1 Department of Bacteriology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
2 PRESTO, JST, 4-1-8 Honcho Kawaguchi, Saitama, Japan
3 Laboratory of Veterinary Immunology, Department of Veterinary Science, College of Agriculture, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan
4 Department of Food Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2422, Japan
5 Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan
6 CREST, JST, 4-1-8 Honcho Kawaguchi, Saitama, Japan
Correspondence
Keiji Oguma
kuma{at}md.okayama-u.ac.jp
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ABSTRACT |
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INTRODUCTION |
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The NTX blocks, specifically and with high potency, the release of acetylcholine from the peripheral nerve terminus. This poisoning mechanism was characterized as the cleavage of the proteins involved in the fundamental process of exocytosis (reviewed by Jahn & Niemann, 1994; Montecucco & Schiavo, 1994
; Schiavo et al., 2000
). Human and animal food-borne botulism are caused by ingestion of food or feed containing the progenitor toxin. The orally ingested progenitor toxin is absorbed from the upper small intestine into the lymphatic system (Sakaguchi et al., 1984
), then enters the bloodstream, and reaches peripheral nerves, where neurological dysfunction is elicited.
It has been reported that the progenitor toxin possesses greater potential for oral toxicity than does NTX alone, since the former is more resistant to low pH and proteases in the digestive tract than the latter (Sakaguchi et al., 1984). Thus the nontoxic components are presumed to have the role of protecting the neurotoxin against acidity and proteases in the digestive tract. In addition, we showed that HA functions as an adhesin in the attachment of 16S and 19S toxins to the microvilli of the upper small intestine. (Fujinaga et al., 1997
, 2000
). In type A, HA1 and HA3b are the binding subcomponents; HA1 recognizes galactose like HA-positive progenitor toxin (16S and 19S toxins), whereas HA3b recognizes sialic acid (Fujinaga et al., 2000
). On the other hand, type C 16S toxin recognizes sialic acid (Fujinaga et al., 1997
. Inoue et al., 1999
). In this study, we produced all of the type C HA-subcomponents, including some of their precursor forms, as proteins fused with glutathione S-transferase (GST), to identify and characterize the type C HA-subcomponents exhibiting binding activity toward intestinal epithelial cells and erythrocytes.
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METHODS |
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GST-free HA-subcomponents were prepared by Factor Xa protease treatment of the GST-fusion proteins as described previously (Fujinaga et al., 2000).
Purification of 16S toxin.
The type C 16S toxin was purified from the culture fluid of C. botulinum type C strain Stockholm (C-ST) according to the procedure employed for purifying type A toxins (Inoue et al., 1996). Briefly, the toxin in the culture fluid was precipitated by 50 % saturation of ammonium sulfate. The precipitate was dialysed against 50 mM sodium acetate buffer (pH 4·2), and then applied on to an SP-Toyopearl 650M (Tosoh) column. The 16S toxin separated by this column chromatographic procedure was further purified by gel filtration on a Sephacryl S-300 (Amersham) column.
SDS-PAGE and immunoblot analysis.
SDS-PAGE was performed by the method of Laemmli (1970), using a 4 % stacking gel and a 12·5 % separating gel under reducing conditions. Proteins were stained with Coomassie brilliant blue R-250 (Merck). For immunoblot analysis, proteins were electrotransferred and reacted with 104-fold-diluted rabbit polyclonal anti-type C 16S toxin serum (Oguma et al., 1980
), and then reactive bands were visualized using the ECL detection system (Amersham).
Binding assay with intestinal tissue sections.
The binding of the 16S toxins and recombinant proteins to paraformaldehyde-fixed sections of guinea pig upper small intestine was assayed as described previously (Fujinaga et al., 2000).
Binding assay with erythrocytes.
The binding of the 16S toxins and recombinant proteins to human erythrocytes (type O) was assayed as described previously (Hoschützky et al., 1989; Fujinaga et al., 2000
).
Neuraminidase treatment of erythrocytes.
Human type O erythrocytes (10 % suspension in PBS) were treated with Arthrobacter ureafaciens neuraminidase (a highly purified preparation containing no protease, N-acetylneuraminic acid aldolase or glycosidase; Nacalai Tesque) at a final concentration of 0·01 unit ml1, at 37 °C for 1 h. The erythrocytes were then washed twice with PBS and suspended in PBS (50 % neuraminidase-treated erythrocytes).
Preparation of glycosphingolipid (GSL) fractions from erythrocyte membranes.
Packed erythrocytes (human, type A) were lysed in 10 vols 0·5 % acetic acid, and then the stroma was collected and washed five times with water by centrifugation at 2600 g for 20 min. Lipids were extracted sequentially from the stroma with 20 vols each of chloroform/methanol (successively 2 : 1, 1 : 1, and 1 : 2, v/v), and chloroform/methanol/water (30 : 60 : 8, by vol.) at room temperature overnight. The lipid extracts were combined and evaporated to dryness in vacuo, then the residue was dissolved in chloroform/methanol (1 : 1, v/v). The redissolved extracts were subjected to mild alkaline hydrolysis by addition of 1 M sodium methylate (Wako Pure Chemical) at room temperature for 3 h. The solution was neutralized with acetic acid, and then dialysed against distilled water and evaporated. The GSL fraction was dissolved in a minimum amount of a mixture of chloroform/methanol/water (30 : 60 : 8, by vol.), and applied to a DEAE-Sephadex A-25 column (acetate form) using the method of Ledeen et al. (1973). The neutral GSL fraction was eluted with chloroform/methanol/water (30 : 60 : 8, by vol.) and dried on a rotary evaporator. The ganglioside fraction was eluted with chloroform/methanol/0·8 M sodium acetate (30 : 60 : 8, by vol.), dialysed against distilled water, and evaporated to dryness in vacuo. Standard GSLs were as follows. Lactosylceramide (LacCer), GM3 and sialosylparagloboside (SPG) were purified from human erythrocytes (Hakomori & Siddiqui, 1974
; Ledeen et al., 1982
). Paragloboside (PG) was isolated from bovine erythrocytes (Uemura et al., 1978
). The structures of these GSLs are shown in Table 2
.
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Determination of protein concentration.
Protein concentration was determined using the Lowry method with BSA as a standard.
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RESULTS |
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DISCUSSION |
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In the TLC binding assay, GST-HA3b and GST-HA3 were reactive to GM3 and SPG, which contain NeuAc2-3Gal
1-4Glc
1- and NeuAc
2-3Gal
1-4GlcNAc
1-3Gal
1-4Glc
1- residues, respectively, but not with the neutral GSL fraction, LacCer (an asialo- derivative of GM3) and PG (an asialo- derivative of SPG). We reported previously that type C 16S toxin exhibits the same ligand specificity toward these GSLs (Inoue et al., 1999
). These findings indicate that the epitope recognized by GST-HA3b, GST-HA3 and 16S toxin may be the terminal (at least) disaccharide, NeuAc
2-3Gal
1-, sequence of glycoconjugates, and that NeuAc is crucial for the binding of GST-HA3b, GST-HA3 and 16S toxin to guinea pig intestinal epithelial cells and human erythrocytes. GST-HA1 recognized GM3 and SPG, but not the neutral GSL fraction or LacCer, like GST-HA3b and GST-HA3. This protein, however, could bind to PG. LacCer and PG have a Gal
1-4Glc
1- structure and Gal
1-4 GlcNAc
1-3Gal
1-4Glc
1- structure, respectively. These results suggest that GST-HA1 recognizes the terminal (at least) disaccharide (NeuAc
2-3Gal
1-) structures in GM3 and SPG and the terminal (at least) disaccharide (Gal
1-4GlcNAc
1-) sequence of PG. In the present study, the binding of GST-HA1 to guinea pig intestinal epithelial cells and human erythrocytes was not stopped by neuraminidase treatment. Thus, GST-HA1 recognized the Gal
1-4GlcNAc
1- structure which is the terminal disaccharide sequence of PG (asialo- derivative of SPG) as a receptor. In addition, binding to neuraminidase-treated erythrocytes was inhibited by the addition of Gal, GalNAc or Lac, suggesting that the Gal moiety of the Gal
1-4GlcNAc
1- residue is important for the binding of GST-HA1 to the neuraminidase-treated erythrocytes.
The binding assays with guinea pig intestinal tissue sections, with human erythrocytes, and with GSLs extracted from erythrocytes showed that GST-HA3 and GST-HA3b have the same ligand specificity as 16S toxin. It is suggested, therefore, that HA3b in the 16S toxin contributes directly to the binding of these ligands. In contrast, GST-HA1 showed a different ligand specificity to 16S toxin: GST-HA1 recognizes the galactose moiety in addition to the sialic acid moiety. At this time, we have no experimental data to explain why the ligand specificity of HA1 is different from that of 16S toxin. We speculate that the binding of HA1 to the galactose moiety is disrupted by the tertiary structure of the 16S toxin. Sagane et al. (2001) surmised that HA1 in the 16S toxin has an essential role in the binding of 16S toxin to erythrocytes based on the analyses of 16S toxins that have truncated HA1 toxins produced by type D strain 1873 and type C strain Yoichi. In agreement with this idea, our previous data showed that monoclonal antibodies against type C HA1 inhibit the binding of 16S toxin to intestinal epithelial cells and erythrocytes (Mahmut et al., 2002
). These studies may indicate that HA1 in 16S toxin also interacts directly with cellular ligands. Further study of the crystal structure and/or reconstitution of 16S toxin containing mutated HA-subcomponents will be required to understand the role of HA1 and HA3b in the binding activity of 16S toxin.
Analyses of deletion mutants of type C HA3b showed that the removal of 13 amino acids from the carboxy-terminus completely abolished the binding activity. This result indicates that the carboxy-terminal region of type C HA3b is important for binding. In the carboxy-terminal region, we found a sequence that has significant similarity with that of the carbohydrate recognition domain of sialic-acid-binding immunoglobulin-like lectins (Siglecs) (Angata & Brinkman-Van der Linden, 2002), as illustrated in Fig. 7
. A conserved arginine residue among Siglecs, which is essential for sialic acid binding (Angata & Brinkman-Van der Linden, 2002
), also exists in the sequence of HA3b. Presumably, the 13 amino acid deletion at the carboxy-terminus disrupts this domain in type C HA3b and causes the loss of binding activity. Site directed mutagenesis of the conserved arginine residue is in progress to further characterize this domain in HA3b.
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Maksymowych & Simpson (1998) reported that type A and B botulinum neurotoxins alone bound to human intestinal cell lines and entered the basolateral compartment via transcytosis. In the case of type C, 16S toxin binds strongly to intestinal epithelial cells of guinea pigs, leading to an efficient absorption of the toxin, whereas the neurotoxin alone and 12S hardly bind to the cells, resulting in a much less efficient absorption (Fujinaga et al., 1997
). This means that the HA has a crucial role in the intestinal binding and absorption of the neurotoxin and/or neurotoxin complexes at least in type C botulism. It is worth investigating which glycoconjugate(s) are the intestinal receptor(s) for the HA. In the binding assay with guinea pig intestinal tissue sections, protease treatment of the sections greatly reduced the binding of native type C 16S, GST-HA3, GST-HA3b and GST-HA1 (data not shown). This result may indicate that these proteins recognize glycoproteins rather than glycolipids on the intestinal cells. Further study is needed to identify the intestinal receptor(s) for HA.
In conclusion, our study found that HA of type C 16S toxin has two distinct binding subcomponents, HA1 and HA3b, which recognize carbohydrates with different specificities. The differences in receptor binding between HA1 and HA3b lead us to speculate that a multi-step dependent activation of 16S toxin binding is needed for the toxin to bind and pass through the barrier of intestinal epithelial cells. This study provides fundamentally important information on the interaction of the 16S toxin with intestinal epithelial cells.
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ACKNOWLEDGEMENTS |
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Received 7 October 2003;
revised 12 January 2004;
accepted 14 January 2004.
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