1 Department of Food Science and Technology, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493, Japan
2 Hokkaido Institute of Public Health, N19, W12, Kita-Ku, Sapporo 060-0819, Japan
3 The Sars International Centre for Marine Molecular Biology, Thormøhlensgt 55, N-5008 Bergen, Norway
4 Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita 565-0871, Japan
5 Department of Bacteriology, Okayama University, Medical School, 2-5-1 Shikata-Cho, Okayama 700-8558, Japan
Correspondence
Tohru Ohyama
t-oyama{at}bioindustry.nodai.ac.jp
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
In culture, NT is part of a large complex (toxin complex; TC) through association with non-toxic components by non-covalent binding, such as the non-toxic non-haemagglutinin (NTNHA; 130 kDa) and haemagglutinin (HA) components. HA, which has haemagglutination activity, is composed of three subcomponents, HA-70, -33 and -17 (respectively 70, 33 and 17 kDa). C. botulinum strains produce different forms of TC, the 280 kDa M-TC (a complex of NT and NTNHA), the 650 kDa L-TC (a complex of NT, NTNHA and HAs) and the LL-TC (dimer of L-TC) (Sakaguchi, 1982). Thus, both L-TC (serotypes AD and G) and LL-TC (serotype A) are haemagglutination-positive, while M-TC (serotypes AF) is haemagglutination-negative. Recently, haemagglutination-negative TC species (410, 540 and 610 kDa TC), which have a smaller number of HA-33/HA-17 molecules than those of L-TC, have also been found in the culture supernatant of serotype D strains (Mutoh et al., 2003
).
The NT, NTNHA and HA-70 subunits of the TC species produced by serotype A, C and D strains have always been found to be nicked at specific sites due to bacterial proteases, yielding more protein bands on SDS-PAGE than the number of predicted gene products (Oguma et al., 1999; Sagane et al., 1999
, 2002
; Watanabe et al., 1999
). Thus, the appearance of many fragments on SDS-PAGE may further complicate the analysis of the botulinum TC structure. Fortunately, we found that the botulinum toxin of the serotype D unique strain 4947 (D-4947) can be purified from the culture supernatant as an L-TC and an M-TC, which consisted only of unnicked components (Kouguchi et al., 2002
). Using the isolated components of the D-4947 TC, we have demonstrated in vitro reconstitution of the 650 kDa L-TC with properties that are indistinguishable from those of the parent L-TC (Kouguchi et al., 2002
). In addition, we have shown that the complete subunit composition of D-4947 L-TC is a dodecamer composed of a single NT, a single NTNHA, two HA-70, four HA-33 and four HA-17 molecules (Mutoh et al., 2003
).
However, little is known about the relative configuration of individual components in the botulinum TC subunit structure or the interaction between subunit components. In this study, the interaction domains between subunit components of the D-4947 TC were examined. Assays for individual toxin component susceptibilities to limited proteolysis with trypsin using single components and complex forms, which were reconstituted in vitro by various combinations of individual components, were performed. Based on these results, we propose a dodecamer model of the subunit structure of the D-4947 TC. The model enhances understanding of the three-dimensional subunit structure of botulinum TC, which will be explored with X-ray crystallographic analysis in the future and may explain how the non-toxic components protect the NT from proteolytic attack.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
PAGE and N-terminal amino acid sequence analysis.
PAGE under non-denaturing conditions (native PAGE) was carried out using the method of Davis (1964) at pH 8·8 using a 512·5 % polyacrylamide linear gradient gel. SDS-PAGE was performed as described by Laemmli (1970)
using a 15 % polyacrylamide gel in the presence of 2-mercaptoethanol (2-ME). The molecular size markers were phosphorylase b (97 kDa), BSA (66 kDa), egg ovalbumin (45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20·1 kDa) and
-lactalbumin (14·4 kDa). The separated protein bands were detected with Coomassie brilliant blue R-250 (CBB). The CBB-staining intensities of the bands were analysed with ImageJ 1.30v (http://rsb.info.nih.gov/ij/). The bands separated by SDS-PAGE were transferred onto a PVDF membrane according to a previously described method (Hirano & Watanabe, 1990
) and the amino acid sequences of the components were determined using an automated protein sequence analyser (model 492HT; Applied Biosystems).
Isolation of individual components from D-4947 TCs.
Isolation of HA-70 and HA-33/HA-17 from D-4947 TCs was performed according to the method described by Kouguchi et al. (2002). The purified L-TC (250 mg) dissolved in 0·7 ml 20 mM Tris/HCl (pH 7·8), containing 4 M guanidine hydrochloride, was incubated at 21 °C for 4 h. The sample was then applied to a HiLoad 16/60 Superdex 200 pg gel-filtration column equilibrated with guanidine hydrochloride buffer. Two peak fractions containing HA-70 and HA-33/HA-17 were eluted and pooled separately. After removal of guanidine hydrochloride buffer by dialysis against 20 mM Tris/HCl (pH 7·8) at 4 °C for 15 h, each component was concentrated using appropriate ultrafiltration membranes.
The separation of NT and NTNHA/HAs (NTNHA/HA-70/HA-33/HA-17) from L-TC was achieved by the method developed by Hasegawa et al. (2004) using a HiLoad 16/60 Superdex 200 pg gel-filtration column equilibrated with 20 mM Tris/HCl (pH 8·8) containing 0·4 M NaCl. Each separated sample was applied to a Mono Q HR5/5 anion-exchange column equilibrated with 20 mM Tris/HCl (pH 7·8) and eluted by a linear gradient of NaCl (01·0 M). NTNHA was also separated from M-TC using the method described above.
Reconstitution of TC-related complexes by various combinations of the components.
Reconstitution of the HA-70/HA-33/HA-17 complex was achieved by mixing isolated HA-70 and HA-33/HA-17 at a protein ratio of 1 : 1 in 20 mM Tris/HCl (pH 7·8), followed by a 30 min incubation at room temperature. For reconstitution of the M-TC/HA-70 complex, M-TC and isolated HA-70 were mixed at a protein ratio of 1 : 3 and then transferred into reconstitution buffer with a final concentration of 5 mM sodium phosphate (pH 6·0), 350 mM KCl, 20 mM MgCl2, 6 mM 2-ME and 0·5 mM PMSF. After incubation at 27 °C for 21 h, the M-TC/HA-70 complex was separated from uncomplexed components using a HiLoad 16/60 Superdex 200 pg gel-filtration column. NTNHA/HA-70 was derived by separating NT from the reconstituted M-TC/HA-70 complex. M-TC/HA-70 was dialysed against 20 mM Tris/HCl (pH 7·8) and applied to a Mono Q HR5/5 column equilibrated with dialysis buffer. The target protein was eluted by a linear gradient of NaCl (01·0 M).
Analytical gel filtration.
A Superdex 200 HR 10/30 (Amersham Bioscience) gel-filtration column (1·0x30 cm) was equilibrated with 50 mM phosphate buffer (pH 6·0) that contained 0·15 M NaCl. The molecular masses of the isolated components and complexes were estimated by calculations with standard proteins thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), BSA (67 kDa), egg ovalbumin (43 kDa), chymotrypsinogen A (25 kDa) and RNase A (13·7 kDa). The molecular masses were estimated from the means of three experiments.
Limited proteolysis with trypsin.
Complex forms (L-TC, NTNHA/HAs, M-TC/HA-70, NTNHA/HA-70, M-TC, HA-70/HA-33/HA-17 and HA-33/HA-17) and the single forms (NT, NTNHA and HA-70) were incubated with TPCK-trypsin (0·06 U mg1 using N--benzoyl-DL-arginine-p-nitroanilide as substrate) (Sigma) in 50 mM phosphate buffer (pH 6·0) containing 0·15 M NaCl at 37 °C. For each experiment, 1 : 100 protein ratio of trypsin against each preparation was used. The reaction was terminated by the removal of aliquots from the mixture at 10, 30, 60, 120 and 360 min, which were then subjected to SDS-PAGE.
Far-UV circular dichroism (CD) spectroscopy.
The secondary structure of HA-70 was analysed using a far-UV CD spectrophotometer (model J-720WI; JASCO). CD spectra were recorded using a 10 mm path length cuvette at room temperature. The sample was dialysed against 50 mM phosphate buffer (pH 6·0) containing 0·15 M NaCl and 0·32 mg protein ml1. The secondary structure content was estimated by the method of Yang et al. (1986) and Manavalan & Johnson (1987)
using software provided with the instrument.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
To characterize the molecular composition of various reconstituted complexes, each component responsible for the reconstituted complexes was purified by gel filtration (Fig. 1a). After mixing the components in various combinations in the appropriate molar ratios, products were analysed by SDS-PAGE (Fig. 1b
). The molecular composition of single components or complexes was estimated based on the determined molecular masses by gel filtration and calculated from the deduced sequences from D-4947 TC, as summarized in Table 1
. However, the molecular masses of isolated NTNHA/HAs and NTNHA/HA-70 that were estimated based on their elution volumes were 3040 % greater than the expected molecular masses. In contrast, the estimated molecular mass of the isolated NT was much lower than the expected 150 kDa (Hasegawa et al., 2004
), probably due to hydrophobic interactions of the high-ionic-strength buffer employed between unique D-4947 NT and the Superdex 200 gel matrix, which is composed of highly cross-linked agarose and dextran (Lee & Whitaker, 2004
).
|
|
|
Tryptic susceptibility of single and complex forms of HA subcomponents
Since it was predicted that the HA-70 subcomponent plays a key role as an adherent protein linking M-TC and HA-33/HA-17, leading to the formation of L-TC, the tryptic characteristics of the HA-70 molecule were examined. Interestingly, unlike other components, an isolated form of HA-70 was highly sensitive to trypsin digestion, yielding many fragments within a 10 min incubation, as shown in Fig. 4(a). Similarly, in complexes containing one molecule of HA-70 such as HA-70/HA-33/HA-17, HA-70 was still degraded into many fragments by trypsin digestion (Fig. 4b
). On the other hand, when two molecules of HA-70 formed complexes with NTNHA, such as NTNHA/HA-70 and NTNHA/HA-70/HA-33/HA-17 (see Table 1
), HA-70 protein in both complexes was protected from random cleavage with trypsin, although it was cleaved at specific nicking sites, leading to 22 to 23 kDa N-terminal and 55 kDa C-terminal fragments, as shown in Fig. 5(a, b)
. Similarly, in the M-TC/HA-70 (NT/NTNHA/HA-70) and L-TC (NT/NTNHA/HA-70/HA-33/HA-17) complexes, HA-70 protein was also cleaved at specific sites (Fig. 3b, c
). This suggests that two HA-70 molecules are buried in the interior of L-TC, adhering to NTNHA and HA-33/HA-17, and expose their specific cleavage sites on the surface of L-TC.
|
|
|
Similarly, the secondary structure of serotype C strain 6814 HA-33, which closely resembles D-4947 HA-33, with 96 % amino acid sequence identity, showed a predominantly -sheet (70·371·1 %) and random coil (29·230·1 %) structure as estimated from the CD spectrum (Kouguchi et al., 2001
). A similar
-sheet-rich structure was observed for serotype A HA-33, which was also absolutely resistant to trypsin digestion (Sharma et al., 1999
). Recently, Inoue et al. (2003)
demonstrated that HA-33 from serotype C is composed of two
-trefoil domains linked by an
-helix, according to crystal structure analysis.
Representation of a possible arrangement of the subunit structure of the botulinum TC
Based on the combined results of the experiments described here and the subunit composition of L-TC (a single NT, a single NTNHA, two HA-70, four HA-33 and four HA-17) in our previous report (Mutoh et al., 2003), we propose the model for the arrangement of the individual components in the botulinum L-TC shown in Fig. 7
. The model was deduced from the following experimental observations.
|
HA-70 seems to adhere to NTNHA with the other HA-70 molecule, leading to the protection of the specific cleavage site of NTNHA (15 kDa N-terminal and 115 kDa C-terminal fragments) by HA-70. Simultaneously, HA-70 adhering to NTNHA also escapes from random degradation by trypsin.
HA-17, with its N-terminal region covered by HA-70, is interposed between HA-70 and HA-33 molecules.
Four HA-33 molecules, which are absolutely resistant to proteolysis, are exposed on the surface of TC.
Previously, a partial model for the botulinum NT complex was proposed by Chen et al. (1997) based on antibody mapping to the domain of type A NT in complexed and uncomplexed forms. However, their model demonstrated merely the interaction between the NT and non-toxic portion in the complex, since they failed to distinguish individual non-toxic components such as NTNHA and the HAs. X-ray crystallography had indicated the most probable set of three-dimensional coordinates for nearly all of the atoms of serotype A and B NT proteins (Lacy et al., 1998
; Swaminathan & Eswaramoorthy, 2000
) and serotype C HA-33 (Inoue et al., 2003
). Currently, the three-dimensional structures of NTNHA, HA-70 and HA-17 proteins are not available; thus, it is not possible to know their structural features. Therefore, our model may be subject to a few caveats. However, results on the subunit interaction of the botulinum TC raise the possibility that the botulinum non-toxic complex could be used as a novel drug delivery vehicle in the future, according to the current understanding that HA subcomponents may increase the internalization of the NT into the bloodstream via binding to intestinal membrane (Fujinaga et al., 1997
, 2000
, 2004
), in addition to protecting the structural integrity of NT from protease digestion.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Davis, B. J. (1964). Disc electrophoresis. II. Method and application to human serum proteins. Ann N Y Acad Sci 121, 404427.[Medline]
Fu, F. N., Sharma, S. K. & Singh, B. R. (1998). A protease-resistant novel hemagglutinin purified from type A Clostridium botulinum. J Protein Chem 17, 5360.[CrossRef][Medline]
Fujinaga, Y., Inoue, K., Watanabe, S., Yokota, K., Hirai, Y., Nagamachi, E. & Oguma, K. (1997). The haemagglutinin of Clostridium botulinum type C progenitor toxin plays an essential role in binding of toxin to the epithelial cells of guinea pig small intestine, leading to the efficient absorption of the toxin. Microbiology 143, 38413847.[Medline]
Fujinaga, Y., Inoue, K., Nomura, T., Sasaki, J., Marvaud, J. C., Popoff, M. R., Kozaki, S. & Oguma, K. (2000). Identification and characterization of functional subunits of Clostridium botulinum type A progenitor toxin involved in binding to intestinal microvilli and erythrocytes. FEBS Lett 467, 179183.[CrossRef][Medline]
Fujinaga, Y., Inoue, K., Watarai, S. & 10 other authors (2004). Molecular characterization of binding subcomponents of Clostridium botulinum type C progenitor toxin for intestinal epithelial cells and erythrocytes. Microbiology 150, 15291538.[CrossRef][Medline]
Hasegawa, K., Watanabe, T., Sato, H. & 10 other authors (2004). Characterization of toxin complex produced by a unique strain of Clostridium botulinum serotype D 4947. Protein J 23, 371378.[CrossRef][Medline]
Hirano, H. & Watanabe, T. (1990). Microsequencing of proteins electrotransferred onto immobilizing matrices from polyacrylamide gel electrophoresis: application to an insoluble protein. Electrophoresis 11, 573580.[Medline]
Inoue, K., Sobhany, M., Transue, T. R., Oguma, K., Pedersen, L. C. & Negishi, M. (2003). Structural analysis by X-ray crystallography and calorimetry of a haemagglutinin component (HA1) of the progenitor toxin from Clostridium botulinum. Microbiology 149, 33613370.[CrossRef][Medline]
Kouguchi, H., Watanabe, T., Sagane, Y. & Ohyama, T. (2001). Characterization and reconstitution of functional hemagglutinin of the Clostridium botulinum type C progenitor toxin. Eur J Biochem 268, 40194026.
Kouguchi, H., Watanabe, T., Sagane, Y., Sunagawa, H. & Ohyama, T. (2002). In vitro reconstitution of the Clostridium botulinum type D progenitor toxin. J Biol Chem 277, 26502656.
Krieglstein, K. G., DasGupta, B. R. & Henschen, A. H. (1994). Covalent structure of botulinum neurotoxin type A: location of sulfhydryl groups, and disulfide bridges and identification of C-termini of light and heavy chains. J Protein Chem 13, 4957.[Medline]
Lacy, D. B., Tepp, W., Cohen, A. C., DasGupta, B. R. & Stevens, R. C. (1998). Crystal structure of botulinum neurotoxin type A and implications for toxicity. Nat Struct Biol 5, 898902.[CrossRef][Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685.[Medline]
Lee, S. C. & Whitaker, J. R. (2004). Are molecular weights of proteins determined by Superose 12 column chromatography correct? J Agric Food Chem 52, 49484952.[CrossRef][Medline]
Manavalan, P. & Johnson, W. C., Jr (1987). Variable selection method improves the prediction of protein secondary structure from circular dichroism spectra. Anal Biochem 167, 7685.[CrossRef][Medline]
Montecucco, C. & Schiavo, G. (1993). Tetanus and botulism neurotoxins: a new group of zinc proteases. Trends Biochem Sci 18, 324327.[CrossRef][Medline]
Mutoh, S., Kouguchi, H., Sagane, Y., Suzuki, T., Hasegawa, K., Watanabe, T. & Ohyama, T. (2003). Complete subunit structure of the Clostridium botulinum type D toxin complex via intermediate assembly with nontoxic components. Biochemistry 42, 1099110997.[CrossRef][Medline]
Oguma, K., Inoue, K., Fujinaga, Y., Yokota, K., Watanabe, T., Ohyama, T., Takeshi, K. & Inoue, K. (1999). Structure and function of Clostridium botulinum progenitor toxin. J Toxicol Toxin Rev 18, 1734.
Sagane, Y., Watanabe, T., Kouguchi, H., Sunagawa, H., Inoue, K., Fujinaga, Y., Oguma, K. & Ohyama, T. (1999). Dichain structure of botulinum neurotoxin: identification of cleavage sites in types C, D, and F neurotoxin molecules. J Protein Chem 18, 885892.[CrossRef][Medline]
Sagane, Y., Watanabe, T., Kouguchi, H., Sunagawa, H., Obata, S., Oguma, K. & Ohyama, T. (2002). Spontaneous nicking in the nontoxic-nonhemagglutinin component of the Clostridium botulinum toxin complex. Biochem Biophys Res Commun 292, 434440.[CrossRef][Medline]
Sakaguchi, G. (1982). Clostridium botulinum toxins. Pharmacol Ther 19, 165194.[CrossRef][Medline]
Schiavo, G., Matteoli, M. & Montecucco, C. (2000). Neurotoxins affecting neuroexocytosis. Physiol Rev 80, 717766.
Sharma, S. K., Fu, F. N. & Singh, B. R. (1999). Molecular properties of a hemagglutinin purified from type A Clostridium botulinum. J Protein Chem 18, 2938.[CrossRef][Medline]
Shone, C. C., Hambleton, P. & Melling, J. (1985). Inactivation of Clostridium botulinum type A neurotoxin by trypsin and purification of two tryptic fragments. Proteolytic action near the COOH-terminus of the heavy subunit destroys toxin-binding activity. Eur J Biochem 151, 7582.[Abstract]
Simpson, L. L. (2000). Identification of the characteristics that underlie botulinum toxin potency: implications for designing novel drugs. Biochimie 82, 943953.[CrossRef][Medline]
Sugiyama, H. (1980). Clostridium botulinum neurotoxin. Microbiol Rev 44, 419448.[Medline]
Swaminathan, S. & Eswaramoorthy, S. (2000). Crystallization and preliminary X-ray analysis of Clostridium botulinum neurotoxin type B. Acta Crystallogr D Biol Crystallogr 56, 10241026.[CrossRef][Medline]
Watanabe, T., Sagane, Y., Kouguchi, H., Sunagawa, H., Inoue, K., Fujinaga, Y., Oguma, K. & Ohyama, T. (1999). Molecular composition of progenitor toxin produced by Clostridium botulinum type C strain 6813. J Protein Chem 18, 753760.[CrossRef][Medline]
Yang, J. T., Wu, C. S. & Martinez, H. M. (1986). Calculation of protein conformation from circular dichroism. Methods Enzymol 130, 208269.[Medline]
Received 1 December 2004;
revised 28 January 2005;
accepted 1 February 2005.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |