Biomacromolecules 2002, 3, 411-414
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A Quartz Crystal Microbalance Method for Rapid Detection and Differentiation of Shiga Toxins by Applying a Monoalkyl Globobioside as the Toxin Ligand Hirotaka Uzawa,*,†,‡ Shoko Kamiya,†,‡ Norihiko Minoura,†,‡ Hirofumi Dohi,§ Yoshihiro Nishida,§ Kazuhiro Taguchi,† Shin-ichiro Yokoyama,| Hiroshi Mori,| Toshimi Shimizu,†,‡ and Kazukiyo Kobayashi§ Nanoarchitectonics Research Center (NARC), National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8565, Japan, CREST, Japan Science and Technology Corporation (JST), Tsukuba Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305 8562, Japan, Department of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University, Chikusa-ku, Nagoya 464-8603, Japan, and Department of Public Health Pharmacy, Gifu Pharmaceutical University, Mitabora-higashi, Gifu 502-8585, Japan Received November 12, 2001
A simple globobiosyl (Gb2) ceramide mimic carrying a monoalkyl chain (C18) was applied for a monolayer Langmuir-Blodgett (L-B) technique to detect Shiga toxins (Stxs) by a quartz crystal microbalance (QCM) method. The artificial glycolipid, synthesized from penta-O-acetyl-D-galactopyranose via a conventional glycosidation pathway, was developed at the air-water surface for the formation of the monolayer film. Then, the film was transferred onto a QCM cell surface modified with alkanethiols. Upon the addition of each of Stx-1 and Stx-2, the decrease of frequency reached saturation within 45 min at a few nanogram order per quartz cell. Binding constants (Ka) estimated for each of Stx-1 and Stx-2 showed little difference between the two toxins. On the other hand, in the presence of an artificial acrylamido Gb2 copolymer as a competitive inhibitor, the two toxins showed a large difference in the binding behavior to the L-B monolayer. Shiga toxins (Stxs) produced by Escherichia coli O-157: H-7 and other enterohemorrhagic E. coli species cause serious hemolytic uremic syndrome (HUS). Millions of people worldwide have suffered from Stxs every year.1-3 To diagnose and treat these dreadful toxins immediately, rapid and simple detection of Stxs is essential. Extensive studies have been carried out to develop analytical tools, in which PCR and immunological methods seem to be most widely employed.4-7 Recently, an alternative approach applying a species-specific binding interaction between Stxs and globosyl (Gb2 and Gb3) ceramides has attracted much interest. Nilsson and Mandenius proposed a scanning electron microscopy (SEM) sensor using Gb2 mimics for the detection of uropathogenic bacteria.8 They suggested that the Gb2 analogue may be applicable for Stxs detection by using, for example, piezoelectric crystal microbalance, ellipsometry, and plasmon resonance spectroscopy. Kitov et al. reported a similar system using 16-mercaptohexadecanyl Gb2 glycosides.9 In this communication, we propose a quartz crystal microbalance (QCM) system for rapid detection of Shiga toxins. Two types of Shiga toxins, Stx-1 and Stx-2, have multisubunit proteins made up of a single A-subunit with * To whom correspondence should be addressed. E-mail: h.uzawa@ aist.go.jp (H. Uzawa). Telephone: +81-298-61-4431. Fax: +81-298-614680. † National Institute of Advanced Industrial Science and Technology. ‡ CREST. § Nagoya University. | Gifu Pharmaceutical University.
N-glycanase activity and five copies of a B-subunit with carbohydrate binding domains.10 The B-subunits are reported to recognize a Pk antigenic Gb3 trisaccharide10,11 and a simpler Gb2 disaccharide.12 It has been, therefore, accepted that the toxins utilize the nonreducing terminal RGal1-4Gal linkage for the host cell adhesion. On the other hand, we had previously reported that a neoglycoconjugate carrying a Gb3 cluster could possess potent activity to neutralize Stx-1, while the Gb3 cluster model was inactive for Stx-2.13,14 The result suggests that Stx-2 may show carbohydrate recognition different from the case of Stx-1. This speculation prompted us to establish an analytical tool to detect the carbohydrate binding of Stx-1 and Stx-2 and to investigate the possible difference. In this communication, we propose a QCM system as a simple way to detect and differentiate the Shiga toxins. The system applies a Langmuir-Blodgett (L-B) monolayer constructed of a synthetic monoalkyl globobioside A which serves as a common carbohydrate probe for both Stx-1 and Stx-2. The globobioside A was prepared from penta-O-acetylD-galactopyranose by a conventional chemical pathway (Scheme 1). A key intermediate 115 carrying an OH-4 group was derived in six steps and coupled with tetra-O-benzylR-D-galactopyranosyl chloride (2)16 to afford a mixture of R1-4 globobioside analogue 317 and β1-4 bioside18 (ca. R:β ) 4:1). The R-galabioside 3 was isolated by silica gel column chromatography and subjected to catalytic hydrogenolysis with H2 and Pd to afford the desired monoalkyl Gb2 mimic A.19
10.1021/bm010161a CCC: $22.00 © 2002 American Chemical Society Published on Web 04/18/2002
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Scheme 1a
Figure 2. Schematic drawing of the quartz crystalline surface.
a Reagents and conditions: i, 2,3,4,6-tetra-O-benzyl-R-D-galactopyranosyl chloride (2), AgOTf, MS4A, Et2O, 0 °C, R (46%), β (11%); ii, H2, Pd, MeOH, AcOEt, 50-60 °C, 82%.
Figure 1. π-A isotherms of (A) monoalkyl globobioside A and (B) its isomer β1-4 galabioside monolayer in subphase at 20 °C.
The QCM technique has provided a powerful way to quantify intermolecular interactions20,21 and to investigate the energy dissipation kinetics for biomacromolecules such as proteins and polynucleotides.22 The method has also been coupled with an L-B monolayer technique and widely applied to glycolipids to determine their binding to proteins and neoglycoconjugates.20,23-25 Though the formation of a stable L-B monolayer glycolipids is thought to require dialkyl or diacyl chains at the aglycon,26 the monoalkyl globobioside A was found to arrange a stable monolayer film on the air-water surface reproducibly. Comparing the respective π-A curve of globobioside A and its regioisomer β1-4 bioside, the isotherm of the latter was more expanded than that of globobioside A as shown in Figure 1, possibly due to the steric hindrance at the carbohydrate moiety. This means that the R1-4 linkage of Gb2 is suitable for the formation of the L-B monolayer. Then, the film was transferred at a surface pressure of 24 mN m-1 onto the surface of a quartz crystal modified with 1-dodecanethiol monolayer according to the L-B transfer technique, the schematic structure of which was depicted in Figure 2. We applied the present system to the analysis of Stxs in the way reported for biomacromolecules,27,28 after we evaluated the validity of the present system with a standard lectin
Figure 3. Typical time courses of frequency changes (∆F) of a QCM on the monolayer of globobioside A and inhibition assay of Stx-1 and Stx-2 in the presence of acrylamido Gb2 copolymer as an inhibitor: (A) Stx-1 (2.0 nM); (B) Stx-2 (2.0 nM) + Gb2 copolymer (ca. 1000 times molar equiv Gb2 unit); (C) Stx-2 (2.0 nM); (D) Stx-1 (2.0 nM) + Gb2 copolymer (ca. 10 times molar equiv Gb2 unit); (E) the toxinfree protein extracts (0 nM of Stx-1 and 2) as a negative control. The measurements were carried out in phosphate buffer solution (0.01 M, pH 7.2, 20 mL) containing 0.15 M saline at 25 °C. The structure of Gb2 copolymer was shown in Figure 5.
protein.29 Each of the two toxins, Stx-1 and Stx-2, was prepared from the cultivates of E. coli O-157: H-7 mutant strains.30 Crude protein of precipitates (purity ca. 2-3 w/w % of total proteins as described in Table 1) were used for the QCM analysis without further purification to demonstrate the simplicity of the experimental protocol. As a key reference to evaluate background response, we used a crude protein fraction without toxins derived from cultivates of E. coli O-157: H-7 strains producing no Shiga toxins. In Figure 3, the frequency change (∆F) was plotted versus time (h) after the addition of each of the protein fractions. Clear ∆F could be observed for both Stx-1 and Stx-2 (Figure 3A,C). The change reached saturation within 45 min after the addition of Stxs. This result indicates that the simple monoalkyl Gb2 monolayer serves as a common ligand of Stx-1 and Stx-2. It is also significant that the toxin-free protein extracts showed no apparent response showing that a nonspecific binding interaction is negligibly small (Figure 3E). Thus, the QCM response for Stx-1 and Stx-2 can be ascribed to their specific toxin adhesion onto the Gb2 monolayer. All these data support that the present QCM
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Communications Table 1. Selected QCM Data and Binding Parametersa,b of Stx-1 and Stx-2 sample Stx-1 Stx-2
µgc
∆F (Hz)
2.8 2.8
-75 -31
MW (kDa)
Kaa (M-1)
k1b (M-1 s-1)
70 70
5.5 × 1.2 × 108
1.8 × 1.5 × 105
108
105
k-1b (s-1) 10-3
0.5 × 1.1 × 10-3
Ka ) k1/k-1b (M-1) 4.1 × 108 1.3 × 108
-1 b a Determined by reciprocal plotting as shown in Figure 4 with the following equation:23,24 ∆m-1 ) Ka∆m -1 + ∆m max[Stx] max . Obtained from the courses of the binding process by using the following equation according to the ref 23: [ligand‚toxins]t ) [ligand‚toxins]∞[1 - exp(-t/τ)], where [ligand‚toxins]t ) ∆mt, [ligand‚toxins]∞ ) ∆mmax and 1/τ ) k1[toxins] + k-1. The binding (k1) and dissociation constants (k-1) were calculated from the time course binding experiments shown in Figure 3 by the above equation, using several different concentrations as follows: Stx-1, 1.0, 1.5, 1.75, 2.0, 2.5, 3.1 nM; Stx-2, 1.0, 2.0, 3.0, 6.0, 10.0 nM. The error of the data is estimated within ca. 5%. c The amounts of active proteins were calculated by an immunochemical method. See ref 7.
revealed that Stx-1 did not show apparent binding in the presence of the Gb2 copolymer (ca. 10 times the molar equiv Gb2 unit). This means that the plural binding sites of Stx-1 (at least 10 sites per the toxin) are blocked effectively by the Gb2 copolymer. On the other hand, Stx-2 could keep adhesion to the Gb2 monolayer even in the presence of a large excess amount of the copolymer (>1000 times). This behavior matches well with our previous result14 and gives a significant basis to discriminate between Stx-1 and Stx-2. In summary, we have demonstrated a rapid way to detect Shiga toxins by applying a simple monoalkyl Gb2 for an L-B monolayer-based QCM technique. The simple glycolipid was found to serve as a common carbohydrate probe to detect both types of Shiga toxins. It could be also demonstrated that the competitive assay using the Gb2 copolymer provides information useful to discriminate between Shiga toxins 1 and 2. Figure 4. The reciprocal plots of (A) Stx-2 and (B) Stx-1 after being calculated from saturation binding experiments of both of two toxins.
Acknowledgment. This work was partly supported by the Japan Science and Technology Corporation (JST) through the CREST (Core Research for Evolutional Science and Technology). Supporting Information Available. Preparation of Stx-1 and 2, synthesis of A, preparation of Gb2 carbohydrate probe monolayer, QCM measurements, and calculation of binding constants and dissociation rate constants. This material is available free of charge via the Internet at http://pubs.acs.org.
Figure 5. Structure of Gb2 acrylamido copolymer [Mn ) 2.5 × 105, Gb2:acrylamide ) 1:10 (molar ratio)].
system provides a rapid tool to detect both Stx-1 and Stx-2 in the crude protein fractions of E. coli O-157: H-7 cultivates. To compare the binding behavior of the two toxins, we calculated binding constants and kinetic parameters of each Stx-1 and Stx-2 in a reported way23,24 as cited in Figure 4 and Table 1. Since the QCM measurement was carried out for crude Stxs fractions without the calibration for energy dissipation and other possible effects,22 the derived values may be deviated from the accurate ones and of little quantitative significance.31 The derived data are, however, highly suggestive of little difference in the biding property of Stx-1 and Stx-2 to the Gb2 monolayer. This result was contrary to our expectation that the two toxins would have different binding property to the carbohydrate epitopes such as in the case of binding to Gb3 neoglycoconjugates.14 To obtain more precise information on carbohydrate recognition, we applied a Gb2 neoglycoconjugate32 (Figure 5, Mn ) 2.5 × 105, Gb2:acrylamide ) 1:10 (molar ratio)) for a competitive binding assay (Figure 3B,D). The results
References and Notes (1) Holmgren, J.; Svennerholm, A. M. Gastroenterology Clinics of North America 1992, 21, 283. (2) Yamasaki, S.; Takeda, Y. J. Toxicol. Toxin ReV. 1997, 16, 229. (3) Karmali, M. A. Clin. Microbiol. ReV. 1989, 2, 15. (4) Tsen, H.-Y.; Jian, L.-Z. J. Appl. Microbiol. 1998, 84, 585. (5) Weeratna, R. D.; Doyle, M. P. Appl. EnViron. Microbiol. 1991, 57, 2951. (6) Acheson, D. W. K.; Jacewicz, M.; Kane, A. V.; D.-Rolfe, A.; Keusch, G. T. Microb. Pathog. 1993, 14, 57. (7) Ashkenazi, S.; Cleary, T. G. J. Clin. Microbiol. 1989, 27, 1145. (8) Nilsson, K. G. I.; Mandenius, C.-F. Bio/Technol. 1994, 12, 1376. (9) (a) Kitov, P. I.; Railton, C.; Bundle, D. R. Carbohydrate Res. 1998, 307, 361. (b) Kitov, P. I.; Sadowska, J. M.; Mulvey, G.; Armstrong, G. D.; Ling, H.; Pannu, N. S.; Read R. J.; Bundle, D. R. Nature 2000, 403, 669. (10) Ling, H.; Boodhoo, A.; Hazes, B.; Cummings, M. D.; Armstrong, G. D.; Brunton, J. L.; Read, R. J. Biochemistry 1998, 37, 1777. (11) Bock, K.; Breimer, M. E.; Brignole, A.; Hansson, G. C.; Karlsson, K.-A.; Larson, G.; Leffler, H.; Samuelsson, B. E.; Stromberg, N.; Eden, C. S.; Thurin, J. J. Biol. Chem. 1985, 260, 8545. (12) Magnusson, G.; Ahlfors, S.; Dahme´n, J.; Jansson, K.; Nilsson, U.; Noori, G.; Stenvall, K.; Tjo¨rnebo, A. J. Org. Chem. 1990, 55, 3932. (13) Nishida, Y.; Dohi, H.; Uzawa, H.; Kobayashi, K. Tetrahedron Lett. 1998, 39, 8681. (14) Dohi, H.; Nishida, Y.; Mizuno, M.; Shinkai, M.; Kobayashi, T.; Takeda, T.; Uzawa, H.; Kobayashi, K. Bioorg. Med. Chem. 1999, 7, 2053.
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(15) Synthesis of 1. Commercially available penta-O-acetyl-D-galactose was converted to octadecyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside using 1-octadecanol and trimethylsilyltrifluoromethanesulfonate. Deacetylation with MeOH-NaOMe followed by 3,4-Oisopropylidenation and 2,6-di-O-benzylation gave octadecyl 2,6-diO-benzyl-3,4-O-isopropylidene-β-D-galactopyranoside. De-O-isopropylidenation under usual acidic conditions afforded octadecyl 2,6O-benzyl-β-D-galactopyranoside, which was selectively benzylated at OH-3 with Bu2SnO, n-Bu4NBr, and BnBr to give octadecyl 2,3,6tri-O-benzyl-β-D-galactopyranoside (1). Selected data for 1: δH (300 MHz, CDCl3) 4.35 (H-1, d, J ) 7.8 Hz). δC (75 MHz, CDCl3) 104.4 (C-1). (16) Austin, P. W.; Hardy, F. E.; Buchanan, J. G.; Baddiley, J. J. Chem. Soc. 1965, 1419. (17) Selected data for 3: δH (300 MHz, CDCl3) 5.03 (H-1′, bs), 4.313 (H-1, d, J ) 7.5 Hz). δC (75 MHz, CDCl3) 104.8 (C-1), 101.3 (C1′). (18) Selected data for β(1-4)-galabioside: δH (300 MHz, CDCl3) 4.92 (H-1′, d, J ) 7.2 Hz); 4.36 (H-1, d, J ) 7.5 Hz). δC (75 MHz, CDCl3) 104.6 (C-1); 103.6 (C-1′). Subsequent hydrogenolysis to give fully deprotected β(1-4) bioside; δH (300 MHz, DMF-d7) δ 4.41 (H-1′, d, J ) 7.8 Hz); 4.20 (H-1, d, J ) 7.5 Hz); 3.97 (H-4′, bd, J ) 3.3 Hz). (19) Selected data for A: δH (300 MHz, THF-d8): δ 5.00 (H-1′, d, J ) 3.6 Hz); 4.22 (H-1, d, J ) 6.6 Hz). δC (75 MHz, THF-d8) 105.0 (C-1); 102.5 (C-1′). (20) Alfonta, L.; Willner, I.; Throckmorton, D. J.; Singh, A. K. Anal. Chem. 2001, 73, 5287. (21) Mori, T.; Naito, M.; Irimoto, T.; Okahata, Y. Chem. Commun. 2000, 45. (22) Ho¨o¨k, F.; Rodahl, M.; Brzezinski, P.; Kasemo, B. Langmuir 1998, 14, 729. Rodahl, M.; Ho¨o¨k, F.; Fredriksson, C.; Keller, C. A.; Krozer,
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(23) (24) (25) (26) (27) (28) (29)
(30)
(31)
(32)
A.; Brzezinski, P.; Voinova, M.; Kasemo, B. Faraday Discuss 1997, 107, 229. Ebata, Y.; Okahata, Y. J. Am. Chem. Soc. 1994, 116, 11209. Matsuura, K.; Tsuchida, A.; Okahata, Y.; Akaike, T.; Kobayashi, K. Bull. Chem. Soc. Jpn. 1998, 71, 2973. Matsuura, K.; Kitakouji, H.; Tsuchida, A.; Sawada, N.; Ishida, H.; Kiso, M.; Kobayashi, K. Chem. Lett. 1998, 1293. Mingotaud, A.-F.; Mingotaud, C.; Patterson, L. K. In Handbook of Monolayer; Academic Press: New York, 1993. Hasegawa, T.; Matsuura, K.; Ariga, K.; Kobayashi, K. Macromolecules 2000, 33, 2772. Sato, T.; Serizawa, T.; Okahata, Y. Biochim. Biophys. Acta 1996, 1285, 14. Prior to Stx analysis, we evaluated the validity of the present QCM system with an R-galactoside binding protein (Maclura pomifera lectin: MPL). Upon the addition of this lectin, the QCM system soon showed frequency decrease (∆F) reflecting mass increase at the surface, while another lectin derived from RCA120 specific to β-galactoside did not show apparent frequency change (data not shown). Mori, H.; Yokoyama, Y. In preparation. An immunological assay could prove that the Stx-1 was not contaminated with Stx-2 and vice versa. We did not determine the dissipation factor in this study. However, these binding parameters obtained here were almost consistent with those determined by surface plasmon resonance (data not shown here). Therefore, the dissipation factor, in our case, might be small, not enough to change the order of 108 in Ka. Dohi, H.; Nishida, Y.; Tanaka, H.; Kobayashi, K. Synlett 2001, 1446.
BM010161A
