Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada, and 2Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 481090606, USA
Received on July 13, 2000; revised on September 25, 2000; accepted on September 25, 2000.
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Abstract |
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Key words: binding specificity/dissociation constant/frontal affinity chromatography coupled to electrospray mass spectrometry/lectin/Neu5Ac26Galß14Glc/GlcNAc containing oligosaccharides
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Introduction |
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Frontal affinity chromatography coupled online to an electrospray mass spectrometer (FAC/MS) is a recently developed screening method for high-throughput screening of synthetic combinatorial libraries and compound mixtures (Schriemer and Hindsgaul, 1998; Schriemer et al., 1998
). It is a chromatographic technique in which an affinity column is prepared by immobilizing a biological receptor (antibody, enzyme, etc.). A sample consisting of a mixture of compounds is then continuously infused through the column. The order of elution parallels the order of affinity, with the "strongest" ligands eluting the latest. FAC/MS incorporates two-dimensional (intensity vs. m/z) electrospray mass spectrometry for effluent monitoring, allowing the analysis of compound mixtures in a single run. The dissociation constants (Kd) of active ligands in the mixtures can be determined as described by Kasai and coworkers (Kasai et al., 1986
). We present herein the application of FAC/MS to quantitate the binding properties of PSL.
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Results |
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The column capacity was determined experimentally based on Equation 1, which governs the relationship between the retention volume (Vx V0), the dissociation constant (Kd), the concentration of a ligand [X]0 and the column capacity (Bt) (Kasai et al., 1986).
(1)
Neu5Ac23Galß14GlcNAcß-O-MCO (5) and Neu5Ac-
26Galß14Glcß-O-MCO (2) were selected as the "void volume marker and ligand indicator." The void volume of the PSL column was also confirmed by using non-sialylated sugars such as, GlcNAcß-O-MCO and Galß14Glcß-O-MCO. They all broke through at the same time as compound 5, indicating that 5 was not retarded by the PSL column. A series of solutions containing varying concentrations of 2 (from 1 to 20 µM) and a constant concentration of 5 (1 µM) were prepared and infused into the PSL affinity column. The corresponding V-V0 values were measured. A plot of {[X]0(V-V0)}1 versus [X]0-1 was generated (Figure 2) where the reciprocal of the y-intercept indicates a Bt of 556 pmol, the number of active sites. This number corresponds to 278 pmol of active immobilized dimer (Mo et al., 2000
). The dissociation constant of 2 was thus calculated by slope to be 12.2 ± 0.6 µM.
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Figure 3 shows two examples of specificity studies performed using FAC/MS. Figure 3a shows that a mixture of two human milk oligosaccharides, LST b (9) and LST c (10), can be separated by the PSL affinity column. In this case, both LST b and c are Neu5Ac26 terminated pentasaccharides with the same molecular weight. The retarded phase is due to the binding of LST c (10), which has a Neu5Ac
26Galß14GlcNAc non-reducing end. LST b (9) did not show significant binding activity because the sialic acid group is on the 6-position of GlcNAc, instead of the required 6-position of Gal. The Kdmix for the isomer LST c (10) was estimated as 16.8 µM.
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It can be seen that the front of 22 is more diffuse (less steep) than that of 21, suggesting that 22 may be composed of two major unresolved isomeric species with similar affinity toward PSL. This is supported by the results of studies using high performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) on these compounds (Townsend et al., 1988, 1989). The HPAE-PAD chromatograms showed that sample 21 is composed of one major oligosaccharide (
90%) and three minor ones, and that the 22 sample contains two major structures (3:2 ratio) and five or six other minor compounds. The front of 21 broke through later than that of 22. This result suggests that there might be cooperativity in the multiple binding of
26 linkages. Such cooperativity would not exist when both
23 and
26-linked structures are present in the same molecule. This is supported by the finding that fetuin, which contains both
23 and
26-linked N-acetylneuraminic acids, did not form a precipitate with the lectin until the Neu5Ac groups were removed and resialylated with 2,6-sialyltransferase (Mo et al., 2000
).
The dissociation constants of all 22 oligosaccharides were then determined individually. That is, each oligosaccharide was mixed with the void volume marker 5 and then injected into the PSL affinity column for FAC/MS analysis. Table I lists the Kd values determined individually. Neu5Ac23 terminated oligosaccharides used in this study include Neu5Ac
23Galß14Glc/GlcNAc (4 and 5), Neu5Ac
23Galß13GlcNAc (12) and their fucosylated derivatives (7, 11, and 13). One synthetic ß anomer, Neu5Acß23Galß14GlcNAc (6), was also used. All Neu5Ac2-3 terminated oligosaccharides eluted at the void volume from the PSL affinity column (no retardation observed compared to the void volume marker 5). Therefore, in consideration of the measurement accuracy, their dissociation constants were estimated to be greater than 1000 µM, if indeed they were recognized at all. Only the Neu5Ac
26Galß14Glc/GlcNAc sequence-containing oligosaccharides showed high binding activity against the immobilized PSL; among them, Neu5Ac
26Galß14GlcNAc displayed the highest affinity with a Kd value of 10 µM. However, if the Neu5Ac group is
26 linked to a non-terminal sugar, such as in 8 and 9, the structure is not bound.
The binding affinity between 23 and
26 linked isomers differs by at least 100-fold (Table I). The affinity order of Neu5Ac2-6 terminated trisaccharides was in the order: Neu5Ac
26Galß14GlcNAc > Neu5Ac
26Galß14Glc-O-MCO > Neu5Ac
26Galß14Glc, indicating only minor contributions to binding by the NAc and aglycone groups. The dominant recognition of the terminal disaccharide unit is further confirmed by the fact that the tetrasaccharide 10 binds as well as the trisaccharides.
Six sulfated LacNAc (Galß14GlcNAc) derivatives were also evaluated individually using FAC/MS. Among them, 6-O-sulfo LacNAc had the strongest binding affinity toward the lectin (Kd = 259 ± 19 µM). This is 20 times weaker (G = 8.0 KJ·mol1) than Neu5Ac
26Galß14GlcNAc, demonstrating the importance of the sialic acid residue in PSL binding. Interestingly, the internal 6-O-sulfo LacNAc 16 also binds, though with lower affinity (Kd = 578 ± 64 µM). The remaining sulfated compounds were inactive.
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Discussion |
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Another advantage of FAC/MS is the miniaturization of the column to the 220 µl scale, which reduces the time of the experiment to 10 min and consumes less protein and valuable oligosaccharides. In general, not more than 1 nmol of protein is used in the preparation of a micro-scale column. A column prepared by this method can be used repeatedly with careful handling. For example, the activity of the PSL column used in the present study still remained at 95% after about 300 runs over a period of 6 months. Only micrograms of ligands are needed for an FAC/MS experiment. A single run consumed only about 0.1 µg of oligosaccharide.
From the dissociation constant data shown in Table I, it can be concluded that PSL possesses combining sites that recognize an N-acetylneuraminic acid 26 linked to a ß-galactosyl group (Mo et al., 2000
) that can be further attached to Glc or GlcNAc. Surprisingly, both 6- and 6'-O-sulfo LacNAc derivatives 15 and 16 bound to the lectin, though 20 times weaker than the 6'-O-sialyllated structure. Caution should therefore be exercised when using PSL alone as a structure-determination tool.
To our knowledge, this Polyporus squamosus lectin has the highest specificity toward 26 linked sialo-oligosaccharides among the known lectins. A lectin from the fruiting body of Psathyrella velutina mushroom has recently been reported to be specific for non-reducing terminal N-acetylneuraminic acid (Ueda et al., 1999
); however, it cannot distinguish
23 and
26 linkages. This lectin also strongly binds to non-reducing terminal N-acetylglucosamine residues (Kobata et al., 1994
). Another lectin isolated from tuberous roots of Trichosanthes japonica was reported to have an affinity similar to that of PSL (Yamashita et al., 1992
). That lectin also recognized Neu5Ac
26Galß14GlcNAc, but was totally inactive toward the
23 sialylated Galß14GlcNAc. However, the authors claimed that the lectin is highly specific to both HSO3-6Galß14GlcNAc and Neu5Ac
26Galß14GlcNAc.
In summary, we have shown that FAC/MS can be applied for the rapid determination of the carbohydrate-binding specificity of a lectin. The results confirmed and extended earlier study using quantitative precipitation, hapten inhibition, and quenching of specific intrinsic fluorescence (Mo et al., 2000). This method can be applied to studies of any ligandreceptor binding, but we focused here only on a carbohydrate-binding protein. As the dissociation constant of each ligand can be estimated from a single FAC/MS run, the structureactivity relationships (SAR) of ligands can be performed using mixtures. We suggest that the micro-scale FAC/MS screening method will be a valuable asset in biological affinity studies, in addition its main application of high throughput screening of combinatorial libraries.
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Materials and methods |
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Two micro-scale columns (Schriemer et al., 1998) with identical column volumes (9.8 µl) were prepared by packing controlled porous glass beads covalently coupled to streptavidin (CPG-SA, CPG Inc., USA) into orange PEEK tubings (ID, 0.50 mm; length, 50 mm). One column was saturated with d-biotin (1 ml, 0.2 mg/ml in PBS buffer) and served as a blank column for control experiments. The other column was saturated with biotinylated PSL in PBS buffer (0.5 mg/ml) by infusion at flow rate of 8 µl/min for 120 min. The PSL affinity column was then blocked by infusion of d-biotin and washed with PBS buffer, then kept refrigerated at 4°C for later use.
The FAC/MS apparatus was set up by connecting three syringes that were placed on a multi-syringe pump (PHD 2000, Harvard Apparatus) and a switching valve (Rheodyne, model 9725) with the PSL affinity column to the sample inlet of a Hewlett-Packard series 1100 MSD single quadruple mass spectrometer. The three syringes (each 1 ml volume) contained sample, ammonium acetate buffer (2 mM, 0.1 mM Ca(OAc)2, pH 7.2) and makeup (acetonitrile) solution, respectively. All solutions were infused simultaneously with the syringe pump at a flow rate of 8 µl/min per syringe. The column effluent from the sample was combined with the makeup flow (acetonitrile) in a tee to give a total flow rate of 16 µl/min on entering the mass spectrometer. After each run the column was re-equilibrated with buffer by switching the loading valve (Schriemer et al., 1998). For characterization of the eluent the spectrometer scanned from m/z 100 to 1500 in 1.5 s in the negative-ion mode. For screening of mixtures, the spectrometer was operated in selected-ion-monitoring (locked on the m/z values of the individual ligands) and negative ion mode. A chamber voltage of 3500 V with a grounded electrospray needle, N2 drying gas flow rate of 4 l/min, and N2 nebulizer pressure of 480 mbar were used. Breakthrough volumes were measured as midpoints in the extracted ion chromatograms. All data were processed with Microsoft Excel software, and figures are presented as IGOR program files.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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