Selective Expansion of the GM3 Glycolipid Monolayer Induced by

Bioorganic Chemistry, Gifu University, Gifu 501-1193, Japan, and Division of ... Nagoya University Bioscience Center, Chikusa, Nagoya 464-8601, Japan...
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Selective Expansion of the GM3 Glycolipid Monolayer Induced by Carbohydrate-Carbohydrate Interaction with Gg3 Trisaccharide-Bearing Glycoconjugate Polystyrene at the Air-Water Interface Kazunori Matsuura, Hiromoto Kitakouji, Ryuichi Oda, Yuki Morimoto, Hiroki Asano, Hideharu Ishida,† Makoto Kiso,† Ken Kitajima,‡ and Kazukiyo Kobayashi* Department of Molecular Design and Department of Biotechnology, 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 April 12, 2002. In Final Form: June 11, 2002 Carbohydrate-carbohydrate interactions between GM3 and Gg3 were investigated using surface pressure-area (π-A) isotherms of glycolipid monolayers in contact with glycoconjugate polystyrenes at the air-water interface. The GM3 monolayer was greatly expanded by the Gg3-trisaccharide (GalNAcβ14Galβ1-4Glc)-bearing glycoconjugate polystyrene [PN(Gg3)] and moderately expanded by lactose-bearing polystyrene, while little or no expansion was observed with GalLac (Galβ1-4Galβ1-4Glc)- or cellobiosebearing polystyrenes or Gg3-trisaccharide-bearing styrene monomer. The PN(Gg3)-induced expansion of π-A isotherms of sphingolipid monolayers was maximal with GM3 and decreased in the following order: (KDN)GM3 > GM2 ≈ LacCer > GD3 ≈ 2,6-isoGM3 ≈ GM4 ≈ GM1 ≈ sphingomyelin (SM). The extent of expansion reflected the strength of carbohydrate-carbohydrate interactions with Gg3, which were amplified by the clustering effect of carbohydrates in the glycoconjugate polystyrenes. The expansion of various proportions of GM3/SM mixed monolayers by PN(Gg3) was smaller than that of the GM3 monolayer, indicating the importance of clustered carbohydrates in the monolayer. The PN(Gg3)-induced expansion of the GM3 monolayer required no calcium ion, and the expansion was strongly inhibited in the presence of urea and N-acetamido sugars. These observations reveal a new aspect of the mechanism of the carbohydrate-carbohydrate interactions where the N-acetyl groups and hydrogen bonds between the carbohydrate moieties play important roles. The π-A isotherms using glycoconjugate polystyrenes have been demonstrated to be useful as a molecular probe for the carbohydrate-carbohydrate interactions.

Introduction Increasing attention has been paid to the biological functions of oligosaccharide chains of glycolipids and glycoproteins at plasma membranes in various biological processes including cell-cell communications.1,2 It was recently demonstrated that these oligosaccharide chains interact not only with carbohydrate-binding proteins but also with complementary oligosaccharide chains.3-14 Ha* To whom correspondence should be addressed. Phone: +81 52 789 2488. Fax: +81 52 789 2528. E-mail: kobayash@ mol.nagoya-u.ac.jp. † Gifu University. ‡ Nagoya University Bioscience Center. (1) Fukuda, M.; Hindsgaul, O. Molecular Glycobiology; IRL: Oxford, 1994. (2) Varki, A.; Cumming, R.; Esko, J.; Freeze, H.; Hart, G.; Marth, J. Essentials of Glycobiology; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1999. (3) Hakomori, S. Pure Appl. Chem. 1991, 63, 473-482. (4) Eggenst, I.; Fenderson, B.; Toyokuni, T.; Dean, B.; Stroud, M.; Hakomori, S. J. Biol. Chem. 1989, 264, 9476-9484. (5) Hakomori, S.; Igarashi, Y.; Kojima, N.; Okoshi, K.; Handa, K.; Fenderson, B. Glycoconjugate J. 1991, 8, 178. (6) Stewart, R. J.; Boggs, J. M. Biochemistry 1993, 32, 10666-10674. (7) Kojima, N.; Hakomori, S. J. Biol. Chem. 1989, 264, 20159-20162. (8) Kojima, N.; Hakomori, S. J. Biol. Chem. 1991, 266, 17552-17558. (9) Kojima, N.; Shiota, M.; Sadahira, Y.; Handa, K.; Hakomori, S. J. Biol. Chem. 1992, 267, 17264-17270. (10) Iwabuchi, K.; Yamamura, S.; Prinetti, A.; Handa, K.; Hakomori, S. J. Biol. Chem. 1998, 273, 9130-9138. (11) Song, Y.; Withers, D. A.; Hakomori, S. J. Biol. Chem. 1998, 273, 2517-2525. (12) Brewer, G. J.; Matinyan, N. Biochemistry 1992, 31, 1816-1820.

komori et al. reported that carbohydrate-carbohydrate interactions have important roles in cellular recognition: for example, the LeX-LeX interaction in compaction in embryogenesis,4 the Gal-3SO3Gal interaction in the formation of compacted myelin membrane,5,6 and the Gg3GM3 interaction between lymphoma and melanoma cells.7-9 The Gg3-GM3 interaction has also been suggested to initiate signal transduction through inhibition or activation of transducer molecules.10,11 The mechanism underlying these carbohydrate-carbohydrate interactions at the molecular level has not yet been sufficiently elucidated. There have been many attempts to investigate the carbohydrate-carbohydrate interaction systematically with various model systems.15-19 An attempt was made to estimate the interaction using a simple LeX model in water by NMR spectroscopy,15 but the interaction was too small to elucidate the carbohydrate-carbohydrate inter(13) Gupta, D.; Arango, R.; Sharon, N.; Brewer, C. F. Biochemistry 1994, 33, 2503-2508. (14) 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. 1993, 270, 5089-5097. (15) Wormald, M. R.; Edge, C. J.; Dwek, R. A. Biochem. Biophys. Res. Commun. 1991, 180, 1214-1221. (16) Geyer, A.; Gege, C.; Schmidt, R. R. Angew. Chem., Int. Ed. 1999, 38, 1466-1468. (17) Yu, Z. W.; Calvert, T. L.; Leckband, D. Biochemistry 1998, 37, 1540-1550. (18) Koshy, K. M.; Boggs, J. M. J. Biol. Chem. 1996, 271, 3496-3499. (19) Koshy, K. M.; Wang, J.; Boggs, J. M. Biophys. J. 1999, 77, 306318.

10.1021/la0258289 CCC: $22.00 © 2002 American Chemical Society Published on Web 07/30/2002

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Scheme 1. Synthesis and Abbreviations of Glycoconjugate Polystyrenes

action in biological recognition events. Recently, Geyer et al. devised a molecular assembly made of a LeX-bearing lipid bilayer that interacts with a LeX trisaccharide in the presence of Ca2+, but the affinity constant estimated by NMR spectroscopy was still low (Ka ∼ 2 M-1).16 Leckband et al. reported an attractive force between lactosylceramide-containing lipid membranes using a surface force apparatus.17 Boggs et al. presented evidence for a calciummediated carbohydrate-carbohydrate interaction between galactosylceramide and a 3-sulfated galactosylceramide using electrospray ionization mass spectrometry 18,19 and IR spectroscopy.20 To date, there are no model systems that reproduce the strong interaction between carbohydrates in biological recognition events. Various types of artificial glycoconjugate polymers carrying clustered oligosaccharide side chains have been developed to make use of the cluster effect to induce effective binding to the corresponding lectins, toxins, and viruses.21-29 We developed glycoconjugate polystyrenes in which highly concentrated oligosaccharide chains are attached to every repeating unit along the hydrophobic polystyrene main chain.23-27 We expected that the oligosaccharide-clustered array, combined with a clustered glycolipid Langmuir monolayer, would be a powerful tool to detect carbohydrate-carbohydrate interactions. Lipid monolayers at the air-water interface have recently received much attention not only as a model of molecular recognition on plasma membranes30-32 but also as a field to enhance the effectiveness of hydrogen bonding and electrostatic interaction in water.33 Sato et al. reported (20) Boggs, J. M.; Menikh, A.; Rangaraj, G. Biophys. J. 2000, 78, 874-885. (21) Roy, R. Trends Glycosci. Glycotechnol. 1996, 8, 79-99. (22) Bovin, N. V. Glycoconjugate J. 1998, 15, 431-446. (23) Kobayashi, K.; Tsuchida, A.; Usui, T.; Akaike, T. Macromolecules 1997, 30, 2016-2020. (24) Kobayashi, K.; Tawada, E.; Akaike, T.; Usui, T. Biochim. Biophys. Acta 1997, 1336, 117-122. (25) Matsuura, K.; Akasaka, T.; Hibino, M.; Kobayashi, K. Bioconjugate Chem. 2000, 11, 202-211. (26) Hasegawa, T.; Kondoh, S.; Matsuura, K.; Kobayashi, K. Macromolecules 1999, 32, 6595-6603. (27) Ohno, K.; Tsujii, Y.; Miyamoto, T.; Fukuda, T.; Goto, M.; Kobayashi, K.; Akaike, T. Macromolecules 1998, 31, 1064-1069. (28) Sigal, G. B.; Mammem, M.; Dahmann, G.; Whitesides, G. M. J. Am. Chem. Soc. 1996, 118, 3789-3800. (29) Kamitakahara, H.; Suzuki, T.; Nishigori, N.; Suzuki, Y.; Kanie, O.; Wong, C.-H. Angew. Chem., Int. Ed. 1998, 37, 1524-1528. (30) Maggio, B. Prog. Biophys. Mol. Biol. 1994, 62, 55-117. (31) Sato, T.; Serizawa, T.; Okahata, Y. Biochim. Biophys. Acta 1996, 1285, 14-20. (32) Sato, T.; Serizawa, T.; Otake, F.; Nakamura, M.; Terabayashi, T.; Kawanishi, Y.; Okahata, Y. Biochim. Biophys. Acta 1998, 1380, 82-92. (33) Ariga, K.; Kunitake, T. Acc. Chem. Res. 1998, 31, 371-378.

that a ganglioside monolayer on the air-water interface functioned as a cell surface model to recognize specific lectins and viruses.31,32 Recently, we found that the artificial glycoconjugate polymers and glycolipid monolayers at the air-water interface are useful molecular probes to investigate carbohydrate-carbohydrate interactions. We reported in the preliminary form highly sensitive detection of GM3Gg3 interactions by surface pressure-area (π-A) isotherms34 and also quantitative estimation of the interaction by surface plasmon resonance.35 The present paper describes the detailed study of a π-A isotherm of the GM3Gg3 interaction using various glycolipids (Figure 1), various glycoconjugate polystyrenes (Scheme 1), and some additives. As illustrated in Figure 2, the π-A isotherm of the glycolipid monolayer was greatly expanded when strong carbohydrate-carbohydrate interaction was induced between the glycolipid monolayer at the air-water interface and the glycoconjugate polystyrene in the subphase. Important information on the structureaffinity relationship in the GM3-Gg3 interaction has been obtained. Materials and Methods Materials. Deionized water with high resistivity (>18 MΩ cm) purified with a Milli-Q purification system (Millipore, Bedford, MA) was used as the subphase in a Langmuir trough. Spectral grade chloroform and methanol were used to spread lipids. GM1, GM2, GM3, GD3, lactosylceramide, and sphingomyelin were purchased from Sigma Chemical Co. (St. Louis, MO). KDN(GM3) was obtained from rainbow trout testis.36 GM437 and 2,6-GM338 were chemically prepared by Kiso et al. PN(Lac) and PN(GalLac) were synthesized according to the previously described procedure.23 Synthesis of Gg3 Trisaccharide Substituted Styrene Monomer. Gg3 trisaccharide was prepared by deprotection of the 2-(trimethylsilyl)ethyl glycoside derivative.39 The trisaccharide (50 mg, 0.092 mmol) and ammonium hydrogen carbonate were stirred in water (3.0 mL) in an open vessel at 37 °C for 1 (34) Preliminary report: Matsuura, K.; Kitakouji, H.; Tsuchida, A.; Sawada, N.; Ishida, H.; Kiso, M.; Kobayashi, K. Chem. Lett. 1998, 14931494. (35) Matsuura, K.; Kitakouji, H.; Sawada, N.; Ishida, H.; Kiso, M.; Kitajima, K.; Kobayashi, K. J. Am. Chem. Soc. 2000, 122, 7406-7407. (36) Yu, S.; Kitajima, K.; Inoue, S.; Khoo, K.-H.; Morris, H. R.; Dell, A.; Inoue, Y. Glycobiology 1995, 5, 207-218. (37) Murase, T.; Kameyama, A.; Kartha, K. P. R.; Ishida, H.; Kiso, M.; Hasegawa, A. J. Carbohydr. Chem. 1989, 8, 265-283. (38) Hasegawa, A.; Ogawa, M.; Kiso, M. Biosci. Biotechnol. Biochem. 1992, 56, 535-536. (39) Hotta, K.; Komba, S.; Ishida, H.; Kiso, M.; Hasegawa, A. J. Carbohydr. Chem. 1994, 13, 665-677.

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Figure 1. Structures and abbreviations of lipids used in this study.

Matsuura et al. water (3.0 mL). The mixture was stirred magnetically at 0-5 °C for 1 h, and then p-vinylbenzoyl chloride (0.15 mL, 1.1 mmol) in tetrahydrofuran (1.5 mL) was added. After 3 h, the mixture was washed with chloroform to remove unreacted p-vinylbenzoyl chloride. The solution was concentrated to 5-10 mL, and then the product was chromatographed on a TSKgel HW-40S column (eluent, water). The fractions containing Gg3 trisaccharide substituted styrene monomer were collected, desalted, and lyophilized. The yield was 55 mg (89%). 1H NMR (D2O): δ 2.21 (3H, s, NHCOCH3), 4.07-3.36 (18H, m, sugar), 4.58 and 4.42 (2H, m, J1′,2′ ) J1′′,2′′ ) 7.8 Hz, H-1′ and H-1′′), 5.18 (1H, d, J1,2 ) 9.2 Hz, H-1), 5.41 (1H, d, J ) 10.8 Hz, CH2dCH (cis)), 5.93 (1H, d, J ) 17.9 Hz, CH2dCH (trans)), 6.80 (1H, dd, J ) 10.8 and 17.9 Hz, CH2dCH), 7.56 and 7.79 (4H, d, J ) 8.3 Hz, C6H4). 13C NMR (D2O): δ 24.5 (CH3), 119.8 (C-2′′), 63.4-63.8 (C-6, C-6′, and C-6′′), 73.8-80.4 (the other pyranose carbons), 82.3 (C-1), 105.8 (C-1′ and C-1′′), 119.8 (CH2dCH), 135.2 and 144.3 (ipso-phenyl), 128.5 and 130.4 (meta- and ortho-phenyl), 138.8 (CH2dCH), 174.3 (CdO), 178.0 (CdO of sugar). Synthesis of PN(Gg3). A solution of the Gg3 trisaccharide substituted styrene monomer (55 mg, 0.082 mmol) in deionized water (0.5 mL) was degassed with an aspirator, and N,N,N′,N′tetramethylethylenediamine (12.5 µL, 1.0 mol %) and ammonium peroxodisulfate (2.5 mg, 13 mol %) were added. The mixture was stirred at room temperature for 2 h. The product was precipitated from its aqueous solution into methanol. The precipitate was dissolved in water, dialyzed in a cellulose tube (cutoff molecular weight ) 3500; Nacalai Tesque, Kyoto) against water for 3 days, and freeze-dried to give a white powdery polymer, 52 mg (95%). 1H NMR (D O): δ 2.26-1.02 (3H, CH CH), 2.09 (3H, br-s, 2 2NHCOCH3), 4.71-3.20 (20H, sugar), 5.19 (1H, br-s, H-1), 6.52 and 7.41 (4H, C6H4). The number-average molecular weights (Mn) of PN(Gg3), PN(Cel), PN(Lac), and PN(GalLac) were estimated by size exclusion chromatography (SEC) using pullulans as standards and water as the eluent. π-A Isotherms. Lipid solution (typically 0.243 mg/mL) in spectroscopic grade chloroform/methanol (4/1 v/v, 20 µL) was spread on pure water and on aqueous solutions (100 mL) of glycoconjugate polymers ([oligosaccharide] ) 1 × 10-12 to 10-6 M) in a computer-controlled Miyata type moving wall trough (Nippon Laser & Electronics Lab., Nagoya). After the organic solvents were evaporated for 20 min, compression was started at a rate of 6.9 mm2 s-1. The temperature of the subphase was maintained at 25 ( 0.2 °C.

Results

Figure 2. Schematic illustration of detection of carbohydratecarbohydrate interaction at the air-water interface using a Langmuir trough. day. Ammonium hydrogen carbonate (total amount, 5.6 g) was added at intervals to ensure saturation. When thin-layer chromatography (TLC) indicated no more conversion, the mixture was diluted with water (10 mL) and then concentrated in a rotary evaporator. Excess ammonium hydrogen carbonate was removed by repeating dilution of the residue with water and concentration twice. Sodium carbonate (100 mg) and methanol (3.0 mL) were added to the resulting crude N-acetylgalactosaminyllactosylamine in

Syntheses of Glycoconjugate Polystyrenes. Scheme 1 shows the synthesis of N-glycoside-bearing polystyrenes. The trisaccharide GalNAcβ1-4Galβ1-4Glc of Gg3 was prepared by deprotection of the 2-(trimethylsilyl)ethyl glycoside derivative.39 β-Amino function was derived to the reducing terminal of GalNAcβ1-4Galβ1-4Glc, Galβ14Galβ1-4Glc, lactose, and cellobiose with ammonium hydrogen carbonate and then allowed to react with p-vinylbenzoyl chloride without isolation of the intermediate glycosamine in 59-89% yield. These monomers were identified by 1H NMR, 1H-1H correlation spectroscopy (COSY), 13C NMR, and mass spectroscopy. The polymerization was performed using a redox initiator (ammonium peroxodisulfate and N,N,N′,N′-tetramethylethylenediamine) in degassed water to afford the corresponding glycoconjugate polystyrenes, as summarized in Table 1. The polymers were soluble in water and dimethyl sulfoxide (DMSO). 1H NMR spectra of the polymers in D2O became broad, and the vinyl signals of the monomer disappeared. The number-average molecular weights (Mn) of the glycopolymers estimated by size exclusion chromatography were on the order of 104, as listed in Table 1. Interaction of GM3 Monolayers with Glycoconjugate Polystyrenes. Figure 3A shows π-A isotherms of GM3 in the absence and presence of PN(Gg3) and its

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Table 1. Synthesis of Glyconjugate Polymers polymer PN(Gg3) PN(Lac) PN(Cel) PN(GalLac)

monomer 0.055 (0.082) 1.00 (2.1) 0.23 (0.49) 0.10 (0.16)

AP,a equiv 0.14 0.10 0.10 0.10

TMEDA,b equiv 1.0 0.79 0.51 0.51

water, mL 0.5 25d 12 12

time, h 5 2 5 5

yield, g (%)

Mnc

Mw/Mnc

0.052 (95) 0.90 (90) 0.21 (93) 0.076 (76)

2.9 × 2.0 × 104 3.4 × 104 5.2 × 104

1.5 1.8 3.0 4.0

a Ammonium peroxodisulfate. b Tetramethylethylenediamine. c Estimated by SEC (pullulan standard, in water). (5 mL).

Figure 3. (A) π-A Isotherms of GM3 in the presence of PN(Gg3) (a) and its monomer (b) in the subphase and on pure water (c) at 25 °C. The concentration of oligosaccharide units in the subphase is 1 × 10-8 M. (B) π-A Isotherms of GM3 in the presence of PN(Gg3) (a), PN(GalLac) (b), PN(Lac) (c), and PN(Cel) (d) in the subphase and on pure water (e) at 25 °C. The concentration of oligosaccharide units in the subphase is 1 × 10-8 M. (C) Concentration dependence of the oligosaccharide units of glycopolymers on the expansion amount of the surface area of GM3 monolayers at 30 mN m-1.

monomer (1 × 10-8 M) in the subphase at 25 °C. The π-A isotherm (curve c) of the GM3 monolayer on pure water gave a monotonic and smooth curve to collapse at 57 mN/m without passing through a phase of liquid-expanded film.30 The limiting molecule-occupied area of the GM3 monolayer was calculated to be 62 Å2. Addition of 1 × 10-8 M PN(Gg3) (curve a) into the subphase caused significant expansion of the GM3 monolayer over the whole range of surface pressure, and the apparent limiting moleculeoccupied area of GM3 was increased to 88 Å2. The expansion was observed even at the collapsing pressure

104

d

Containing DMSO

at which the monolayer exists as a solid condensed film. Because the GM3 monolayer was minimally expanded by the Gg3 monomer (curve b), a large expansion of the GM3 monolayer with PN(Gg3) was induced by specific binding due to the carbohydrate-carbohydrate interactions between GM3 trisaccharides and clustered Gg3 trisaccharides along the polymer chain. Figure 3B shows π-A isotherms of the GM3 monolayer in the absence and presence of various glycoconjugate polystyrenes (1 × 10-8 M) in the subphase. PN(GalLac), in which Gal is substituted in place of GalNAc in the nonreducing terminal of PN(Gg3), expanded the GM3 monolayer a little, suggesting that the NHAc group in Gg3 has an important role. PN(Lac), in which GalNAc is deleted from PN(Gg3), also expanded the GM3 monolayer, but the increment of the area was about half that with PN(Gg3). This observation is consistent with the previous reports on the weak interaction between GM3 and lactosylceramide.8,9 On the other hand, PN(Cel), in which the configuration at the 4′ position is inverted from PN(Lac), expanded the GM3 monolayer a little. Therefore, the GM3 monolayer specifically recognized the carbohydrate structures in the glycoconjugate polystyrenes in the subphase. π-A Isotherms of GM3 were measured in the presence of a wide range of concentrations (10-12 to 10-6 M) of these glycoconjugate polymers, and the increments of area per molecule ∆A of GM3 at 30 mN/m, which is reported to be the surface pressure of plasma membranes,30 were plotted against sugar concentrations in Figure 3C. PN(Gg3) and PN(Lac) expanded the GM3 monolayer, depending on the sugar concentration, whereas PN(GalLac) and PN(Cel) expanded the monolayer a little, even at 1 µM. PN(Gg3) expanded the GM3 monolayer specifically, even at a very low concentration (1 pM). The concentration dependency produced a gentle slope over a wide concentration range. This tendency is not consistent with the Langmuir type isotherm but suggestive of the interaction responsible for indefinite binding sites. Interaction of Various Glycolipid Monolayers with Glycoconjugate Polystyrenes. Figure 4A summarizes the plots of the increments of area per molecule of various lipids at 30 mN/m against the Gg3 concentration. The sphingomyelin (SM) monolayer was little expanded by PN(Gg3) at 30 mN/m. The expansion of (KDN)GM3, which is substituted with OH instead of the NHAc group in NeuNAc of GM3, was much smaller than that of GM3 and similar to that of LacCer over the entire range of concentrations of PN(Gg3). Thus, not only the NHAc group in GalNAc of Gg3 but also the NHAc group in NeuNAc of GM3 has an important role in the GM3-Gg3 interaction. The expansion of monolayers of other gangliosides (GM1, GM2, GM4, GD3, and 2,6-isoGM3) by PN(Gg3) in the subphase was also examined and summarized in Figure 4B. The GM2 monolayer was expanded by about half as much as the GM3 monolayer and similarly to the LacCer and (KDN)GM3 monolayers. The GM1 expanded a little, even at 1 µM of PN(Gg3). The interaction of GM3 with PN(Gg3) was decreased by substitution of the 3-position of Gal in GM3 with a monosaccharide and

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Figure 4. Increment of area per molecule of the monolayers of various glycolipids (A) and gangliosides (B) at 30 mN m-1 in the presence of PN(Gg3) in the subphase.

Figure 5. Increment of area per molecule at 30 mN/m induced by 1 × 10-8 M PN(Gg3) in the subphase at 25 °C against GM3 content in the mixed monolayers of GM3 and SM.

further with a disaccharide. GM4, GD3, and 2,6-isoGM3 monolayers were only slightly expanded by PN(Gg3), indicating that PN(Gg3) recognizes the trisaccharide structure of GM3 as a whole. π-A Isotherms of mixed monolayers of GM3 with SM were measured in the presence and absence of PN(Gg3) at 25 °C, and the increments of area per molecule at 30 mN/m were plotted against the GM3 content (Figure 5). If the mixed monolayers interact with PN(Gg3) at the same intensity independently of the GM3 content, the increment should be proportional to the GM3 content (broken line in Figure 5). The plots produced a downward curve up to the largest expansion of the GM3 monolayer (100%). The carbohydrate-carbohydrate interaction is suggested to be sensitive to the extent of clustering of the GM3.

Matsuura et al.

Figure 6. (A) Effect of Ca2+ on the expansion of the GM3 monolayer induced by PN(Gg3): (a) 1 × 10-8 M PN(Gg3), (b) 1 × 10-8 M PN(Gg3) and 1 mM CaCl2, (c) 1 mM CaCl2, and (d) on pure water. (B) Effect of urea on expansion of the GM3 monolayer induced by PN(Gg3): (a) 1 × 10-8 M PN(Gg3), (b) 1 × 10-8 M PN(Gg3) and 1 mM urea, (c) 1 mM urea, and (d) on pure water.

Effect of Ca2+ on the GM3-Gg3 Interaction. The effect of Ca2+ on the π-A isotherms of the GM3 monolayer was investigated by adding 1 mM CaCl2 to the subphase in the absence and presence of PN(Gg3). As shown in Figure 6A, the expansion of the GM3 monolayer induced by PN(Gg3) was not affected by Ca2+, indicating that Ca2+ was not required for the GM3/Gg3 interaction in the present system. Effect of Urea on the GM3-Gg3 Interaction. As shown in Figure 6B, the expansion of the GM3 monolayer by PN(Gg3) (10-8 M) was suppressed unequivocally by the addition of 1 mM urea to the subphase. The suppression effect with urea was also observed at the concentrations of 10-12 and 10-10 M PN(Gg3). These results suggest that hydrogen bonding is important in the GM3-Gg3 interaction. Inhibition of the GM3-Gg3 Interaction with Monosaccharides. π-A Isotherms of the GM3 monolayer induced by PN(Gg3) were produced by adding various kinds of monosaccharide derivatives (1 mM) to the subphase. Figure 7 summarizes the expansivity of the GM3 monolayer at 30 mN/m as a measure of the interaction. The GM3/PN(Gg3) interaction was strongly inhibited by all the acetamido monosaccharides tested: GlcNAc, GalNAc, ManNAc, and NeuNAc. In contrast, the interaction was affected little by Glc, Gal, glucosamine (GlcN), and galactosamine (GalN). The presence of an acetamide group is essential in the interaction. The interaction was intermediately inhibited by Man and the methyl ester of NeuNAc. Esterification of NeuNAc reduced the inhibitory effect of free NeuNAc on the interaction,

Carbohydrate Interaction at Air-Water Interface

Figure 7. Effect of 1 mM monosaccharides, urea, and Ca2+ on the expansion ratio of the GM3 monolayer at 30 mN/m in the presence of 10 nM PN(Gg3).

although the methyl ester still moderately inhibited, therefore suggesting the participation of the carboxyl group of GM3 in the interaction. The inhibition by mannose was unexpected, and the cause is not clear. Discussion We present a model system that is useful for investigating carbohydrate-carbohydrate interactions in biological recognition events. The model system is constructed from clustered oligosaccharide chains along the polymer backbone and also in the glycolipid Langmuir monolayer at the air-water interface to amplify carbohydratecarbohydrate interactions. Remarkable expansion of the π-A isotherm of GM3 monolayers was induced by specific carbohydrate-carbohydrate interaction with the Gg3bearing glycoconjugate polystyrene [PN(Gg3)]. The significance of the glyco-cluster effect in carbohydratecarbohydrate interactions is also demonstrated by the experiments using the Gg3-substituted styrene monomer and the mixed GM3/SM glycolipid monolayers. The importance of the NHAc groups in the GM3-Gg3 interaction is suggested by the π-A isotherms using various combinations of glycolipids and glycoconjugate polymers and by inhibition with N-acetamide sugars. It is also suggested on the basis of the expansion of monolayers of various gangliosides ((KDN)GM3, GM1, GM2, GM4, GD3, and 2,6-isoGM3) that PN(Gg3) in the subphase recognizes not only some specified portions of GM3 but also the trisaccharide as a whole. It is also

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important to note that the expansion of GM3 monolayers against a wide range of concentrations (10-12 to 10-6 M) of PN(Gg3) was not consistent with the Langmuir type isotherm but suggestive of the interaction responsible for indefinite binding sites. Ca2+ was not required for the GM3/Gg3 interaction in the present system. This is in contrast to Hakomori’s report:7 the interaction of GM3 liposomes with a Gg3coated polystyrene surface is strongly promoted by Ca2+ and inhibited by EDTA. One important difference between the two systems is the aqueous environment in which the molecular recognition is induced: at the air-water interface in the present system and at the solid-water interface in Hakomori’s system. It was recently reported33 that hydrogen bonding is reinforced at the air-water interface rather than in bulk water. It is probable that the hydrogen bonds in the GM3/PN(Gg3) interaction at the air-water interface are strong enough to cause the expansion of the monolayer, even in the absence of Ca2+. The presence of complementary hydrophobic surfaces between the respective trisaccharides may be a driving force of the GM3-Gg3 interaction, as proposed by Hakomori.7 The present study demonstrated the important role of NHAc groups in both GM3 and Gg3, the inhibition effect of urea, and also no requirement of Ca2+. It is reasonable to assume that hydrogen bonds of NHAc groups may also participate in the GM3-Gg3 interaction. It is also interesting to compare this result with carbohydrateprotein interactions,40 which are discussed on the importance of hydration and reorganization of water molecules in addition to the roles of key polar interactions, intraand intermolecular hydrogen bondings, and polyamphiphilicity in aqueous solution. Although π-A isotherms are not sufficiently quantitative to determine the affinity constant of the interaction, the method will be useful to elucidate the mechanism and to detect novel carbohydrate combinations in carbohydrate-carbohydrate interaction. Acknowledgment. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, and Culture. We are grateful to Professor Yoshio Okahata and Dr. Mineo Hashizume of the Tokyo Institute of Technology and Professor Toshinori Sato of Keio University for their useful suggestions. LA0258289 (40) Lemieux, R. U. Acc. Chem. Res. 1996, 29, 373-380.