Biomedical subjects

Hirokazu Kouguchi

Publications and source records attributed to Hirokazu Kouguchi.

11 recordsLinked to original sources

PubMed2007

Effect of nicking the C-terminal region of the Clostridium botulinum serotype D neurotoxin heavy chain on its toxicity and molecular properties.

A unique strain of Clostridium botulinum serotype D 4947 produces toxin complexes that are composed of un-nicked components, including a neurotoxin (BoNT) and auxiliary proteins. This BoNT showed aberrant elution upon Superdex gel filtration, indicating a much lower molecular weight, due to hydrophobic interaction with the column. Limited trypsin proteolysis of BoNT produces two nicks; first nick yielded a BoNT 50 kDa light chain disulfide linked to a 100 kDa heavy chain (Hc), and a second nick arose in Hc C-terminal 10 kDa. The second nick occurred in the putative binding domain of the BoNT molecule and induced alterations in its secondary structure, leading to a significant reduction of mouse toxicity in comparison with that of the fully-activated singly nicked BoNT. These results help to clarify the role of the C-terminal half of the Hc in the oral toxicity of single-chain and more complex forms of BoNT.

Animals↗

PubMed2006

Quantitative detection of gene expression and toxin complex produced by Clostridium botulinum serotype D strain 4947.

Botulinum toxin is produced by Clostridium botulinum as a large toxin complex (L-TC) non-covalently assembled with a neurotoxin (NT), a non-toxic non-hemagglutinin (NTNHA) and hemagglutinin subcomponents (HA-70, HA-33, and HA-17). In this study, the gene expressions of five individual L-TC components were examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in C. botulinum serotype D strain 4947 (D-4947) during cell growth. Transcripts for the five component genes were successfully detected in the mid-exponential growth phase (6.5 h), reaching a maximum at the early stationary growth phase (12 h). The ratio of the mRNA transcripts of nt and ntnha was approximately 1:1, suggesting that nt and ntnha are bicistronically transcribed. On the other hand, the transcript levels of the ha genes were several-fold higher than those of nt and ntnha, although the mRNA transcript level of ha-33 was less than the other two ha subcomponent genes. The results based on qRT-PCR indicate that a shortage of HA-33 among the proteins associated with botulinum TC could explain the production by D-4947 of other smaller-sized L-TCs (610, 540 and 410 kDa) with fewer HA-33 molecules than the mature 650 kDa L-TC. Western blot analysis demonstrated that TC species in cell lysate were initially observed in the mid-exponential phase, while extracellular TCs were detected subsequently in the early stationary phase.

Bacterial Proteins↗

PubMed2005

Characterization of various recombinant antigens from Echinococcus multilocularis for use in the immunodiagnosis.

Using four clones isolated from Echinococcus multilocularis cDNA library with alveolar echinococcosis (AE) patient sera, various antigens were expressed as ThioHis tag-fused protein. Recombinant EmII/3 antigen was produced as the five fragments divided into the N-terminal (#5 and #5s), the central (#6 and #6s) and the C-terminal domain (#7). Immunoblot analysis revealed that the #7 showed significant reactivity whereas those of #5 and #5s were relatively low. The #6 and #6s also showed lower reactivity than that of #7, although the two minor bands of #6 reacted with every serum. These results suggested that an immunodominant region of EmII/3 locate within the C-terminal one third. The #8s recombinant antigen, Ser23-Glu176 of actin filament fragmenting protein (AFFP), apparently reacted with the AE patient sera, while the #1 antigen synthesized as a full-length antigen BI did not show such high reactivity. Thus, #7 and #8s antigens showed significant potential for use in immunodetection of AE. In addition, the specific antibodies against #7 and #8s reacted with specific antigens in crude extract of E. multilocularis cyst, indicating that these antigens retained antigenicity common to native EmII/3 and AFFP, respectively.

Animals↗

PubMed2005

The role of exoproteases in governing intraneuronal metabolism of botulinum toxin.

Botulinum toxin type A has a long duration of action, and thus it can block transmitter release for several weeks to several months. However, little is known about the precise mechanism that accounts for termination of toxin action. Therefore, experiments were done to gauge the effects of aminopeptidases and carboxypeptidases on the structure and function of the toxin. Exoproteases were added to the holotoxin, the native light chain, and a recombinant light chain. Treated toxin and light chain were examined for their effects on neuromuscular transmission and on isolated substrate. The data showed that aminopeptidase attack did not alter the N-terminus of the toxin/light chain, nor did it produce losses in biological activity. Carboxypeptidase attack did alter the C-terminus of the light chain, but not sufficiently to alter biological activity. The data suggest that the tertiary structure of the light chain confers upon the molecule substantial resistance to exoproteases.

Amino Acid Sequence↗

PubMed2005

Characterization of the interaction between subunits of the botulinum toxin complex produced by serotype D through tryptic susceptibility of the isolated components and complex forms.

The 650 kDa large toxin complex (L-TC) produced by Clostridium botulinum serotype D strain 4947 (D-4947) has a subunit structure composed of unnicked components, i.e. neurotoxin (NT), non-toxic non-haemagglutinin (NTNHA) and three haemagglutinin subcomponents (HA-70, HA-33 and HA-17). In this study, subunit interactions were investigated through the susceptibilities of the toxin components to limited trypsin proteolysis. Additionally, complex forms were reconstituted in vitro by various combinations of individual components. Trypsin treatment of intact D-4947 L-TC led to the formation of mature L-TC with nicks at specific sites of each component, which is usually observed in other strains of serotype D. NT, NTNHA and HA-17 were cleaved at their specific sites in either the single or complex forms, but HA-33 showed no sign of proteolysis. Unlike the other components, HA-70 was digested into random fragments as a single form, but it was cleaved into two fragments in the complex form. Based on the relative position of exposed or hidden regions of the individual components in the complex derived from their tryptic susceptibilities, an assembly model is proposed for the arrangement of individual subunits in the botulinum L-TC.

Botulinum Toxins↗

PubMed2005

Four molecules of the 33 kDa haemagglutinin component of the Clostridium botulinum serotype C and D toxin complexes are required to aggregate erythrocytes.

Normally, large-sized botulinum toxin complexes (L-TC) of serotype C and D are composed of a single neurotoxin, a single non-toxic non-haemagglutinin, two HA-70 molecules, four HA-33 molecules and four HA-17 molecules that assemble to form a 650 kDa L-TC. The 540 and 610 kDa TC species (designated here as L-TC2 and L-TC3, respectively) were purified in addition to the 650 kDa L-TC from the culture supernatants of serotype D strains (D-4947 and D-CB16) and serotype C strains (C-6814 and C-Yoichi). The 650 kDa L-TC from D-4947, D-CB16 and C-6814 showed haemagglutination and erythrocyte-binding activity, but their L-TC2 and L-TC3 species had only binding activity. In contrast, every TC species from C-Yoichi having the C-terminally truncated variant of HA-33 exhibited neither haemagglutination activity nor erythrocyte-binding activity. Four strain-specific HA-33/HA-17 complexes were isolated from the 650 kDa L-TC of each strain. The 650 kDa HA-hybrid L-TCs were reconstituted by various combinations of isolated HA-33/HA-17 complexes and haemagglutination-negative L-TC2 or L-TC3 from each strain. HA-hybrid 650 kDa L-TC, including at least one HA-33/HA-17 complex derived from C-Yoichi, lost haemagglutination activity, leading to the conclusion that the binding of four HA-33 molecules is required for haemagglutination activity of botulinum L-TC. The results of the modelling approach indicated that the structure of a variant C-Yoichi HA-33 molecule reveals clear deformation of the beta-trefoil domain responsible for the carbohydrate recognition site.

Botulinum Toxins↗

PubMed2004

Characterization of toxin complex produced by a unique strain of Clostridium botulinum serotype D 4947.

A unique strain of Clostridium botulinum, serotype D 4947 (D-4947), produces a considerable amount of a 650 kDa toxin complex (L-TC) and a small amount of a 280 kDa M-TC, a 540 kDa TC, and a 610 kDa TC. The complexes are composed of only un-nicked components, including neurotoxin (NT), nontoxic nonhemagglutinin (NTNHA) and hemagglutinin subcomponents (HA-70, HA-33 and HA-17). Unlike other NTs from all serotype strains, separation of D-4947 NT from L-TC, except for M-TC, during chromatography required highly alkaline conditions around pH 8.8. The separated NT and NTNHA/HAs complex can be reconstituted to L-TC that is indistinguishable from the parent L-TC with respect to toxicity, hemagglutination activity and gel filtration profile. The isoelectric points of NT and NTNHA/HAs were close together depending on the number of HA-33/17 molecules. We have established a new method to separate the unique D-4947 NT from the complex, which will yield valuable information on structure of botulinum toxin.

Animals↗

PubMed2003

Complete subunit structure of the Clostridium botulinum type D toxin complex via intermediate assembly with nontoxic components.

Clostridium botulinum serotype D strains usually produce two types of stable toxin complex (TC), namely, the 300 kDa M (M-TC) and the 660 kDa L (L-TC) toxin complexes. We previously proposed assembly pathways for both TCs [Kouguchi, H., et al. (2002) J. Biol. Chem. 277, 2650-2656]: M-TC is composed by association of neurotoxin (NT) and nontoxic nonhemagglutinin (NTNHA); conjugation of M-TC with three auxiliary types of hemagglutinin subcomponents (HA-33, HA-17, and HA-70) leads to the formation of L-TC. In this study, we found three TC species, 410, 540, and 610 kDa TC species, in the culture supernatant of type D strain 4947. The 540 and 610 kDa TC species displayed banding patterns on SDS-PAGE similar to that of L-TC but with less staining intensity of the HA-33 and HA-17 bands than those of L-TC, indicating that these are intermediate species in the pathway to L-TC assembly. In contrast, the 410 kDa TC species consisted of M-TC and two molecules of HA-70. All of the TC species, except L-TC, demonstrated no hemagglutination activity. When the intermediate TC species were mixed with an isolated HA-33/17 complex, every TC species converted to 650 kDa L-TC with full hemagglutination activity and had the same molecular composition of L-TC. On the basis of titration analysis with the HA-33/17 complex, the stoichiometry of the HA-33/17 complex molecules in the L-TC, 610 kDa, and 540 kDa TC species was estimated as 4, 3, and 2, respectively. In conclusion, the complete subunit composition of mature L-TC is deduced to be a dodecamer assembled by a single NT, a single NTNHA, two HA-70, four HA-33, and four HA-17 molecules.

Amino Acids↗

PubMed2003

Molecular characterization of GroES and GroEL homologues from Clostridium botulinum.

We report novel findings of significant amounts of 60- and 10-kDa proteins on SDS-PAGE in a culture supernatant of the Clostridium botulinum type D strain 4947 (D-4947). The N-terminal amino acid sequences of the purified proteins were closely related to those of other bacterial GroEL and GroES proteins, and both positively cross-reacted with Escherichia coli GroEL and GroES antibodies. Native GroEL homologue as an oligomeric complex is a weak ATPase whose activity is inhibited by the presence of GroES homologue. The 2634-bp groESL operon of D-4947 was isolated by PCR and sequenced. The sequence included two complete open reading frames (282 and 1629 bp), which were homologous to the groES and groEL gene family of bacterial proteins. Southern and Northern blot analyses indicate that the groESL operon is encoded on the genomic DNA of D-4947 as a single copy, and not on that of its specific toxin-converting phage.

Amino Acid Sequence↗

PubMed2002

Spontaneous nicking in the nontoxic-nonhemagglutinin component of the Clostridium botulinum toxin complex.

The nontoxic-nonhemagglutinin (NTNHA) component, in both isolated form and the neurotoxin (NT)/NTNHA complexed form, was prepared protease-free from toxin complexes produced by Clostridium botulinum type D strain 4947. NTNHA in both preparations was found to be spontaneously converted to the nicked NTNHA form leading to 15- and 115-kDa fragments with the excision of several amino acid residues at specific sites on SDS-PAGE during long-term incubation, while that of the NT/NTNHA/hemagglutinin complexed form remained unnicked single-chain polypeptides under the same conditions. Considering that the NTNHA preparation contained small amounts of the nicked form of NTNHA and the addition of trypsin accelerated the cleavage, it is speculated that a nicked form of NTNHA remaining after the purification and/or NTNHA itself catalyzes the cleavage of intact NTNHA.

Amino Acid Sequence↗

PubMed2001

In vitro reconstitution of the Clostridium botulinum type D progenitor toxin.

Clostridium botulinum type D strain 4947 produces two different sizes of progenitor toxins (M and L) as intact forms without proteolytic processing. The M toxin is composed of neurotoxin (NT) and nontoxic-nonhemagglutinin (NTNHA), whereas the L toxin is composed of the M toxin and hemagglutinin (HA) subcomponents (HA-70, HA-17, and HA-33). The HA-70 subcomponent and the HA-33/17 complex were isolated from the L toxin to near homogeneity by chromatography in the presence of denaturing agents. We were able to demonstrate, for the first time, in vitro reconstitution of the L toxin formed by mixing purified M toxin, HA-70, and HA-33/17. The properties of reconstituted and native L toxins are indistinguishable with respect to their gel filtration profiles, native-PAGE profiles, hemagglutination activity, binding activity to erythrocytes, and oral toxicity to mice. M toxin, which contained nicked NTNHA prepared by treatment with trypsin, could no longer be reconstituted to the L toxin with HA subcomponents, whereas the L toxin treated with proteases was not degraded into M toxin and HA subcomponents. We conclude that the M toxin forms first by assembly of NT with NTNHA and is subsequently converted to the L toxin by assembly with HA-70 and HA-33/17.

Amino Acid Sequence↗