Glycobiology, 2001, Vol. 11, No. 2 141-147
© 2001 Oxford University Press
Rapid determination of the binding affinity and specificity of the mushroom Polyporus squamosus lectin using frontal affinity chromatography coupled to electrospray mass spectrometry
Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada, and 2Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109–0606, 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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 Top Abstract IntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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The binding affinity and specificity of the mushroom Polyporus squamosus lectin has been determined by the recently developed method of frontal affinity chromatography coupled to electrospray mass spectrometry (FAC/MS). A micro-scale affinity column was prepared by immobilizing the lectin (
25 µg) onto porous glass beads in a tubing column (9.8 µl column volume). The column was then used to screen several oligosaccharide mixtures. The dissociation constants of 22 sialylated or sulfated oligosaccharides were evaluated against the immobilized lectin. The lectin was found to be highly specific for Neu5Ac
2–6Galß1–4Glc/GlcNAc containing oligosaccharides with Kd values near 10 µM. The FAC/MS assay permits the rapid determination of the dissociation constants of ligands as well as a higher throughput screening of compound mixtures, making it a valuable tool for affinity studies, especially for testing large numbers of compounds.
Key words: binding specificity/dissociation constant/frontal affinity chromatography coupled to electrospray mass spectrometry/lectin/Neu5Ac2–6Galß1–4Glc/GlcNAc containing oligosaccharides
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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Sialic acids are ubiquitous components of mammalian cell surface glycoproteins and glycolipids. Because of their terminal position and their charge, sialylated oligosaccharide sequences have long been considered to be critical determinants in mammalian cell–cell recognition and in viral and bacteria adhesion (Rademacher et al., 1988
; Wenneras et al., 1990; Corfield, 1992; von Itzstein et al., 1993). The overexpression of cell surface sialic acid is highly correlated with a malignant phenotype in many cancers (Waibel et al., 1988; Cho et al., 1994; Dohi et al., 1994; Okada et al., 1994; Sawada et al., 1994a,b; Yang et al., 1994; Jorgensen et al., 1995) and, as a result, a chemical approach to engineering cell surface sialic acids has been recently established for tumor targeting (Mahal et al., 1997). Lectins are extremely useful probes not only for the detection and preliminary characterization of glycoconjugates present on cell surface and tissues, but also for their purification. A number of sialic acid binding lectins have been isolated and characterized, principally from invertebrates (e.g., lobsters, crabs, snails) (Mandal and Mandal, 1990; Zeng and Gabius, 1992). However, very few sialic acid–binding lectins have been reported from plant sources, from which lectins are most readily purified (Goldstein, 1999). Recently, Goldstein and coworkers isolated a lectin from the fruiting body of the polypore mushroom Polyporus squamosus (PSL, Polyporus squamosus lectin) that recognized sialylated oligosaccharides and reported the initial specificity characterization by hemagglutination and quantitative precipitation inhibition assays (Mo et al., 2000). In order to further characterize this potentially useful lectin, the binding affinity and specificity of the protein have here been examined in further detail.
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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Twenty-two sialylated and sulfated oligosaccharides were selected in this work to study the binding affinity of PSL. Their structures are shown in Table I. Their selection was based on the previous study showing that four lactose derivatives with a negative charged N-acetylneuraminic acid as the terminal group had stronger inhibitory activity than the other neutral mono- and disaccharides (Mo et al., 2000
). A mixture containing eight 2–3 and 2–6 sialylated lactose derivatives (Table I, compounds 1–8, each 2.5 µM) was prepared in 2 mM NH4OAc and 0.1 mM Ca(OAc)2, pH 7.2. This mixture was used to test the activity of the PSL affinity column. The m/z value of each compound in the mixture was characterized by ESI-MS in the negative and scan mode. Their m/z values were then programmed for selected ion-monitoring (SIM) and the mixture was continuously infused through the PSL column while the elution profile was monitored by ESI-MS detection in the negative mode. A plot of signal intensity versus m/z of the mixture in SIM mode is presented in Figure 1c, showing that peaks derived from all of the compounds are detected. The disialylated compound 8 displayed a strong signal at (M-2H+)2–/2 while the others showed signals at their monobasic (M-H+)– values. Two sets among them: 1 and 4, 3 and 6, each have the same m/z value and are isomers, respectively. Compounds 2 and 5, which contain a methoxycarbonyloctyl group, showed higher signal intensity than the others.
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Figure 1a shows the time dependence of each of the peaks in Figure 1c as the mixture is run through the PSL column. The fronts of the elution of oligosaccharides 5, 7, and 8 (
2–3 linked) appeared first, whereas the front of the 2–6 linked oligosaccharide (2) came later. Significantly, the chromatograms of ions at m/z 632.2 (1 and 4) and 673.3 (3 and 6) clearly show biphasic characteristics, suggesting that the 2–3 and 2–6 linked isomers were separated on the PSL affinity column with the 2–3 isomers barely retarded in the mixture. This resolution was later confirmed by individual experiments demonstrating that the 2–6 linked isomers (1 and 3) were the retarded ones. When the 2–3 linked isomers 4 and 6 were run individually, no retardation was observed (data not shown). When the blank column was infused with the same mixture, the negative control spectrum, Figure 1b, confirmed that non-specific binding to the column was negligible. All eight compounds (1–8) appeared at the void volume in the blank column, confirming that the retardation of the three 2–6 linked saccharides is due to the specific binding to the immobilized PSL. The binding affinities of oligosaccharides 1–8 against PSL parallel their elution order as shown in Figure 1a. However, in order to determine the binding constants of the ligands, the binding capacity (number of active PSL binding sites) of the column had to be evaluated.
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)
Neu5Ac2–3Galß1–4GlcNAcß-O-MCO (5) and Neu5Ac-2–6Galß1–4Glcß-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 Galß1–4Glcß-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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With the known Bt, the dissociation constants of each ligand in a mixture can be estimated from a single FAC/MS run. The Kdmix values of 1–3 in the mixture were calculated to be 22.6, 20.2, and 16.2 µM, respectively, based on the EIC shown in Figure 1a. Because ligands 1–3 in the mixture are competing for the same combining site of PSL, their Kdmix values determined in a mixture should be an underestimate of their individual Kd values, though they should be good approximations.
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 Neu5Ac2–6 terminated pentasaccharides with the same molecular weight. The retarded phase is due to the binding of LST c (10), which has a Neu5Ac2–6Galß1–4GlcNAc 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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The second example shown in Figure 3b is the FAC/MS analysis of two complex-type N-linked oligosaccharides (Table I, 21 and 22), which were isolated from human fibrinogen (Townsend et al., 1982
) and bovine fetuin (Townsend et al., 1989), respectively. Both samples consist of a number of structural isomers. Both of 21 and 22 analyte solutions were prepared by dissolving 20 µg of solid directly in 2 mM NH4OAc (pH 7.2), 0.1 mM Ca(OAc)2 and the void volume marker 5 was added. Their EICs were recorded separately, and plotted together as shown in Figure 3b. Both 21 and 22 display monotone and retarded elution fronts in relation to the front of the void volume marker 5, indicating that the major isomers of the two samples may contain at least one Neu5Ac2–6Galß1–4GlcNAc terminal. Their apparent dissociation constants were determined to be 20.6 ± 1.5 µM (21) and 88.4 ± 4.4 µM (22), respectively.
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 2–6 linkages. Such cooperativity would not exist when both 2–3 and 2–6-linked structures are present in the same molecule. This is supported by the finding that fetuin, which contains both 2–3 and 2–6-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. Neu5Ac2–3 terminated oligosaccharides used in this study include Neu5Ac2–3Galß1–4Glc/GlcNAc (4 and 5), Neu5Ac2–3Galß1–3GlcNAc (12) and their fucosylated derivatives (7, 11, and 13). One synthetic ß anomer, Neu5Acß2–3Galß1–4GlcNAc (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 Neu5Ac2–6Galß1–4Glc/GlcNAc sequence-containing oligosaccharides showed high binding activity against the immobilized PSL; among them, Neu5Ac2–6Galß1–4GlcNAc displayed the highest affinity with a Kd value of 10 µM. However, if the Neu5Ac group is 2–6 linked to a non-terminal sugar, such as in 8 and 9, the structure is not bound.
The binding affinity between 2–3 and 2–6 linked isomers differs by at least 100-fold (Table I). The affinity order of Neu5Ac2-6 terminated trisaccharides was in the order: Neu5Ac2–6Galß1–4GlcNAc > Neu5Ac2–6Galß1–4Glc-O-MCO > Neu5Ac2–6Galß1–4Glc, 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 (Galß1–4GlcNAc) 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·mol–1) than Neu5Ac2–6Galß1–4GlcNAc, 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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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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In this study, we have shown that FAC/MS is an efficient screening methodology that can be applied to assess the specificity of a novel lectin. As shown in Figure 1a and Figure 3a, FAC/MS is a useful technique to identify the active species from a mixture that are complicated by the presence of stereoisomers. Also, the apparent dissociation constants of the ligands in the mixtures can be easily estimated based on the theory of frontal affinity chromatography using Equation 1 (Kasai et al., 1986
). Although in this method target proteins must be immobilized and therefore ligand–protein interaction takes place at a solution/solid interface, the immobilized proteins may be good models for membrane-bound proteins. The capability of screening mixtures of compounds and rapid Kd determination makes FAC/MS an attractive alternative to other screening methods. Another advantage of FAC/MS is the miniaturization of the column to the 2–20 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 2–6 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 2–6 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 2–3 and 2–6 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 Neu5Ac2–6Galß1–4GlcNAc, but was totally inactive toward the 2–3 sialylated Galß1–4GlcNAc. However, the authors claimed that the lectin is highly specific to both HSO3–-6Galß1–4GlcNAc and Neu5Ac2–6Galß1–4GlcNAc.
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 ligand–receptor 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 structure–activity 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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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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The polypore mushroom Polyporus squamosus lectin (PSL) was isolated from the fruiting body of the mushroom and biotinylated as previously reported (Mo et al., 2000
). All the oligosaccharides with a methoxycarbonyloctyl (abbreviated as MCO) aglycone (see Table I) were available from previous experiments (Palcic et al., 1989; Field et al., 1995). Other oligosaccharides used were purchased from Sigma, Calbiochem, Toronto Research Chemicals Inc. or Glyco, Inc. PEEK (polyetheretherketone) tubing, union, tee, and frit were from Fisher Scientific.
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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This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC grants to O.H. and to M.M.P.) and by SYNSORB Biotech, Calgary. B.Z. was supported by a postdoctoral fellowship from the Alberta Heritage Foundation for Medical Research (AHFMR). We thank the United States National Institutes of Health for a grant (GM29470 to I.J.G.) in support of this research.
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Abbreviations |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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EIC, extracted ion chromatogram; ESI-MS, electrospray ionization mass spectrometry; FAC/MS, frontal affinity chromatography coupled to mass spectrometry; HPAE-PAD, high performance anion-exchange chromatography with pulsed amperometric detection; MCO, methoxycarbonyloctyl; PBS, phosphate buffer saline; PSL, Polyporus squamosus lectin; SIM, selected ion monitoring.
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1 To whom correspondence should be addressed
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References |
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Cho, S.H., Sahin, A., Hortobagyi, G.N., Hittelman, W.N., and Dhingra, K. (1994) Sialyl-Tn antigen expression occurs early during human mammary carcinogenesis and is associated with high nuclear grade and aneuploidy. Cancer Res., 54, 6302–6305.
Corfield, T. (1992) Bacterial sialidases–roles in pathogenicity and nutrition. Glycobiology, 2, 509–521.
Dohi, T., Hashiguchi, M., Yamamoto, S., Morita, H., and Oshima, M. (1994) Fucosyltransferase-producing sialyl Lea and sialyl Lex carbohydrate antigen in benign and malignant gastrointestinal mucosa. Cancer, 73, 1552–1561.[Web of Science][Medline]
Field, R.A., Otter, A., and Hindsgaul, O. (1995) Synthesis and 1H NMR characterization of the six isomeric mono-O-sulfates of 8-methoxycarbonyloct-1-yl O-ß-D-galactopyranosyl-(1
4)-2-acetamido-2-deoxy-ß-D-glucopyranoside. Carbohydr. Res., 276, 347–363[Web of Science][Medline]
Goldstein, I.J. (1999) Sialic acid-binding plant lectins. In Inoue, Y., Lee, Y.C., and Troy, F.A. (eds.), Sialobiology and Other Novel Forms of Glycosylation. Gakushin Publishing Co., Osaka, pp. 95–103.
Jorgensen, T., Berner, A., Kaalhus, O., Tveter, K.J., Danielsen, H.E., and Bryne, M. (1995) Up-regulation of the oligosaccharide sialyl Lewis X: a new prognostic parameter in metastatic prostate cancer. Cancer Res., 55, 1817–1819.
Kasai, K., Oda, Y., Nishikata, M., and Ishii, S. (1986) Frontal affinity chromatography: theory for its application to studies on specific interactions of biomolecules. J. Chromatogr. Biomed. Appl., 376, 33–47.
Kobata, A., Kochibe, N., and Endo, T. (1994) Affinity chromatography of oligosaccharides on Psathyrella velutina lectin column. Methods Enzymol., 247, 228–237.[Web of Science][Medline]
Mahal, L.K., Yarema, K.J., and Bertozzi, C.R. (1997) Engineering chemical reactivity on cell surfaces through oligosaccharide biosynthesis. Science, 276, 1125–1128.
Mandal, C., and Mandal, C. (1990) Sialic acid binding lectins. Experientia, 46, 433–41.[Web of Science][Medline]
Mo, H., Winter, H.C., and Goldstein, I.J. (2000) Purification and characterization of a Neu5Ac2–6Galß1–4Glc/GlcNAc-specific lectin from the fruiting body of the polypore mushroom Polyporus squamosus. J. Biol. Chem., 275, 10623–10629.
Okada, Y., Jin-no, K., Ikeda, H., Sakai, N., Sotozono, M., Yonei, T., Nakanishi, S., Moriwaki, S., and Tsuji, T. (1994) Changes in the expression of sialyl-Lewis X, a hepatic necroinflammation-associated carbohydrate neoantigen, in human hepatocellular carcinnomas. Cancer, 73, 1811–1816.[Web of Science][Medline]
Palcic, M.M., Venot, A.P., Ratcliffe, R.M., and Hindsgaul, O. (1989) Enzymatic synthesis of oligosaccharides terminating in the tumor-associated sialyl-Lewis A determinant. Carbohydr. Res., 190, 1–11.[Web of Science][Medline]
Rademacher, T.W., Parekh, R.B., and Dwek, R.A. (1988) Glycobiology. Annu. Rev. Biochem., 57, 785–838.[Web of Science][Medline]
Sawada, R., Tsuboi, S., and Fukuda, M. (1994) Differential E-selectin-dependent adhesion efficiency in sublines of a huamn colon cancer exhibiting distinct metastatic potentials. J. Biol. Chem., 269, 1425–1431.
Sawada, T., Ho, J.J., Chung, Y.S., Sowa, M., and Kim, Y.S. (1994) E-selectin binding by pancreatic tumor cells is inhibited by cancer sera. Int. J. Cancer, 57, 901–907.[Web of Science][Medline]
Schriemer, D.C., Bundle, D.R., Li, L., and Hindsgaul, O. (1998) Micro-scale frontal affinity chromatography with mass spectrometric detection: a new method for the screening of compound libraries. Angew. Chem. Int. Ed., 37, 3383–3387.
Schriemer, D.C., and Hindsgaul, O., (1998) Deconvolution approaches in screening compound mixtures. Combinatorial Chem. High Throughput Screening, 1, 155–170.[Web of Science][Medline]
Townsend, R.R., Hardy, M.R., Cumming, D.A., Carver, J.P., and Bendiak, B. (1989) Separation of branched sialylated oligosaccharides using high-pH anion-exchange chromatography with pulsed amperometric detection. Anal. Biochem., 182, 1–8.[Web of Science][Medline]
Townsend, R.R., Hardy, M.R., Hindsgaul, O., and Lee, Y.C. (1988) High-performance anion-exchange chromatography of oligosaccharides using pellicular resins and pulsed amperometric detection. Anal. Biochem., 174, 459–470.[Web of Science][Medline]
Townsend, R.R., Hilliker, E., Li, Y.-T., Laine, R.A., Bell, W.R., and Lee, Y.C. (1982) Carbohydrate structure of human fibrinogen. J. Biol. Chem., 257, 9704–9710.
Ueda, H., Kojima, K., Saitoh, T., and Ogawa, H. (1999) Interaction of a lectin from Psathyrella velutina mushroom with N-acetylneuraminic acid. FEBS Lett., 448, 75–80.[Web of Science][Medline]
von Itzstein, M., Wu W.Y., Kok, G.B., Pegg, M.S., Dyason, J.C., Jin, B., Van Phan, T., Smythe, M.L., White, H.F., and Oliver, S.W. (1993) Rational design of potent sialidase-based inhibitors of influenza virus replication. Nature, 363, 418–423.[Medline]
Waibel, R., O’Hara, C.J., Smith, A., and Stahel, R.A. (1988) Tumor-associated membrane sialoglycoprotein on human small cell lung carcinoma identified by the IgG2a monoclonal antibody SWA20. Cancer Res., 48, 4318–4323.
Wenneras, C., Holmgren, J., and Svennerholm, A.-M. (1990) The binding of colonization factor antigens of enterotoxigenic Escherichia coli to intestinal cell membrane proteins. FEMS Microbiol. Lett., 54, 107–112.[Medline]
Yamashita, K., Umetsu, K., Suzuki, T., and Ohkura, T. (1992) Purification and characterization of a Neu5Ac2–6Galß1–4GlcNAc and HSO3–-6Galß1–4GlcNAc specific lectin in tuberous roots of Trichosanthes japonica. Biochemistry, 31, 11647–11650.[Medline]
Yang, J.M., Byrd, J.C., Siddiki, B.B., Chung, Y.S., Okuno, M., Sowa, M., Kim, Y.S., Matta, K.L., and Brockhausen, I. (1994) Alterations of O-glycan biosynthesis in human colon cancer tissues. Glycobiology 4, 873–884.
Zeng, F.-Y., and Gabius, H.J. (1992) Sialic acid-binding proteins: characterization, biological function and application. Z. Naturforsch., 470, 641–653.
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