Department of Bacteriology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan1
Department of Molecular Medicine, Research Institute, Osaka Medical Center for Maternal and Child Health, 840 Murodo-cho, Izumi, Osaka 594-1101, Japan2
Hokkaido Institute of Public Health, N19, W12, Sapporo 060-0819, Japan3
Department of Food Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196, Yasaka, Abashiri 099-2422, Japan4
Author for correspondence: Keiji Oguma. Tel: +81 86 235 7162. Fax: +81 86 235 7162. e-mail: kuma{at}med.okayama-u.ac.jp
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ABSTRACT |
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Keywords: Clostridium botulinum, haemagglutinin, glycolipid, glycoprotein
Abbreviations: CBB, Coomassie brilliant blue; HA, haemagglutinin; MLD, minimal lethal dose
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INTRODUCTION |
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We purified different-sized progenitor toxins from C. botulinum type A and C culture fluids, and demonstrated that the HAs consist of subcomponents that have molecular masses of 5253, 3335, 1923 and 1517 kDa, which are designated here HA3b, HA1, HA3a and HA2, respectively (Fujinaga et al., 1994 ; Inoue et al., 1996
). In the case of the type A strain, the HA and its one subcomponent protein, which had a molecular mass of 35 kDa (HA1), were purified in addition to the progenitor toxins. This indicated that HA and HA1 exist in a free state in the culture fluid. The free HA showed the same HA activity as the HA-positive toxins (16S and 19S toxins), whereas the free HA1 did not show HA activity (Inoue et al., 1996
).
Experimental results obtained using the Ouchterlony test suggest that the antigenicity of the non-toxic components of types C and D is identical (Oguma et al., 1980 ). This hypothesis was confirmed by a comparison of the entire nucleotide sequence of the non-toxic components, which indicated that the two sequences were essentially identical (Fujinaga et al., 1994
; Hauser et al., 1994
; Ohyama et al., 1995
;Tsuzuki et al., 1990
, 1992
). The HA activity of type C and D cultures were investigated by Suzuki et al. (1986
) and Balding et al. (1973
) by employing crude HA or 16S toxin preparations. Suzuki et al. (1986
) reported that the HA activity of type C crude HA was inhibited by adding gangliosides and fetuin to the reaction mixture, and also reduced by employing erythrocytes which had been treated with neuraminidase. Balding et al. (1973
) reported that the HA activities of type C and D toxins were inhibited in the presence of MN active glycoprotein, as well as employing the neuraminidase-treated erythrocytes. Therefore, it is postulated that the sugar (N-acetylneuraminic acid; NeuAc) on the surface of erythrocytes is most likely the receptor for type C and D HA binding. However, it has yet to be established that purified erythrocyte glycolipids and glycoproteins will bind to HAs.
In this paper, we established a new and simple method for purifying type D 16S toxin. It was also possible to purify free HA1 using the new purification method. We performed haemagglutination and haemagglutination-inhibition tests, and the direct-binding tests, by employing purified type C and D 16S toxins and type D free HA1, in order to determine how they bind to erythrocytes.
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METHODS |
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SDS-PAGE and electroblotting.
SDS-PAGE was performed by the method of Laemmli (1970 ) using 12·5% acrylamide linear gel. Protein bands were stained with Coomassie brilliant blue (CBB) R-250. The bands separated by SDS-PAGE were electroblotted to PVDF membranes (ProBlott; Applied Biosystems) with a semidry blotting apparatus (Nippon Eido) according to the methods described by Hirano & Watanabe (1990
).
Preparation of antisera and immunoblotting.
Preparation of antisera against type C non-toxic components (a complex of non-toxic non-HA and HA) that was prepared previously (Oguma et al., 1980 ) in a rabbit was used. The protein bands separated by SDS-PAGE were electroblotted to a PVDF membrane as described above. The membrane was immersed overnight in PBS (pH 7·4) containing 10% (w/v) skimmed milk (blocking buffer 1). The membrane was incubated for 1 h with 1:1000-diluted anti-type C non-toxic components serum in blocking buffer 1 and then washed in PBS (pH 7·4) containing 0·05% (v/v) Tween 20 (PBS-Tween). After incubation with 1:1000-diluted peroxidase-labelled pig anti-rabbit IgG (Dako A/S) in blocking buffer 1 for 1 h, immunoreactive bands were detected by the ECL Western blotting system (Amersham).
Enzyme treatment of human erythrocytes.
Ten millilitres of 10% (v/v) washed erythrocytes were incubated with 0·01 unit Arthrobacter ureafaciens neuraminidase (Nacalai tesque), 80 mg trypsin (Difco), 80 mg papain (Wako Pure Chemical Industries), 8 mg proteinase K (Wako Pure Chemical Industries), or 100 mU endoglycoceramidase (EGCase II ACT, Takara Shuzo) in PBS (pH 6·0) at 37 °C for 1 h. After treatment, the erythrocytes were washed three times in PBS (pH 6·0).
Determination of HA titre and inhibition tests.
The HA titre was obtained by microtitration methods using multiwell plates. Type C and D 16S toxins or HA1 solution (100 µg ml-1, 35 µl) were diluted in serial twofold steps with PBS (pH 6·0) and mixed with an equal volume of 1% (v/v) suspension of washed human erythrocytes or enzyme-treated erythrocytes. After incubation at room temperature for 2 h, the reciprocal of the highest dilution that showed haemagglutination was used as the HA titre. All tests were performed in duplicate and repeated three times. Inhibition of HA activity of the type C and D 16S toxins with several different saccharides were examined as follows. Each diluted preparation (20 µl) was mixed with 20 µl of an appropriate concentration of saccharide solutions and incubated at 37 °C for 1 h. Thereafter, 40 µl 1% (v/v) suspension of erythrocytes was added to the mixtures. Following incubation at room temperature for 2 h, haemagglutination was assessed. All tests were performed in duplicate and repeated twice.
Binding to erythrocytes.
The binding of 16S toxins and HA1 to erythrocytes were analysed by two different methods. The first method was conducted using 96-well microtitre plates. Fifty microlitres of 16S toxin (50 µg ml-1) or HA1 (50 µg ml-1) were incubated in microtitration plates, and left to stand overnight at 4 °C. The plates were washed three times with 200 µl PBS (pH 6·0), and 150 µl 1% (w/v) BSA in PBS (pH 6·0) was added to the plates. After 2 h incubation at room temperature, 100 µl 1% (v/v) native or neuraminidase-treated erythrocytes in PBS (pH 6·0) containing 1% (w/v) BSA was added to the plates. For the inhibition tests, 100 µl 1% (v/v) native or neuraminidase-treated erythrocytes in PBS (pH 6·0) containing 1% (w/v) BSA and 100 mM monosaccharide were added to the plates. Plates were incubated for 30 min at room temperature and washed with 200 µl PBS (pH 6·0) six or seven times. Bound erythrocytes were lysed by adding 50 µl distilled water and the A405 was analysed. All tests were performed in duplicate and repeated three times. The second method used to assess the binding of the 16S toxin and HA1 to erythrocytes was performed using an anti-type C non-toxic components serum. Twenty microlitres of 16S toxin (20 µg ml-1) or HA1 (10 µg ml-1) were mixed with 20 µl PBS (pH 6·0), 20 µl 4% (w/v) BSA in PBS (pH 6·0) and 20 µl 1% (v/v) native or neuraminidase-treated erythrocytes. After incubation at 37 °C for 1 h, the erythrocytes were precipitated by centrifugation and washed three times with 150 µl PBS, pH 6·0. Precipitated erythrocytes were mixed with 10 µl SDS-PAGE sample buffer and heated at 100 °C for 5 min. Electrophoresis was performed on a 12·5% polyacrylamide linear gel. After the protein bands were electroblotted to a PVDF membrane, 16S toxins or HA1 bound to erythrocytes were detected by immunostaining with anti-type C non-toxic components serum. For the inhibition tests, 20 µl 16S toxins (20 µg ml-1) were mixed with 20 µl monosaccharide (400 mM) solution, 20 µl 4% (w/v) BSA in PBS (pH 6·0). After incubation at 37 °C for 1 h, 20 µl 1% (v/v) native or neuraminidase-treated erythrocytes were added to the reaction mixture, and the binding to the erythrocytes was analysed using the methods described above.
Preparation of glycolipids from erythrocytes.
Human blood (type A, 500 ml) was centrifuged and the serum was discarded. The erythrocytes were washed twice in 0·9% (w/v) NaCl. The washed cells were lysed by adding 1500 ml distilled water and centrifuged to sediment ghosts. The wet ghosts were suspended in distilled water and the ratio of chloroform/methanol/water was adjusted to 30:60:8 (by vol.) by the addition of chloroform and methanol. The lipid contents were extracted by sonication for 30 min. The solution was filtered through filter paper and evaporated. The lipids were dissolved in a minimum amount of chloroform/methanol/water (30:60:8, by vol., as above) and applied to a DEAE-Sephadex A-25 column (acetate form, Pharmacia Biotechnology) equilibrated with chloroform/methanol/water (30:60:8, by vol.). The neutral glycolipids were eluted with chloroform/methanol/water (30:60:8, by vol.). The monosialo-, disialo- and polysialoglycolipids were eluted with chloroform/methanol/water (30:60:8, by vol.) containing 40 mM, 100 mM and 300 mM ammonium acetate, respectively. Each evaporated fraction was dissolved in methanolwater (1:1, v/v) and applied to Sep Pak 18C (Waters). After washing the column with distilled water, the desalted glycolipids were eluted with methanol and a mixture of chloroform/methanol (2:1, v/v), and evaporated.
TLC immunostaining.
Binding of 16S toxins and free HA1 to the glycolipids prepared from erythrocytes or the glycolipids purified as described above were analysed by TLC immunostaining. The glycolipids were developed on high-performance TLC aluminium sheets silica gel 60 plates (Merk) with chloroform/methanol/water (65:35:8, by vol.). The developed plates were dipped in 0·2% (w/v) polyisobutyl-methacrylate in n-hexan for 1 min and dried. The plates were incubated in PBS (pH 6·0) containing 1% (w/v) BSA (blocking buffer 2) for 1 h, followed by incubation with 5 µg ml-116S toxin or 10 µg ml-1 HA1 solution (in blocking buffer 2) for 1 h. The plates were then washed three times with PBSTween and reacted for 1 h with antiserum against type C non-toxic components diluted to 1:1000 with blocking buffer 2. After washing three times with PBSTween, the plates were incubated with a peroxidase-labelled anti-rabbit IgG antibody diluted to 1:1000 with blocking buffer 2 for 1 h. The immunoreactive bands that appeared were detected by ECL (Amersham). All of the procedures were performed at room temperature.
Binding to ghost membrane proteins or glycoproteins.
We analysed the binding of 16S toxins and free HA1 to ghost membrane proteins and glycoproteins. The latter were obtained commercially. The PVDF membranes blotted with proteins were immersed overnight in PBS (pH 6·0) containing 10% (w/v) BSA (blocking buffer 3) at 4 °C. The membranes were incubated in 5 µg ml-1 16S toxin or 10 µg ml-1 HA1 solution (in blocking buffer 3) for 1 h and then washed three times with PBSTween. After the membranes were reacted with antiserum against type C non-toxic components diluted to 1:1000 with blocking buffer 3 for 1 h, followed by a peroxidase-labelled anti-rabbit IgG antibody diluted to 1:1000 for 1 h, the immunoreactive bands were detected by ECL (Amersham).
Determination of amino acid sequence.
The N-terminal amino acid sequences of protein bands of ghost proteins which were bound by type C and D 16S toxins were determined. The protein bands electroblotted to a PVDF membrane were stained with CBB R-250, cut out and sequenced with a pulsed-liquid phase protein sequencer (model 477-A; Applied Biosystems).
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RESULTS |
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Haemagglutination and haemagglutination-inhibition tests
The minimum concentration of type C and D 16S toxins to give haemagglutination was determined by using enzyme-treated and non-treated (native) human erythrocytes (Table 1). The native erythrocytes were agglutinated by 0·05 µg type C and D 16S toxins ml-1, whereas the purified HA1 did not agglutinate the native erythrocytes even though 100 µg ml-1 was used. When the erythrocytes that had previously been treated with neuraminidase (37 °C for 1 h) were used, 2550 µg type C and D 16S toxins ml-1 were needed to cause haemagglutination. No haemagglutination was observed in the erythrocytes that had received a prolonged incubation (23 h) with neuraminidase. The HA titre of type C and D 16S toxins was markedly reduced by employing the erythrocytes that had been treated with papain or proteinase K. Treatment of erythrocytes with trypsin also reduced HA titre of type C and D 16S toxins. However, the effect of the trypsin treatment was small when compared with those of the papain and proteinase K treatments. Endoglycoceramidase treatment of erythrocytes did not affect the HA titre of type C and D 16S toxins (data not shown).
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As shown in Fig. 6, type C and D 16S toxins mainly bound to two bands in the monosialoglycolipid fraction extracted from human erythrocytes (Fig. 6b
, c, lane 2). Purified HA1 also bound to these bands in the monosialoglycolipid fraction (Fig. 7
, lane 2). It is now well-established that GM3 and SPG constitute the major gangliosides found in the membranes of human erythrocytes (Watanabe et al., 1979
). The mobility of two main bands bound by 16S toxins and HA1 was identical to those of GM3 and SPG, respectively.
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DISCUSSION |
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Type C and D 16S toxins agglutinated human erythrocytes. Previously, Suzuki et al. (1986 ) and Balding et al. (1973
) proposed that the NeuAc on the surface of erythrocytes may act as the receptor for types C and D HAs on the basis of haemagglutination and haemagglutination-inhibition tests with crude preparations of types C and D HA or crude type C 16S toxin. They found that haemagglutination titre was reduced by employing erythrocytes pretreated with neuraminidase. In addition, Suzuki et al. (1986
) reported that the type C HA activity was inhibited by adding gangliosides and fetuin to the reaction mixture. Balding et al. (1973
) reported that both type C and type D HA activities were inhibited by adding MN active glycoprotein isolated from human red blood cells, indicating that not only glycolipids, but also glycoproteins containing NeuAc, were potential receptors for HAs. In the present study, we confirm these predictions on the basis of the following tests and their inhibition tests with purified type C and D 16S toxins and type D HA1. The experiments examined haemagglutination activity, binding to erythrocytes, and binding to the glycolipids and glycoproteins prepared from erythrocytes. The HA activities of these toxins were reduced by employing the neuraminidase-pretreated erythrocytes. Furthermore, the HA titres of type C and D 16S toxins were reduced by adding NeuAc to the reaction mixtures (Fig. 4
). Balding et al. (1973
) reported that 20 mM NeuAc did not inhibit the HA activities of the crude type C progenitor toxin and of the crude non-toxic components of type D. In the present study, we demonstrate that the presence of NeuAc at concentrations greater than 20 mM dramatically inhibits the haemagglutination caused by both type C and D 16S toxins (Fig. 4
). HA titres of type C and D 16S toxins were reduced by the treatment of erythrocytes with proteases (Table 1
), but not reduced by endoglycoceramidase treatment. These results suggest that glycoproteins, rather than glycolipids, are critical to the binding of type C and D 16S toxins to the surface of erythrocytes that is associated with haemagglutination. Previously, Balding et al. (1973
) reported that the HA activity of type C crude progenitor toxin was inhibited by the treatment of erythrocytes with papain, while the HA activity of the non-toxic components of type D progenitor toxin was slightly activated. They also reported that the treatment of erythrocytes with trypsin did not change type C and D HA titres. This discrepancy with the findings reported here might be attributable to differences in the concentration of proteases used, difference in the strains and (or) the purity of the samples used in the experiments. Since the sequence of the non-toxic components of C-ST and D-CB16 progenitor toxins were found to be essentially identical (Fujinaga et al., 1994
; Hauser et al., 1994
; Ohyama et al., 1995;Tsuzuki et al., 1990
, 1992
), it seems reasonable to suppose that both type C and D HAs behave in a similar manner. The interaction between type C and D 16S toxins with erythrocytes was reduced by employing neuraminidase-pretreated erythrocytes and by adding the NeuAc to the reaction mixture (Fig. 5
). Binding studies with erythrocyte extracts, purified glycolipids and commercially purchased glycoproteins demonstrated that the type C and D 16S toxins bound to glycolipids containing sialic acid (gangliosides) (Figs 6
, 8
) and sialoglycoproteins (fetuin and glycophorin) (Fig. 9
). However, they did not bind to neutral glycolipids or sulfated glycolipids. The binding of 16S toxin to GM4, GM3, GM2, GD3 and SPG was abolished by treating these gangliosides with neuraminidase. Therefore, it was postulated that the presence of NeuAc in the carbohydrate structure is important for binding of 16S toxins to glycolipids. Furthermore, they did not bind to neuraminidase-treated glycophorin and asialofetuin. These data also support the hypothesis that type C and D 16S toxins bind to both glycolipids and glycoproteins if they contain NeuAc in their carbohydrate structure. Glycophorin is the major sialoglycoprotein of the membrane of human erythrocytes (Marchesi et al., 1972
), and the majority of the sialic acid existing on the surface of the human erythrocytes is carried not by glycolipids but by glycophorin molecules (Alberts et al., 1995
). Therefore, it is speculated that type C and D 16S toxins agglutinate human erythrocytes by binding to glycophorin on the surface of erythrocytes.
Type D free HA1 showed no HA activity. The interaction between type D HA1 and erythrocytes was significantly lower than that observed between the 16S toxin and erythrocytes. Type D HA1 was able to bind to sialylglycolipids, but could not bind to sialoglycoproteins. This also supports the hypothesis that sialoglycoproteins, rather than sialylglycolipids, are important for the binding of type C and D 16S toxin to erythrocytes associated with haemagglutination. Tsuzuki et al. (1990) reported that the gene products from E. coli which were transferred by the plasmid containing HA1 gene showed HA activity. However, this plasmid contained the genes for HA2 and part of HA3 in addition to HA1. To account for these results, we speculate that: 1, the formation of a complex involving HA1, HA2 and part of HA3 may be required to cause haemagglutination; 2, the active binding region of free HA1 to erythrocytes may be obscured by the formation of polymer; 3, free HA1 might be processed at C-terminal region, which is required for its binding to erythrocytes to cause haemagglutination; 4, the higher-order structure of free HA1 may be different from that of HA1 genetically produced by E. coli (or HA1 contained in the 16S toxin). Previously, we purified type A HA1 from culture medium (Inoue et al., 1996 ). The type A HA1 did not show HA activity as also seen for the type D HA1 described in this manuscript. However, Fu et al. (1998
) reported that the type A HA1 possessed HA activity. This discrepancy may be caused by the difference of strain or purification methods. We think the purity of free HA1 is very important because the pooled HA1 fractions after ion-exchange column chromatography showed both weak HA activity and toxic activity (described in Bacteria and toxin section of Methods), which was considered to be caused by contaminated 16S toxin, but these activities were eliminated by purifying this preparation over a hydroxyapatite column (Fig. 2
).
It has been postulated that the orally ingested botulinum progenitor toxins are absorbed from the upper intestine into the lymphatic system (Sugii et al., 1977 ). Recently, we proposed that type C HA plays a critical role in the absorption of the progenitor toxins from the small intestine of guinea pigs (Fujinaga et al., 1997
). On the contrary, Maksymowych & Simpson (1998
) suggested that neurotoxin itself might be absorbed in the case of type A after performing in vitro experiments with human carcinoma cells. In this paper, we demonstrate that the type C and D 16S toxins bound to both sialylglycolipids and sialoglycoproteins. In our experiment with guinea pigs, type C 16S toxin, but neither 12S toxin or neurotoxin, bound to the epithelial cells of the small intestine: this binding was reduced by pretreatment of the epithelial cells with neuraminidase (Fujinaga et al., 1997
). The mechanisms of how HA-positive and -negative toxins (including neurotoxin alone) are absorbed and enter the lymphatic vessels through the epithelial cells should be analysed in the near future.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 6 January 1999;
revised 26 April 1999;
accepted 11 June 1999.