaited. We will discuss the current use of this agent within the urological field.
International Journal of Urology 03/2012; 19(3):202-15. · 1.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Zinc atoms play an essential role in a number of enzymes. Botulinum neurotoxin (BoNT), the most potent toxin known in nature, is a zinc-dependent endopeptidase. Here we identify the nontoxic nonhemagglutinin (NTNHA), one of the BoNT-complex constituents, as a zinc-binding protein, along with BoNT. A protein structure classification database search indicated that BoNT and NTNHA share a similar domain architecture, comprising a zinc-dependent metalloproteinase-like, BoNT coiled-coil motif and concanavalin A-like domains. Inductively coupled plasma-mass spectrometry analysis demonstrated that every single NTNHA molecule contains a single zinc atom. This is the first demonstration of a zinc atom in this protein, as far as we know. However, the NTNHA molecule does not possess any known zinc-coordinating motif, whereas all BoNT serotypes possess the classical HEXXH motif. Homology modeling of the NTNHA structure implied that a consensus K-C-L-I-K-X(35)-D sequence common among all NTNHA serotype molecules appears to coordinate a single zinc atom. These findings lead us to propose that NTNHA and BoNT may have evolved distinct functional specializations following their branching out from a common ancestral zinc protein.
Biochemical and Biophysical Research Communications 02/2012; 419(3):500-4. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Type A neurotoxin (NTX) of Clostridium botulinum was purified by a simple procedure using a lactose gel column. The toxicity of this purified toxin preparation was retained for at least 1 year at -30°C by supplementation with either 0.1% albumin or 0.05% albumin plus 1% trehalose. When purified NTX was used to treat 49 patients with urinary incontinence caused by either refractory idiopathic or neurogenic detrusor overactivity, 36 patients showed significant improvement in symptoms. These beneficial effects were also observed in cases of prostatic hyperplasia. The results obtained with NTX were similar to that of Botox. The effects of NTX on trigeminal neuralgia induced by infraorbital nerve constriction (IoNC) in rats were also studied. Trigeminal ganglion neurons from ipsilateral to IoNC exhibited significantly faster onset of FM4-64 release than sham-operated contralateral neurons. Intradermal injection of NTX in the area of IoNC alleviated IoNC-induced pain behavior and reduced the exaggerated FM4-64 release in trigeminal ganglion neurons.
[Show abstract][Hide abstract] ABSTRACT: Clostridium botulinum serotype C strains produce a neurotoxin (BoNT) along with nontoxic proteins, including nontoxic nonhemagglutinin and three hemagglutinin subcomponents, HA-70, HA-33 and HA-17, to form a large toxin complex (L-TC). While L-TCs produced by serotype C strains usually exhibit hemagglutination (HA) activity via HA-33 binding to sialic acid on erythrocytes, serotype C strain Yoichi (C-Yoichi) L-TC exhibited neither HA nor binding activity towards erythrocytes, probably due to a C-terminal truncation of the HA-33 protein. However, here, we demonstrate that C-Yoichi L-TC newly showed full HA and binding activity towards neuraminidase-treated erythrocytes that was completely inhibited in the presence of galactose (Gal) or lactose (Lac). Binding of C-Yoichi L-TC to rat small intestine epithelial cells (IEC-6) treated with neuraminidase was also significantly enhanced compared with untreated IEC-6 cells. Similarly, the HA-33/HA-17 complex isolated from C-Yoichi L-TC also bound to neuraminidase-treated IEC-6 cells. The binding activity of both L-TC and HA-33/HA-17 was inhibited in the presence of Gal or Lac. Additionally, C-Yoichi L-TC adsorbed tightly to a lactose-affinity gel column. These results strongly suggest that the unusual recognition of the Gal moiety on the cells could be due to a variation and/or a truncation in the C-terminal-half of the unique C-Yoichi HA-33 protein.
[Show abstract][Hide abstract] ABSTRACT: Many of biological system mediated through protein-protein interactions.
Knowledge of protein-protein complex structure is required for
understanding the function. The determination of huge size and flexible
protein-protein complex structure by experimental studies remains
difficult, costly and five-consuming, therefore computational prediction
of protein structures by homolog modeling and docking studies is
valuable method. In addition, MD simulation is also one of the most
powerful methods allowing to see the real dynamics of proteins. Here, we
predict protein-protein complex structure of botulinum toxin to analyze
its property. These bioinformatics methods are useful to report the
relation between the flexibility of backbone structure and the activity.
[Show abstract][Hide abstract] ABSTRACT: A large size botulinum toxin complex (L-TC) is composed of a single neurotoxin (BoNT), a single nontoxic nonhaemagglutinin (NTNHA) and a haemagglutinin (HA) complex. The HA complex is comprised of three HA-70 molecules and three arm structures of HA-33/HA-17 that consist of two HA-33 and a single HA-17. In addition to the mature L-TC, smaller TCs are present in cultures: M-TC (BoNT/NTNHA), M-TC/HA-70 and immature L-TCs with fewer HA-33/HA-17 arms than mature L-TC. Because L-TC displays higher oral toxicity than pure BoNT, it was presumed that nontoxic proteins are critical for food poisoning. In this study, the absorption of TCs across intestinal epithelial cells was assessed by examining the cell binding and monolayer transport of serotype D toxins in the rat intestinal epithelial cell line IEC-6. All TCs, including pure BoNT, displayed binding and transport, with mature L-TC showing the greatest potency. Inhibition experiments using antibodies revealed that BoNT, HA-70 and HA-33 could be responsible for the binding and transport. The findings here indicate that all TCs can transport across the cell layer via a sialic acid-dependent process. Nonetheless, binding and transport markedly increased with number of HA-33/HA-17 arms in the TC. We therefore conclude that the HA-33/HA-17 arm is not necessarily required for, but facilitates, transport of botulinum toxin complexes.
[Show abstract][Hide abstract] ABSTRACT: Microarray analysis is a high-throughput method for analyzing expression
levels of multiple genes, therefore the microarray have been regarded by
many investigators as a powerful method. Treating a huge amount of data
and judgment of differentially expressed genes require appropriate
statistical analysis. When the microarray analysis suggests there are
co-expressed genes under a specific condition, there is high possibility
that the common transcriptional factors (TFs) control them. It is also
difficult to identify the TFs involved in co-expression through only
biochemical experiments. In view of cis-element pattern related to co
expressed genes might be one of the solutions to infer the gene
expression mechanism clearly.
So far, we have constructed Cis-Module database in order to specify
cis-element location and distribution on genome. Using this database and
rat microarray data, we have investigated the TFs network related to
co-expression of genes. If we could also extract the human genes that
are orthologous to co-expressed gene in rat, it will allow us to compare
their cis-elements and TFs and to consider difference of gene expression
profiles between rat and human. It will be very useful to find out
attention to drug discovery targeting gene expression mechanism.
[Show abstract][Hide abstract] ABSTRACT: Recently after human genome sequence has been determined almost perfectly, more and more researchers have been studying genes in detail. Therefore, we are sure that accumulated gene information for human will be getting more important in the near future to develop customized medicine and to make gene interactions clear. Among plenty of information, micro array might be one of the most important analysis method for genes because it is the technique that can get big amount of the gene expressions data from one time experiment and also can be used for DNA isolation. To get the novel knowledge from micro array data, we need to enrich statistical tools for its data analysis. So far, many mathematical theories and definition have been proposing. However, many of those proposals are tested with strict conditions or customized to data for specific species. In this paper, we reviewed existing typical statistical methods for micro array analysis and discussed the repeatability of the analysis, construction the guideline with more general procedure. First we analyzed the micro array data for TG rats, with statistical methods of family-wise error rate (FWER) control approach and False Discovery Rate (FDR) control approach. As existing report, no significantly different gene could be detected with FWER control approach. On the other hand, we could find several genes significantly with FDR control approach even q=0.5. To find out the reliability of FDR control approach with micro array conditions, we have analyzed 2 more pieces of data from Gene Expression Omnibus (GEO) public database on the web site with SAM in addition to FWER and FDR control approaches. We could find a certain number of significantly different genes with BH method and SAM in the case of q=0.05. However, we have to note that the number and kinds of detected genes are different when we compare our result with the one from the published paper. Even if the same approach is used to analyze the same micro array data, we might get a different result because the distinct definition for micro array data has not been set yet. It means that from the same data we will get different results depending on researchers. We are afraid that this problem will have a big effect on developing new medicines and to progress the next step, like a 2nd screening. So, we suggest that we should have certain guidelines to analyze Micro-Array data validly with statistic method and it will surely be helpful for Micro-Array analysis for medical studies in the future.