Biomacromolecules 2004, 5, 937-941
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Surface Plasmon Resonance Study of Carbohydrate-Carbohydrate Interaction between Various Gangliosides and Gg3-Carrying Polystyrene Kazunori Matsuura,†,# Ryuichi Oda,† Hiromoto Kitakouji,† Makoto Kiso,‡ Ken Kitajima,§ and Kazukiyo Kobayashi*,† Department of Molecular Design, Graduate School of Engineering, Nagoya University, Chikusa, Nagoya 464-8603, Japan, Department of Applied Bioorganic Chemistry, Gifu University, Gifu 501-1193, Japan, and Division of Organogenesis, Nagoya University Bioscience Center, Chikusa, Nagoya 464-8601, Japan Received December 7, 2003; Revised Manuscript Received February 8, 2004
Carbohydrate-carbohydrate interactions between Gg3 trisaccharide-carrying polystyrene (PN(Gg3)) and monolayers of several glycosphingolipids (GSLs) were quantitatively investigated by surface plasmon resonance techniques. PN(Gg3) was adsorbed onto a GM3 monolayer strongly and specifically with an apparent affinity constant of Ka ) 2.5 × 106 M-1, and the apparent affinity constants onto GSLs decreased in the following order: GM3 > LacCer > (KDN)GM3 ≈ GlcCer > GM2 ≈ GD3 ≈ GM4 > GM1 ≈ 2,6-isoGM3 > ceramide. These results suggest that PN(Gg3) recognizes not only some specified portions of GM3 but also the trisaccharide as a whole. On the other hand, PN(Lac) and PN(Cel) were bound to GSLs less strongly (Ka ≈ 104 M-1) and less selectively. The kinetic analysis revealed that the selectivity in the adsorption of PN(Gg3) onto the GM3 monolayer is dominated by the faster adsorption rate. Introduction A recent development in glycobiology has revealed that glycosphingolipids (GSLs) form microdomains on the cell surface and they play important roles in cell-cell adhesion and communication.1 It was demonstrated that the GSLs in a microdomain interact not only with carbohydrate-binding proteins but also with complementary GSLs via the carbohydrate-carbohydrate interaction,2 for example, the LeXLeX interaction in compaction in embryogenesis,3 the GalCer-3SO3GalCer interaction in the formation of compacted myelin membrane,4 cell adhesion of marine sponges,5 the GM3-Gg3 interaction between lymphoma and melanoma cells,6 and the (KDN)GM3-Gg3 interaction between rainbow trout sperms and eggs.7 The GM3-Gg3 interaction on GSL microdomains has been suggested to initiate signal transduction though inhibition or activation of transducer molecules.8 Various model systems have been proposed to investigate the carbohydrate-carbohydrate interaction.9-17 Attempts have been made to characterize and quantify this weak interaction in solution with monomeric LeX ligands but have failed.9 Geyer et al. reported that LeX-bearing liposomes interact with LeX trisaccharide in the presence of Ca2+, but * To whom correspondence should be addressed. Phone: +81 52 789 2488. Fax: +81 52 789 2528. E-mail:
[email protected]. † Nagoya University. ‡ Gifu University. § Nagoya University Bioscience Center. # Present address: Department of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu University, Higashi-ku, Fukuoka 812-8581, Japan.
the affinity constants estimated by NMR spectroscopy were still low.10 It is important to note that the carbohydratecarbohydrate interaction in cellular recognition is expressed on the clustered glycosphingolipid microdomains. Recently, model systems using clustered oligosaccharides have been proposed to elucidate the carbohydrate-carbohydrate interaction by us11,12 and other groups.13-17 We have demonstrated that clustered oligosaccharides of glycolipid monolayers and of artificial glycoconjugate polymers are useful to elucidate the GM3-Gg3 interaction using π-A isotherm11 and surface plasmon resonance (SPR).12 Peretz et al. devised a direct measurement of adhesion force between LeX-bearing liposomes using micropipetting techniques.13 Penade´s et al. devised Lex-coated gold nanoparticles that aggregate in the presence of Ca2+ via the homotropic LeX-LeX interaction.14 They also elucidated the interaction quantitatively using SPR to be Ka ≈ 106 M-1.15 These model systems are efficient for LeX-LeX interaction but, as far as we know, no model system has been reported for the GM3-Gg3 interaction except by us. In this paper, we have extended the SPR study of the GM3-Gg3 interaction to the analysis using monolayers of various glycosphingolipids (GM1, GM2, GM4, GD3, (KDN)GM3, and 2,6-isoGM3) to disclose the specificity of the interaction quantitatively. Experimental Section Materials. Deionized water of high resistivity (>18 MΩ cm) purified with a Millipore Purification System (Milli-Q water) was used as a subphase in a Langmuir trough.
10.1021/bm034508g CCC: $27.50 © 2004 American Chemical Society Published on Web 03/18/2004
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Figure 1. Structures of (A) GSLs and (B) glycoconjugate polystyrenes used in this work.
Chloroform and methanol of spectral grade were used to spread lipids. GM1, GM2, GM3, GD3, lactosylceramide, and ceramide were purchased from Sigma. (KDN)GM3 was obtained from rainbow trout testis.18 GM419 and 2,6-GM320 were prepared by Kiso et al.21 PN(Gg3), PN(Lac), and PN(Cel) were synthesized by conventional radical homopolymerization according to the previous procedure.11b,22 The number-average molecular weights (Mn) of PN(Gg3), PN(Lac), and PN(Cel) were estimated respectively to be 2.9 × 104 (Mw/Mn ) 1.5), 2.0 × 104 (Mw/Mn ) 1.8), and 3.4 × 104 (Mw/Mn ) 3.0) by size exclusion chromatography using pullulan as standards and water as eluent. Preparation of LB Film of Gangliosides on a Gold Substrate. A solution of sphingolipid (typically 0.24 mg/mL) in chloroform/methanol (spectral grade) (4/1 v/v, typically 40 µL) was spread on pure water (100 mL) in a computer-controlled Miyata-type moving wall trough (Nippon Laser & Electronics Lab., Nagoya). After the organic solvents were evaporated for 20 min, the Langmuir monolayer was compressed to 30 mN m-1 (compression speed: 6.9 mm2 s-1) and transferred by a vertical dipping method (dipping speed: 10 mm min-1) onto a gold-deposited glass plate which had been hydrophobized with a self-assembled
monolayer of 1-octadecanethiol. The transfer ratio of GSLs monitored by the decrease of area per molecules for the down process was about 1.0 ( 0.1. The temperature of the subphase was maintained at 25 ( 0.2 °C. SPR Measurement. The monolayer-immobilized glass plate was attached to a prism (n ) 1.518) with matching oil and set on a surface plasmon resonance apparatus (SPR 670, Nippon Laser & Electronics Lab., Nagoya). The reflectance was measured with changing incident angle of p-polarized light at 670 nm at 25 °C. Aliquots of a stock solution of glycopolymers were injected into 200 µL of pure water in a batch-cell. The change in the incident angle (∆θ) which provides a constant reflectance (about 0.5) was recorded as a function of time at 25 °C. Results and Discussion Figure 1 shows structures and abbreviations of GSLs and glycoconjugated polystyrenes used in this study. Figure 2A illustrates our SPR setup. A GSL monolayer was prepared by a vertical dipping method onto a hydrophobized golddeposited glass plate. The GSL-immobilized gold substrate was set on a SPR apparatus, and the SPR spectrum was
SPR of Carbohydrate-Carbohydrate Interaction
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Figure 2. Illustration of (A) SPR setup and (B) SPR spectrum for the carbohydrate-carbohydrate interaction between monolayer of gangliosides and glycoconjugate polystyrenes. (a) Hydrophobized gold substrate, (b) monolayer of gangliosides on the substrate, and (c) glycoconjugate polystyrenes on the monolayer.
Figure 4. Binding isotherms of glycoconjugate polystyrenes to gangliosides monolayers depending on the oligosaccharide concentrations at 25 °C. (A) Adsorption of glycoconjugate polystyrenes onto GM3 monolayer. (B) and (C) Adsorption of PN(Gg3) onto various ganglioside monolayers.
Figure 3. (A) Typical SPR time courses of adsorption of PN(Gg3) at 1 µM concentration of Gg3 unit onto various monolayer of gangliosides at 25 °C. (B) Linearized plot according to eq 1 for adsorption of PN(Gg3) onto GM2 monolayer at 10 µM.
measured. As illustrated in Figure 2B, the SPR spectrum of unmodified substrate was shifted to a larger resonance angle (∆θ ≈ 0.31°) by the immobilization of GSL on the substrate and further by the adsorption of glycoconjugate polymer onto GSL. Figure 3A shows typical time courses of the resonance angle changes (∆θ) of monolayers of GM3, GM2, and GM1 responding to the addition of glycoconjugate polystyrenes at 1 µM. PN(Gg3) was significantly adsorbed onto the GM3 monolayer and reached equilibrium within about 10 min,
whereas PN(Gg3) was slightly adsorbed onto the GM2 and GM1 monolayers.23 As previously reported,12 PN(Lac) and PN(Cel) were adsorbed onto the GM3 monolayer more slowly to reach smaller angle changes, and the corresponding Gg3-carrying styrene monomer was not adsorbed at all. The GM3 monolayer recognized strongly the Gg3-trisaccharide clusters of PN(Gg3) in water. These time courses of the angle change can be expressed approximately by pseudo first-order kinetics. The plots of dθ/dt against ∆θ according to eq 1 give adsorption rate constant k1 and dissociation rate constant k-1 dθ/dt ) [sugar]∆θeqk1 - (k1 [sugar] + k-1)∆θ
(1)
where ∆θeq stands for resonance angle change at adsorption equilibrium. A typical dθ/dt vs ∆θ plot for adsorption of PN(Gg3) onto GM2 monolayer is shown in Figure 3B.
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Table 1. Affinity Constants and Kinetic Parameters in Binding of Glycoconjugate Polystyrenes to Sphingolipids polymer
monolayer
Kaa, 104 M-1 c
∆θmax, deg
k1, 102 M-1 s-1
k-1, 10-3 s-1
Kab, 104 M-1 c
PN(Gg3)
GM3 GM2 GM1 GD3 GM4 (KDN)GM3 2,6-isoGM3 LacCer GlcCer Ceramide GM3 (KDN)GM3 LacCer GlcCer Ceramide GM3 (KDN)GM3 LacCer GlcCer Ceramide
250 11 6.1 12 11 25 5.5 65 24 2.9 7.7 5.9 7.7 4.1 0.01 4.4 2.6 6.1 3.9 0.027
0.168 0.420 0.337 0.338 0.342 0.232 0.412 0.139 0.142 0.151 0.152 0.101 0.026 0.186 0.223 0.156 0.104 0.015 0.077 0.066
300 10 3.6 1.5 12 1.5 8.6 71 49 2.9 38 1.1 34 35 0.9 2.0 2.2
3.2 1.5 1.4 1.2 5.4 0.15 4.8 8.5 6.3 5.5 5.2 1.8 62 57 38 6.3 4.3
938 67 26 13 22 100 18 84 78 5.3 73 6.1 5.5 6.1 0.24 3.2 5.1
PN(Lac)
PN(Cel)
4.5 0.5
10 25
4.5 0.20
a Apparent affinity constants obtained from Langmuir eq 2. Each square of the correlation coefficient of linear fitting was over 0.90. All of the standard deviations of Ka (plural measurements) were within 20%. b Apparent affinity constants obtained from kinetic analysis eq 1 (Ka ) k1/k-1). Each square of the correlation coefficient of linear fitting was over 0.80. All of the standard deviations of Ka (plural measurements) were within 40%. c Molarity (mol/L) of the sugar unit.
The ∆θeq values at various sugar concentrations were determined and plotted against the concentration [sugar] to give typical Langmuir-type adsorption isotherms (Figure 4). PN(Gg3) was adsorbed onto the GM3 monolayer at even low concentration (about 10-7-10-6 M), whereas PN(Lac) and PN(Cel) needed much higher concentrations (Figure 4A). The isotherms of PN(Gg3) adsorption onto various ganglioside monolayers (Figure 4, parts B and C) showed that not only the ∆θ dependency on PN(Gg3) concentration but also the saturated maximum angle change (∆θmax) of PN(Gg3) varied with the structure of gangliosides. The difference in saturated maximum angle change may be caused from difference in the stoichiometry of interaction or in the conformation of PN(Gg3) adsorbed on each monolayer.24 Apparent affinity constants per sugar unit (Ka) and maximum angle change (∆θmax) were calculated from the slopes and intercepts according to Langmuir eq 2 [sugar]/∆θ ) [sugar]/∆θmax + 1/∆θmaxKa
(2)
Table 1 summarizes these binding parameters in the carbohydrate-carbohydrate interaction. Comparison of Ka in Langmuir eq 2 shows that PN(Gg3) was bound most strongly to the GM3 monolayer with Ka ) 2.5 × 106 M-1, whereas the affinity constants to monolayers of other gangliosides (GM2, GM1, GD3, and GM4) were about 20-40 times smaller than that to GM3 monolayer. The affinity constant of PN(Gg3) to the sphingolipids was decreased in the order of GM3 > LacCer > (KDN)GM3 ≈ GlcCer > GM2 ≈ GD3 ≈ GM4 > GM1 ≈ 2,6-isoGM3 > ceramide. The affinity constant (Ka) between GM3 monolayer and PN(Gg3) was also decreased to about 1/10 in the presence of 1 mM of sialic acid: Ka ) 2.3 × 105 M-1, ∆θmax ) 0.286°. The following structural considerations on the GM3-Gg3 interaction can be deduced from these results. (1) LacCer and GlcCer: the removal of sialic acid or sialylgalactose from GM3 provides
some decrease of the affinity to Gg3. (2) (KDN)GM3: the substitution of NHAc to OH group in sialic acid of GM3 provides some decrease of the affinity to a similar extent as GlcCer. (3) GM4: the removal of glucose from GM3 significantly decreases the affinity. (4) GM2, GM1, GD3, and 2,6-isoGM3: the addition of sugar moiety to GM3 and rearrangement of sialic acid of GM3 also significantly decrease the affinity. (5) ceramide: the lack of all sugar moiety further decreases the affinity to Gg3. These structural considerations suggest that PN(Gg3) recognizes not only some specified portions of GM3 but also the trisaccharide as a whole. On the other hand, PN(Lac) and PN(Cel) were bound to these GSLs less strongly (Ka ≈ 104 M-1) and less selectively, although these polymers were minimally bound to ceramide (Ka ≈ 102 M-1).25 Apparent affinity constants estimated from the kinetic analysis (Ka ) k1/k-1) according to eq 1 tended to be overestimated in comparison with those from the Langmuir equation (eq 2), although the tendency of selectivity of the carbohydrate-carbohydrate interaction was similar to each other. There is a marked tendency for the larger fluctuation in the adsorption rate constants than in the desorption rate constants. For example, the adsorption rate constant of PN(Gg3) to the GM3 monolayer was larger than that to the GM2 monolayer by about 30-times and that to the GM1 monolayer by about 80-times, whereas the desorption rate constants were comparable. These results suggest that selectivity in the adsorption of PN(Gg3) to the GM3 monolayer is dominated by the faster adsorption rate and that a strong adhesion force may act between PN(Gg3) and GM3 monolayer. Conclusion We have quantitatively estimated the carbohydratecarbohydrate interaction between Gg3-carring polystyrene
SPR of Carbohydrate-Carbohydrate Interaction
(PN(Gg3)) and the monolayer of various gangliosides by SPR. Among the gangliosides used in this work, GM3 was most strongly bound to PN(Gg3) with Ka ) 2.5 × 106 M-1. The affinity between GM3 and PN(Gg3) was significantly decreased by the small structural modification of GM3 (NeuAcR2-3Galβ1-4Glcβ1-1′Cer): that is, removal of NeuAc and/or Gal, rearrangement of NeuAc, and substitution of NHAc group to OH group in NeuAc. We can conclude that the GM3-Gg3 interaction is based on the precise molecular recognition. We believe that the present information on the GM3Gg3 interaction provides an important molecular basis on carbohydrate-carbohydrate interactions to elucidate their biological meanings in glycobiology and cell biology. The present simple model system consists of a single glycolipid monolayer, but this system can be extended to various mixed monolayers to simulate “Glycosignaling Microdomains” of membranes. This achievement is our next target. Acknowledgment. This research was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture. References and Notes (1) (a) Hakomori, S.; Handa, K.; Iwabuchi, K.; Yamamura, S.; Prinetti, A. Glycobiology 1998, 8, xi. (b) Hakomori, S. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 225. (2) (a) Hakomori, S. Pure Appl. Chem. 1991, 63, 473. (b) Spillmann, D.; Burger, M. M. J. Cell Biochem. 1996, 61, 562. (c) Handa, K.; Kojima, N.; Hakomori, S. Methods Enzymol 2000, 312, 447. (3) (a) Eggens, I.; Fenderson, B.; Toyokuni, T.; Dean, B.; Stroud, M.; Hakomori, S. J. Biol. Chem. 1989, 264, 9476. (b) Hakomori, S.; Igarashi, Y.; Kojima, N.; Okoshi, K.; Handa, B.; Fenderson, B. Glycoconjugate J. 1991, 8, 178. (4) (a) Stewart, R. J.; Boggs, J. M. Biochemistry 1993, 32, 10666. (b) Koshy, K. M.; Boggs, J. M. J. Biol. Chem. 1996, 271, 3496. (c) Koshy, K. M.; Wang, J.; Boggs, J. M. Biophys. J. 1999, 77, 306. (d) Boggs, J. M.; Menikh, A.; Rangaraj, G. Biophys. J. 2000, 78, 874. (5) (a) Misevic, G. N.; Burger, M. M. J. Biol. Chem. 1993, 268, 4922. (b) Spillmann, D.; Thomas-Oats, J. E.; van Kuik, J. A.; Vliebenthart, J. F. G.; Misevic, G. N.; Burger, M. M.; Finne, J. J. Biol. Chem. 1995, 270, 5089. (c) Haseley, S. R.; Vermeer, H. J.; Kamerling, J. P.; Vliegenthart J. F. G. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 9419. (6) (a) Kojima, N.; Hakomori, S. J. Biol. Chem. 1989, 264, 20159. (b) Kojima, N.; Hakomori, S. J. Biol. Chem. 1991, 266, 17552. (c) Kojima, N.; Shiota, M.; Sadahira, Y.; Handa, K.; Hakomori, S. J. Biol. Chem. 1992, 267, 17264. (7) Yu, S.; Kojima, N.; Hakomori, S.; Kudo, S.; Inoue, S.; Inoue, Y. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 2854. (8) (a) Iwabuchi, K.; Yamamura, S.; Prinetti, A.; Handa, K.; Hakomori, S. J. Biol. Chem. 1998, 273, 9130. (b) Song, Y.; Withers, D. A.; Hakomori, S. J. Biol. Chem. 1998, 273, 2517.
Biomacromolecules, Vol. 5, No. 3, 2004 941 (9) Wormald, M. R.; Edge, C. J.; Dwek, R. A. Biochem. Biophys. Res. Commun. 1991, 180, 1214. (10) (a) Geyer, A.; Gege, C.; Schmidt, R. R. Angew. Chem., Int. Ed. 1999, 38, 1466. (b) Geyer, A.; Gege, C.; Schmidt, R. R. Angew. Chem., Int. Ed. 2000, 39, 3246. (11) (a) Matsuura, K.; Kitakouji, H.; Tsuchida, A.; Sawada, N.; Ishida, H.; Kiso, M.; Kobayashi, K. Chem. Lett. 1998, 1493. (b) Matsuura, K.; Kitakouji, H.; Oda, R.; Morimoto, Y.; Asano, H.; Ishida, H.; Kiso, M.; Kitajima, K.; Kobayashi K. Langmuir 2002, 18, 6940. (12) Matsuura, K.; Kitakouji, H.; Sawada, N.; Ishida, H.; Kiso, M.; Kitajima, K.; Kobayashi, K. J. Am. Chem. Soc. 2000, 122, 7406. (13) Pincet, F.; Bouar, T. L.; Zhang, Y.; Esnault, J.; Mallet, J. M.; Perez, E, Sinay¨ , P. Biophys. J. 2001, 80, 1354. (14) de la Fuente, J. M.; Barrientos, A. G.; T. C. Rojas, Rojo, J.; Can˜ada, J.; Fernaa´ndez, A.; Penade´s, S. Angew. Chem., Int. Ed. 2001, 40, 2257. (15) Herna´iz, M. J.; de la Fuente J. M., Barrientos, A. G.; Penade´s, S. Angew. Chem., Int. Ed. 2002, 41, 1554. (16) Tromas, C.; Rojo, J.; de la Fuente, J. M.; Barrientos, A. G.; Garcı`a, R.; Penade´s, S. Angew. Chem., Int. Ed. 2001, 40, 3052. (17) Santacroce, P. V.; Basu, A. Angew. Chem., Int. Ed. 2003, 42, 95. (18) Yu, S.; Kitajima, K.; Inoue, S.; Khoo, K.-H.; Morris, H. R.; Dell, A.; Inoue, Y. Glycobiology 1995, 5, 207. (19) Hasegawa, A.; Ogawa, M.; Kiso, M. Biosci. Biotech. Biochem. 1992, 56, 535. (20) Hotta, K.; Komba, S.; Ishida, H.; Kiso, M.; Hasegawa, A. J. Carbohydr. Chem. 1994, 13, 665. (21) The purities of (KDN)GM3 obtained from rainbow trout testis,18 and GM419 and 2,6-GM320 synthesized by Kiso et al. were almost 100%. The purities of glycolipids (Sigma) were determined by TLC as follows: GM3 (98%), GM2 (95%), GM1 (96%), GD3 (90%), LacCer (95%), GlcCer (95%). Although the structures of fatty acid and sphingosine moieties of natural sphingolipids have diverse distribution, we consider that the structural distribution little affects the carbohydrate-carbohydrate interaction. (22) Kobayashi, K.; Tsuchida, A.; Usui, T.; Akaike, T. Macromolecules 1997, 30, 2016. (23) Hakomori reported that the interaction of GM3 liposomes with a Gg3-coated polystyrene surface is strongly promoted by Ca2+ ion.6 We attempted the SPR experiment of GM3-Gg3 interaction in the presence of Ca2+. However, the time course gave an unusual sensorgram and we could not find that the interaction between GM3 monolayer and PN(Gg3) is promoted by Ca2+. At the present state, we cannot discuss on the effect of Ca2+ ion in our system. (24) (a) Matsuura, K.; Tsuchida, A.; Okahata; Y.; Akaike, T.; Kobayashi, K. Bull. Chem. Soc. Jpn. 1998, 71, 2973. (b) Tsuchida, A.; Matsuura, K.; Kobayashi, K. Macromol. Chem. Phys. 2000, 201, 2245. (25) These glycoconjugate polystyrenes were reported to interact with hydrophobic solid surfaces via the polystyrene backbone.24 The interaction of these polymers to the hydrophobic moieties of glycolipid monolayers is possible, but the contribution to the affinity constants should be smaller than the minimum affinity constant (Ka ) 1 × 102 M-1) in Table 1. As shown in Table 1, PN(Gg3) was bound to ceramide monolayer more strongly than the other polymers by a factor of about 102. This may reflect the relatively high hydrophobicity of PN(Gg3).
BM034508G
