2 Faculty of Pharmaceutical Sciences, Kinki University, Kowakae 3-4-1, Higashi-osaka 577-8502, Japan; 3 National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba 305-8602, Japan; and 4 Faculty of Agriculture, Meiji University, Higashi-Mita 1-1-1, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan
Received on March 19, 2004; revised on May 5, 2004; accepted on May 5, 2004
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Abstract |
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Key words: capillary affinity electrophoresis / carbohydrate-binding specificity / 8-aminopyrene-1,3,6-trisulfonate / lectin
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Introduction |
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Most of the assay methods for carbohydrate-binding proteins, such as surface plasmon resonance (Shinohara et al., 1997), fluorescence polarization (Oda et al., 1998
), and time-resolved fluorometry (Lee et al., 1998
; Nakajima et al., 2002
), essentially require carbohydrates in a pure state. However, it is often too laborious to obtain pure carbohydrates in amounts necessary for kinetic studies by either chemical synthesis or isolation from natural sources.
We have developed capillary affinity electrophoresis (CAE) to analyze the molecular interaction between carbohydrates and proteins in solution state (Nakajima et al., 2003). A mixture of oligosaccharides derived from a glycoprotein was labeled with 8-aminopyrene-1,3,6-trisulfonate (APTS) and used without isolation of each oligosaccharide. Interaction between a lectin and each carbohydrate chain in a mixture was determined simultaneously based on their change of electrophoretic mobilities or peak intensities. Furthermore, we found that CAE allowed calculation of affinity constants without determination of the accurate concentrations of carbohydrates (i.e., ligands) that are often difficult to measure. We showed that CAE was applied to classify carbohydrate chains in biological samples using a few lectins (Nakajima et al., 2003
).
In the present study, we show that CAE can examine the presence of a lectin in a crude biological extract using an appropriate set of oligosaccharides. At the initial step, a mixture of oligosaccharides of known compositions is analyzed in the absence of lectin. Then the same mixture is analyzed in the same buffer containing the extract. If we observe changes of migration of oligosaccharides, such changes indicate that a substance (typically lectins) that interacts with the carbohydrates is present in the extract. Another set of a mixture of different oligosaccharides is analyzed in the same manner as described. By repeating these procedures, we can confirm the presence of a lectin and determine the detailed binding specificity of the lectin.
It is important to select an appropriate set of carbohydrates from various glycoproteins. In the previous study, we showed that human 1-acid glycoprotein (AGP), bovine IgGs, porcine thyroglobulin, bovine ribonuclease B, and bovine fetuin were appropriate as sources of N-linked oligosaccharides, because oligosaccharide compositions of these glycoproteins have been well characterized by a number of studies. The N-linked oligosaccharides or their desialylated oligosaccharides derived from these glycoproteins is shown in Table I. These oligosaccharides were previously labeled with APTS before use.
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Results |
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Migrations of the oligosaccharides in the presence of purified SSA and RIP are shown in Figure 2. The purified SSA obviously lowered the peak height of sialo-oligosaccharides and the peaks disappeared at the concentration of 0.8 µM (Figure 2A) as observed for the crude extract, but SSA did not affect the migration of asialo-oligosaccharides (Figure 2B). In contrast, RIP did not cause change of migrations of sialo-oligosaccharides (Figure 2A). However, asialo-oligosaccharides (2 and 4) showed higher affinities to RIP than those having a fucose residue (3 and 5) and were observed gradually later with increase of the concentrations of the lectin. The peaks of these carbohydrates were overlapped at 8 µM RIP (Figure 2B). It should be noted that SSA showed clear binding at 0.8 µM, but RIP required 8 µM concentration for specific binding to asialo-oligosaccharides.
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Interactions between crude protein fraction in tulip bulbs and the carbohydrates derived from a few glycoproteins are shown in Figure 3. In the electrolyte-containing crude extract (5 mg/ml), migrations of asialo-oligosaccharides including (10) derived from porcine thyroglobulin showed dramatic changes, and the major peaks became broad (Figure 3AII). Asialo-oligosaccharides derived from AGP showed interesting binding to the crude extract, and peaks derived from triantennary oligosaccharides (2 and 3) obviously became broader and appeared later on addition of the crude extract in the buffer (Figure 3BII). In contrast, presence of the crude extract of tulip bulbs did not cause obvious changes of migrations of any oligosaccharides derived from ribonuclease B (Figure 3CII). In the previous article, Oda and Minami (1986) reported that TGL showed specific affinity to mannan derived from yeast. Because the structures of mannan from yeast and high-mannose oligosaccharides are different, TGL did not show affinity toward high-mannose oligosaccharides derived from ribonuclease B.
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AGP contains di- (1), tri- (2 and 3), and tetra- (4 and 5) antennary oligosaccharides. Some of the tri- and tetraantennary oligosaccharides are substituted with a fucose residue at one of the lactosamine branch to form sialyl Lewis x structure (3 and 5).
Figure 4 shows the interactions between asialo-oligosaccharides of AGP and TGA at various concentrations. Addition of TGA in the electrolyte caused specific retardation of the migration times of triantennary carbohydrates (2 and 3), but did not affect the migrations of tetraantennary carbohydrates (4 and 5). Peaks of tri- (2 and 3) and tetra- (4 and 5) antennary carbohydrate chains were overlapped at 4.5 µM TGA, and finally, the migration order of them were reversed and the peaks of 2 and 3 were fused to a broad peak at 12.0 µM TGA and observed at 7.7 min.
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Fetuin contains two triantennary oligosaccharides (2 and 6) and a diantennary oligosaccharide (1). One (2) of the triantennary oligosaccharides has three Galß(1-4)GlcNAc branches, and the other (6) contains one Galß(1-3)GlcNAc as well as two Galß(1-4)GlcNAc branches (Table I). TGA clearly distinguished these triantennary oligosaccharides, as shown in Figure 5.
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Bovine IgG contains four major diantennary oligosaccharides (710) that have (1-6) linked fucose residues at chitobiose portion of the reducing end. Interestingly, TGA showed obvious retardation effect on the migration times of these oligosaccharides (710), as shown in Figure 6. The complete form (10) of diantennary oligosaccharide was observed latest, and the one lacking both Gal residues (7) showed the weakest interaction with TGA (Figure 6A).
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Porcine thyroglobulin contains complex type oligosaccharides (10 and 14) and high-mannose type oligosaccharides (HM in Figure 7) as minor oligosaccharides (Kakehi et al., 1999). Addition of TGA decreased the peak intensity of triantennary oligosaccharide (14) even at 0.2 µM TGA in the electrolyte and also retarded diantennary oligosaccharide (10) at higher concentrations than 0.8 µM TGA, as observed for the interactions with oligosaccharides derived from IgG. Migration times of high-mannose oligosaccharides were not changed even in the presence of 12.0 µM TGA.
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Discussion |
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In the crude extract of tulip bulbs, lectin activity of TGA was also easily detected by CAE. However, it was difficult to detect the activity of another lectin in tulip bulbs, TGL. TGL showed only small effect on migration times of high-mannose oligosaccharides derived from ribonuclease B, because TGL preferentially binds manno-oligosaccharides derived from yeast cells.
We also showed that CAE is a powerful method for studies on the detailed carbohydrate-binding specificity using TGA. Because the method allows observation of the interactions of oligosaccharides simultaneously, relative affinities of each oligosaccharide can be easily calculated. TGA showed higher affinity to triantennary oligosaccharides (2 and 3) than diantennary carbohydrate (1), whereas TGA showed no significant interactions with tetraantennary carbohydrates (4 and 5). Among triantennary oligosaccharides, 2 and 3 showed almost the same affinities, but affinity of 6 was higher than that of 2. These indicate that Fuc residue linked through (1-3) linkage to GlcNAc in the outer chain does not interfere the binding with TGA, and the triantennary oligosaccharide with Galß(1-3)GlcNAc at the nonreducing terminus binds more tightly than that having Galß(1-4)GlcNAc. Fucosidase digestion of oligosaccharides derived from bovine IgG reduced their affinities toward TGA, indicating that Fuc residues linked through
(1-6) linkage to the innermost GlcNAc are involved in the lectin binding.
In conclusion, TGA shows the highest affinity to triantennary carbohydrates with three Gal residues, especially, Gal with ß(1-3) linkage at nonreducing termini and diantennary oligosaccharide with a Fuc residue linked through (1-6) linkage to the innermost GlcNAc. These characteristic affinities are well described in Table II. Galactose-binding lectin in the seeds of Tetracarpidium conophorum agglutinin (TCA) has been reported to show similar affinities to those of TGA (Sato et al., 1991
). This lectin shows high affinity to triantennary oligosaccharides but only weak binding to tetraantennary glycans. However, TCA binds more tightly to Galß(1-4)GlcNAc than to Galß(1-3)GlcNAc residue. Furthermore, fucose residue linked through
(1-6) linkage to GlcNAc in diantennary oligosaccharide does not affect the binding for TCA. Datura stramonium seed lectin and Phaseolus vulgaris leukoagglutinin (PHA) also show high affinities to N-glycans of complex type carrying Gal residues at the nonreducing termini (Kaneda et al., 2002
; Yamashita et al., 1987
). However, these lectins bind tetraantennary glycans as well as triantennary glycans. It should be noted that TGA does not recognize tetraantennary oligosaccharides. Thus we could determine the detailed carbohydrate-binding specificity of TGA using four sets of oligosaccharide mixture derived from fetuin, IgG, thyroglobulin, and AGP as glycan libraries.
With the present method, we found that the binding between carbohydrates and a lectin is observed in two different manners. Substantial peak broadening and peak retardation are observed in many of the figures, and in some cases, peak disappearance suggested that ligand exchange rates are comparable or slow compared with electrophoretic migration.
We are collecting the data for carbohydrateprotein interactions and found that ligand exchange rates largely depend on the lectins. For example, lectins such as Concanavalin A, Ricinus communis agglutinin (RCA), and SSA show peak disappearing effect, but those such as TGA, wheat germ agglutinin, and PHA show peak retardation effect. Although precise mechanism showing such difference is not clear, these characteristics are useful for analysis of complex mixture of oligosaccharides.
Some lectins are known to recognize N-linked carbohydrates as well as O-linked carbohydrates. Matsumoto et al. (2001) proposed a dot-blot method for screening lectins on a membrane using glycoproteins coupled with enzyme or biotinyl albumin. This method does not require multivalent binding between protein and ligand, unlike hemagglutination assay, and allows screening of lectins from 16 cultivable mushrooms. In CAE, interactions of a lectin with each carbohydrate chain in a mixture of carbohydrates (glycan library) can be observed at the same time. Therefore when a lectin activity is detected in screening assay, we can know which of carbohydrate chains in glycan libraries are responsible for the binding with the lectin. Furthermore, it should be emphasized that the binding affinity for the interaction can be easily estimated. Thus the present method using CAE may be a powerful tool for finding a new lectin.
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Materials and methods |
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Preparation of a mixture of fluorescent-labeled oligosaccharides from glycoprotein sample
We prepared the fluorescent-labeled oligosaccharides (glycan library) from AGP, fetuin, porcine thyroglobulin, bovine IgG, and ribonuclease B (bovine pancreas) (Kakehi and Honda, 1996; Ma and Nashabeh, 1999
).
Briefly, a sample of glycoprotein (1 mg) was dissolved in 20 mM phosphate buffer (pH 7.0, 50 µl), and N-glycoamidase F (5 mU, 5 µl) was added. After the mixture was incubated at 37°C for 24 h, the solution was kept in a boiling water bath for 5 min and centrifuged at 10,000 x g for 10 min. The supernatant containing the oligosaccharides was evaporated to dryness by a centrifugal vacuum evaporator (Speed Vac, Savant, Farmingdale, NY).
When asialo-oligosaccharides were prepared, the residue was dissolved in 2 M aqueous acetic acid (50 µl), and the mixture was kept at 80°C for 3 h to remove sialic acids (Morimoto et al., 2001). After evaporation of the mixture, the residue was dissolved in 15% aqueous acetic acid (5 µl) containing APTS at the concentration of 100 mM. A freshly prepared solution of 1 M NaBH3CN in tetrahydrofuran (5 µl) was added to the mixture. The mixture was overlaid with mineral oil (100 µl, nD 1.4670, d 0.838; Aldrich) to prevent evaporation of the reaction solvent (Ma and Nashabeh, 1999
; Sei et al., 2002
). The mixture was kept at 55°C for 90 min. Water (200 µl) was added to the mixture, and the fluorescent yellowish aqueous phase (lower layer) was collected. The aqueous layer was applied on a column of Sephadex G-25 (1 cm, 50 cm length) equilibrated with water. The fluorescent fractions eluted earlier were pooled and evaporated to dryness. The dried fluorescent oligosaccharides were stable at least for several months at 25°C. The residue was dissolved in water (100 µl), and a portion (10 µl) was used for CAE.
Preparation of defucosylated oligosaccharides
Defucosylated oligosaccharides were obtained from the oligosaccharide mixture derived from bovine IgG (1 mg) after digestion with -L-fucosidase (200 mU) in 60 µl 50 mM sodium citrate buffer (pH 5.0) at 37°C for 24 h, and then were labeled with APTS as described.
Preparation of crude protein fractions and purified lectins from Japanese elderberry and tulip bulbs
Pulverized bark (50 mg) was suspended in 1 ml of 100 mM Trisacetate buffer (pH 7.4) using a vortex mixer, and the supernatant obtained by centrifugation (3000 x g for 10 min) was used as the crude protein fractions. Crude protein fractions from tulip bulbs were prepared as previously reported (Oda and Minami, 1986). Briefly, two tulip bulbs (about 70 g) were homogenized with a Waring blendor with water (300 ml). After centrifugation of the mixture, solid ammonium sulfate was added to the supernatant to the concentration of 60% saturation. The precipitate collected by centrifugation was dissolved in 100 ml distilled water and used as crude protein fraction after dialysis against distilled water. This brief fractionation with ammonium sulfate was necessary because tulip bulbs contain large amounts of manno-polysaccharides, and binding reactions became unclear. TGL and TGA were purified by affinity chromatography using mannan-Sepharose and thyroglobulin-Sepharose, respectively, as reported previously (Oda and Minami, 1986
).
Protein assay
Protein concentrations were determined based on Lowry method using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA) (Schaffner and Weissmann, 1973).
CAE
CAE was performed using a P/ACE MDQ glycoprotein system (Beckman Coulter) equipped with an eCAP N-CHO capillary (20 cm effective length, 30 cm total length, 50 µm ID, Beckman Coulter) using an argon laser-induced fluorescence detector as described previously (Nakajima et al., 2003). Detection was performed by installing a 520-nm filter for emission with a 488-nm argon laser for excitation. Trisacetate buffer (100 mM, pH 7.4) was used as the electrolyte throughout the work. The sample solution was introduced to the capillary by pressure method (0.5 psi, 5 s). Separation was performed at 25°C at the applied potential of 18 kV. Data were collected and analyzed with a standard 32 Karat software (version 4.0, Beckman Coulter) on Microsoft Windows 2000. The procedures are briefly as follows.
Prior to CAE, a mixture of fluorescent-labeled oligosaccharides was analyzed in the absence of lectin. Then the same electrolyte containing a lectin at the specified concentration was filled in the capillary, and the same mixture of fluorescent oligosaccharides was analyzed. After observing the migrations at different concentrations of the lectin, association constants for the interactions were calculated according to the method as reported previously (Nakajima et al., 2003).
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Footnotes |
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Abbreviations |
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References |
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