1 Department of Food Science and Technology, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493, Japan
2 Department of Applied Biology and Chemistry, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku 156-8502, Japan
3 Hokkaido Institute of Public Health, N19, W12, Kita-Ku, Sapporo 060-0819, Japan
4 The Sars International Centre for Marine Molecular Biology, Thormøhlensgt 55, N-5008 Bergen, Norway
Correspondence
Tohru Ohyama
t-oyama{at}bioindustry.nodai.ac.jp
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ABSTRACT |
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INTRODUCTION |
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In culture supernatants and naturally contaminated foods, the NT molecules exist as part of a large complex (toxin complex; TC) through association with non-toxic non-haemagglutinin (NTNHA; 130 kDa) and haemagglutinin (HA) components. HA is composed of three subcomponents: HA-70, -33 and -17 (70, 33 and 17 kDa, respectively). The C. botulinum serotypes AD and G produce different forms of TC, the 280 kDa M-TC (complex of NT and NTNHA), the 650 kDa L-TC (complex of NT, NTNHA and HAs) and the 900 kDa LL-TC (probable 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. The NT, NTNHA and HA-70 subunits of the TC produced by serotypes A, C and D strains have 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
). Some strains of serotypes C and D produce haemagglutination-negative L-TC due to the deletion of 31 aa residues from the C-terminal region of HA-33 (Sagane et al., 2001
).
We have found that the botulinum toxin of the serotype D unique strain 4947 (D-4947) can be purified from culture supernatant as an L-TC and M-TC, which consists of only unnicked components. Using the isolated components of the D-4947 TC, we have successfully reconstituted in vitro the 650 kDa L-TC with properties that are indistinguishable from those of the parent L-TC (Kouguchi et al., 2002). Additionally, we found haemagglutination-negative TC species (M/HA-70, 540 and 610 kDa L-TC), which have a smaller number of HA-33/HA-17 molecules than those of 650 kDa L-TC and are found in the D-4947 culture supernatant. These TC species are considered to be intermediates in the 650 kDa L-TC assembly pathway. We therefore deduced that the complete subunit composition of D-4947 L-TC is a dodecamer assembled by a single NT, a single NTNHA, two HA-70 molecules, four HA-33 molecules and four HA-17 molecules (Mutoh et al., 2003
). Additionally, we have proposed a model for the arrangement of the individual components in the botulinum L-TC through tryptic susceptibility of the isolated components and complex forms (Suzuki et al., 2005
).
In the gastrointestinal tract of animals, the free NT molecule dissociated from the TC is susceptible to proteolytic and acidic conditions in the digestive environment and is easily degraded into short peptides or amino acids, similar to other proteins. On the other hand, the NTNHA protein and a part of the HA proteins are resistant to proteolytic degradation (Sakaguchi et al., 1984; Fu et al., 1998
). Thus, it is postulated that the non-toxic proteins protect the NT from the host digestive system. Additionally, the HA components have been proposed to recognize sugar chains on intestinal microvilli, thereby possibly providing efficient absorption to the intestinal wall for the botulinum TC (Fujinaga et al., 1997
, 2000
, 2004
). In this study, we examined the role of individual HA components in L-TC for recognition of erythrocytes and haemagglutination activity through the in vitro reconstitution of the HA-hybrid TC species composed of different HA-33/HA-17 isolated from serotype C and D TC strains. Based on these results, we characterized the HA-33 component responsible for haemagglutination caused by botulinum TC, which may lead to clarification of the configuration of the HA-33 molecules in the subunit structure of the botulinum TC.
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METHODS |
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Isolation of the HA-33/HA-17 complex.
Isolation of the HA-33/HA-17 subcomponent complexes from D-4947, D-CB16, C-6814 and C-Yoichi 650 kDa L-TCs were performed according to the method described previously (Kouguchi et al., 2002). A 250 mg pellet of the ammonium sulfate precipitate from the L-TC preparation was dissolved in 0·7 ml 20 mM Tris/HCl (pH 7·8) containing 4 M guanidine hydrochloride (guanidine buffer) and incubated at 21 °C for 4 h. The treated sample was applied to a HiLoad 16/60 Superdex 200 pg gel-filtration column equilibrated with guanidine buffer. The fraction containing HA-33/HA-17 was pooled and diluted to an A280 of 0·1 with guanidine buffer. After dialysis against 20 mM Tris/HCl (pH 7·8) at 4 °C, the fraction containing HA-33/HA-17 was concentrated using Vivapore 10 (Sartorius).
Reconstitution of HA-hybrid L-TC with isolated HA-33/HA-17 from each strain.
For hybridization of L-TCs containing different types of HA-33/HA-17, purified 610 and 540 kDa L-TC from four strains (D-4947, D-CB16, C-6814 and C-Yoichi) and isolated HA-33/HA-17 were mixed at a 1 : 5 molar ratio in phosphate buffer (pH 6·0) containing 0·15 M NaCl. After incubation at room temperature for 10 min, the mixture was applied to a Superdex 200 HR 10/30 gel-filtration column (Amersham Biosciences) equilibrated with the same buffer to remove unbound HA-33/HA-17. The resultant samples were examined by native PAGE.
Haemagglutination assay.
Haemagglutination assays were performed using U-bottom microtitre plates. Samples (35 µl) of each preparation (0·25 µM) were diluted in serial twofold steps with 0·15 M phosphate buffer (pH 7·0) and mixed with an equal volume of 1 % (v/v) equine erythrocytes. After incubation at room temperature for 2 h, the reciprocal haemagglutination titre was determined as 2n.
Western blot erythrocyte binding assay.
The binding of TC species, HA-hybrid TC and isolated HA subcomponents to equine erythrocytes was carried out according to the method described by Inoue et al. (1999) with some modifications. A 60 µl sample of the TC species (0·25 µM) was incubated with 20 µl of a 1 % suspension of equine erythrocytes at room temperature for 30 min, followed by collection of the erythrocytes by centrifugation. The pellets were subjected to SDS-PAGE. The separated bands bound to erythrocytes were blotted to a polyvinylidene difluoride membrane and were probed with rabbit anti-HA of type C toxin complex IgG. The bound antibodies were detected with anti-rabbit IgG-HRP conjugate using a POD immunostaining set (Wako).
PAGE and densitometric analyses.
PAGE under non-denaturing conditions (native PAGE) was carried out at pH 8·8 using a 512·5 % polyacrylamide linear gradient gel, according to the method of Davis (1964). SDS-PAGE was performed as described by Laemmli (1970)
using a 13·6 or 15 % polyacrylamide gel in the presence of 1 % 2-mercaptoethanol. 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 components were analysed with NIH image software, version 1.62 (http://rsb.info.nih.gov/nih-image/). The intensity of the protein band was determined by measuring the area of the peak. The protein concentration was determined by using a BCA protein assay kit (Pierce) with BSA as the standard.
Analytical gel filtration.
A Superdex 200 HR 10/30 gel-filtration column was equilibrated with 50 mM phosphate buffer (pH 6·0) containing 0·15 M NaCl. The molecular masses of the complexes were estimated from calculations with standard proteins aldolase (158 kDa), BSA (67 kDa), egg ovalbumin (43 kDa), chymotrypsinogen A (25 kDa) and RNase A (13·8 kDa). The molecular masses were estimated from the mean of the three experiments.
Isoelectric focusing.
Isoelectric focusing under native conditions was carried out using Immobiline DryStrip (pH 3·010·0, 7 cm; Amersham Bioscience). The sample (30 µg) in 50 mM Tris/HCl buffer (pH 7·4) was applied to a strip holder, then run on an Etten IPGphor II (Amersham Bioscience) electrophoresis apparatus. The separated protein bands were stained with CBB. The isoelectric point (pI) value was estimated by calculations with the Broad pI kit (Amersham Bioscience).
Far-UV circular dichroism (CD) spectroscopy.
The secondary structure of the HA-33/HA-17 complex was analysed using a far-UV CD spectropolarimeter (model J-720WI, JASCO). CD spectra were recorded using a 10 mm path length cuvette at room temperature. The sample (450 µg ml1) was dialysed against 50 mM Tris/HCl buffer (pH 7·4). The secondary structure content was estimated by the methods of Yang et al. (1986) and Manavalan & Johnson (1987)
using software provided with the instrument.
Modelling of the C-Yoichi and D-4947 HA-33 structure.
Structure models of C-Yoichi and D-4947 HA-33 were constructed with the SWISS-MODEL Protein Homology modelling Server (http://swissmodel.expasy.org//SWISS-MODEL.html) (Guex & Peitsch, 1997) using the coordinates of serotype C HA-33 (Protein Data Bank code 1QXM). The images were generated using the Molsoft ICM Browser (available at www.molsoft.com) for displaying stereo ribbon diagrams of the crystal structure of C-Yoichi and D-4947 HA-33 molecules. The models exhibit good geometry according to Ramachandran plots generated using Swiss-PdbViewer (available at http://ca.expasy.org/spdbv/) with 90·2, 88·0 and 87·0 % of the residues in the most favoured regions for HA-33 molecules of D-4947, C-Yoichi and truncated C-Yoichi, respectively.
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RESULTS AND DISCUSSION |
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To characterize the molecular composition of HA-33/HA-17 in each L-TC species, the ratios of the band-staining intensities of HA-33/NT and HA-33/NTNHA on SDS-PAGE in each L-TC species were examined. As summarized in Table 1, ratios of HA-33/NT in D-4947 and HA-33/NTNHA in D-CB16, C-6814 and C-Yoichi were roughly estimated to be 2 : 3 : 4, stepwise, with the 540 kDa L-TC, the 610 kDa L-TC and the 650 kDa L-TC, respectively, as observed in D-4947 L-TCs. Accordingly, we tentatively designated the 540 kDa L-TC as L-TC2 (NT/NTNHA/2.HA-70/2.HA-33/2.HA-17), the 610 kDa L-TC as L-TC3 (NT/NTNHA/2.HA-70/3.HA-33/3.HA-17) and 650 kDa L-TC as just L-TC (complete form containing four HA-33/HA-17 molecules), in the respective strains used in this study. There was no L-TC1 (probably around 500 kDa) corresponding to the L-TC species composed of only one HA-33/HA-17 molecule in the culture supernatants of every strain. However, since a small amount of 410 kDa L-TC0 corresponding to the NT/NTNHA/2.HA-70 complex was found (data not shown), L-TC1 is assumed to immediately convert to L-TC2 or L-TC0 as stable forms in culture supernatants.
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Molecular properties of isolated HA-33/HA-17
Gel filtration elution patterns of the HA-33/HA-17 complexes purified from the L-TC of four strains showed a single peak, as illustrated in Fig. 3(a). The molecular sizes of the isolated HA-33/HA-17 of each strain were estimated to be approximately 40 kDa (but 36 kDa for C-Yoichi) by their elution volumes from gel filtration with 50 mM phosphate buffer (pH 6·0) and 0·15 M NaCl. This molecular size was lower than the calculated mass of 50 471 Da. An apparently lower molecular mass might result from differences in hydrodynamic properties, such as shape and/or hydrophobic interaction between the HA-33/HA-17 complex and the Superdex 200 gel matrix. Thus, every HA-33/HA-17 complex might be present as a monomer complex with a 1 : 1 molecular ratio (Suzuki et al., 2005
). Thus, we obtained pure HA-33/HA-17 complexes from each L-TC, although HA-33 and HA-17 could not be separated from the complex as viable forms due to irreversible precipitation during dialysis. Previously, we demonstrated that the HA-33/HA-17 complex is able to bind to the M-TC/HA-70 complex, but free HA-33 is not. Thus, this observation suggested indirectly that the role of the HA-17 molecule is to adhere to HA-70 and form a linkage between HA-33 and HA-70 (Mutoh et al., 2003
).
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As shown in Fig. 3(c), the isolated HA-33/HA-17s from four strains were purified to homogeneity by native PAGE. However, their gel mobilities were different from each other. To characterize the ionization state of HA-33/HA-17, the pI values were examined by the native isoelectric focusing method. As shown in Table 2
, the pI values of the HA-33/HA-17 complexes from D-4947 and C-6814 were estimated to be neutral, at 6·8 and 7·3, respectively. On the other hand, those of D-CB16 and C-Yoichi were estimated to be alkaline, at 8·0 and 8·4, respectively. Except for the unique results with C-Yoichi HA-33/HA-17, these data seem to reflect the different banding patterns on native PAGE, as shown in Fig. 3(c)
. Although the pI values of the complex form cannot be predicted by protein analysis software, the pI values of each complex seemed intermediary between the predicted alkaline pI values (7·119·07) of the HA-33 molecule rather than the acidic pI values (5·046·49) of the HA-17 molecule. However, only HA-33 among the five components of the botulinum TC was predicted to be a basic protein, while the remaining components were predicted to be acidic, suggesting that the overall TC complex forms will be acidic (Hasegawa et al., 2004
).
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Binding activities of the L-TC species to equine erythrocytes were also analysed by immunoblotting bands, separated by SDS-PAGE, from washed erythrocytes after incubation. As summarized in Table 5, both haemagglutination-negative L-TC2 and L-TC3 species from D-4947, D-CB16 and C-6814 could bind to erythrocytes. Accordingly, the HA-hybrid L-TCs of these strains reconstituted with HA-33/HA-17 molecules from four strains containing C-Yoichi bound to erythrocytes. On the other hand, only when C-Yoichi L-TC2 and L-TC3 were repaired with active HA-33/HA-17 complexes from the other three strains did binding activity appear. However, C-Yoichi L-TC occupied with four truncated HA-33 molecules no longer exhibited binding activity, even if an active HA-33/HA-17 complex was added. Contrary to haemagglutination, it is likely that botulinum TC species containing at least one complete HA-33 molecule can bind to erythrocytes. These results strongly suggest that the lack of the C-terminal region of the HA-33 molecule plays a definite role in the botulinum TC functions of haemagglutination and erythrocyte binding. Kouguchi et al. (2001)
demonstrated that the isolated serotype C HA subcomponents, HA-70, HA-33 and the HA-33/HA-17 complex, bound to erythrocytes, but that each single component showed no aggregation of erythrocytes. They also concluded that a combination of at least HA-33, HA-17 and HA-70 was required for full haemagglutination activity of the botulinum L-TC.
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Molecular modelling of the C-terminal truncated HA-33 of C-Yoichi
Since the X-ray crystallographic analyses of serotypes C and A have been elucidated, the complete HA-33 structure is available (Inoue et al., 2003; Arndt et al., 2005
), thus enabling our efforts in molecular modelling of the C-terminal truncated HA-33 of C-Yoichi and D-4947 using the serotype C HA-33 structure as the modelling template. Both serotype C and A HA-33 models have a dumbbell-shaped structure (70x40x37 Å) with two
-trefoil domains connected by a short
-helix. The HA-33s of serotypes A to D are well known to be members of a carbohydrate recognition protein family, as shown by search results from a characteristic
-trefoil fold previously described in many lectins (Hazes, 1996
; Rutenber et al., 1987
; Transue et al., 1997
). Indeed, the HA-33s of serotype C and D strains employed here were also shown to have two
-trefoil domains, suggesting HA activity and binding activity to the erythrocytes through recognition of N-acetylneuraminic acid or sialylglycolipid (Inoue et al., 1996
). According to the profile created using the hidden Markov model method with Pfam software (Bateman et al., 2000
), one of the putative carbohydrate-binding sites is located in the C-terminal domain. HA-33 molecules commonly consist of a single polypeptide chain of approximately 290 residues across the serotype. However, the amino acid sequence of C-Yoichi HA-33 deduced from the nucleotide sequence was identical to that of the consensus from the N terminus up to residue 145, but showed 46 % aa identity with 76 residue substitutions in the remaining C-terminal region (residues 146285). Despite low sequence identity in this region of the C terminus, Fig. 6
(b) demonstrates a normal structure consisting of two
-trefoil domains that are highly similar to each other.
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ACKNOWLEDGEMENTS |
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Received 6 July 2005;
revised 27 September 2005;
accepted 7 October 2005.
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