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• W4251113918 abstract "Crocus sativus lectin (CSL) is one of the truly mannose-specific plant lectins that has a unique binding specificity that sets it apart from others. We studied sugar-binding specificity of CSL in detail by a solution phase method (fluorescence polarization) and three solid phase methods (flow injection, surface plasmon resonance, and microtiter plate), using a number of different glycopeptides and oligosaccharides. CSL binds the branched mannotriose structure in the N-glycan core. Substitution of the terminal Man in the Manα(1–3)Man branch with GlcNAc drastically decreases binding affinity much more than masking of the terminal Man in the Manα(1–6)Man branch. Most interestingly, the β-Man-linked GlcNAc in N-glycan core structure contributes greatly to the binding. The effect of this GlcNAc is so strong that it can substantially offset the negative effect of substitution on the nonreducing terminal Man residues. On the other hand, the GlcNAc that is usually attached to Asn inN-glycans and the l-Fuc linked at the 6-position of the GlcNAc are irrelevant to the binding. A bisecting GlcNAc neither contributes to nor interferes with the binding. This unique binding specificity of CSL offers many possibilities of its use in analytical and preparative applications. Crocus sativus lectin (CSL) is one of the truly mannose-specific plant lectins that has a unique binding specificity that sets it apart from others. We studied sugar-binding specificity of CSL in detail by a solution phase method (fluorescence polarization) and three solid phase methods (flow injection, surface plasmon resonance, and microtiter plate), using a number of different glycopeptides and oligosaccharides. CSL binds the branched mannotriose structure in the N-glycan core. Substitution of the terminal Man in the Manα(1–3)Man branch with GlcNAc drastically decreases binding affinity much more than masking of the terminal Man in the Manα(1–6)Man branch. Most interestingly, the β-Man-linked GlcNAc in N-glycan core structure contributes greatly to the binding. The effect of this GlcNAc is so strong that it can substantially offset the negative effect of substitution on the nonreducing terminal Man residues. On the other hand, the GlcNAc that is usually attached to Asn inN-glycans and the l-Fuc linked at the 6-position of the GlcNAc are irrelevant to the binding. A bisecting GlcNAc neither contributes to nor interferes with the binding. This unique binding specificity of CSL offers many possibilities of its use in analytical and preparative applications. C. sativus lectin 5-aminomethyl fluorescein concanavalin A flow injection fluorescence polarization Hepes-buffered saline containing 150 mm NaCl, 3 mm EDTA, and 0.005% polysorbate 20 7-hydroxycoumarin-5-carboxylic acid high performance liquid chromatography microtiter plate phosphate-buffered saline PBS containing Triton X-100 surface plasmon resonance “Taka-amylase” (α-amylase fromA. oryzae) T. gesneriana lectin matrix-assisted laser desorption/ionization time-of-flight pyridylaminated Crocus sativus lectin (CSL)1 can be readily purified from bulbs of C. sativus (saffron crocus) (1Oda Y. Tatsumi Y. Biol. Pharm. Bull. 1993; 16: 978-981Crossref PubMed Scopus (16) Google Scholar). Unlike many Man-binding lectins, such as concanavalin A, which recognizes both Man and Glc residues, CSL is truly mannose-specific, binding of which is inhibited only by manno-oligosaccharides, and not by glucose or its oligo/polymers (e.g. glycogen). Other truly mannose-specific lectins from plant bulbs include Tulipa gesneriana lectin (TGL) (2Oda Y. Minami K. Eur. J. Biochem. 1986; 159: 239-245Crossref PubMed Scopus (62) Google Scholar),Galanthus nivalis agglutinin (3Shibuya N. Goldstein I.J. Van Damme E.J.M. Peumans W.J. J. Biol. Chem. 1988; 263: 728-734Abstract Full Text PDF PubMed Google Scholar), Narcissus pseudonarcissus agglutinin, Hippeastrum hybridumagglutinin (4Kaku H. Van Dammer E.J.M. Peumans W.J. Goldstein I.J. Arch. Biochem. Biophys. 1990; 279: 298-304Crossref PubMed Scopus (105) Google Scholar), and Lycoris radiata agglutinin (5Oda Y. Tatsumi Y. Kinoshita M. Kurashimo S. Honda E. Ohba Y. Kakehi K. Bull. Pharm. Res. Technol. Inst. 1997; 6: 45-54Google Scholar). Previously, these mannose-specific lectins of plant bulbs have been shown to exhibit different binding specificities toward various yeast cells of different cell wall mannan structures (6Oda Y. Kinoshita M. Kakehi K. Anal. Biochem. 1997; 254: 41-48Crossref PubMed Scopus (7) Google Scholar), suggesting that each lectin may have its unique sugar-binding specificity. Subsequent studies showed that TGL prefers manno-oligosaccharides with α(1–6) linkage rather than those with α(1–2) or α(1–3) linkage (2Oda Y. Minami K. Eur. J. Biochem. 1986; 159: 239-245Crossref PubMed Scopus (62) Google Scholar), andG. nivalis agglutinin prefers terminal Manα(1–3)Man (3Shibuya N. Goldstein I.J. Van Damme E.J.M. Peumans W.J. J. Biol. Chem. 1988; 263: 728-734Abstract Full Text PDF PubMed Google Scholar). We also discovered that hen ovomucoid is a potent inhibitor of CSL, but it does not inhibit other mannose-specific lectins from plant bulbs. Obviously, CSL has a different sugar-binding specificity from other Man-binding lectins, and this prompted us to engage in more detailed studies of the binding specificity of CSL. Many assay systems are available to analyze carbohydrate-lectin interactions. In this work, we used four different assay systems: (a) flow injection technique (FI), measuring inhibition of the binding between fluorescence-labeled lectins and yeast cells; (b) fluorescence polarization (FP), measuring binding or its inhibition of fluorescence-labeled oligosaccharides by CSL; (c) surface plasmon resonance (SPR), measuring binding of CSL to immobilized ligand; (d) microtiter plate (MP) assay, measuring inhibition of binding of europium-labeled glycopeptide by CSL adsorbed to the wells. Binding studies using these assay systems indicated that CSL can recognize specifically the part ofN-glycan core structure including the trimannosyl group and the GlcNAc residues linked to β-Man (see Fig.1). Oligosaccharides (1–8 and 16),shown in Table I as well as Manβ(1–4)GlcNAc and chitobiose were from Funakoshi (Tokyo, Japan). 2-Aminopyridine, UDP-GlcNAc, β-galactosidase, apotransferrin (human), and γ−globulins (human and bovine) were from Sigma. 7-Hydroxycoumarin-3-carboxylic acid (HCC) succinimidyl ester, 5-aminomethyl fluorescein (AMF), and 6-carboxyfluorescein succinimidyl ester were from Molecular Probes, Inc. (Eugene, OR). β-N-Acetylhexosaminidase (jack bean), β-galactosidase (jack bean), α-mannosidase (jack bean), and endoglycosidase D (Streptococcus pneumoniae) were from Seikagaku Kogyo (Tokyo, Japan). Pronase (Streptomyces griseus) was from Calbiochem-Novabiochem Co.N-Glycanase F (glycoamidase) and α-l-fucosidase were from Roche Molecular Biochemicals. Neuraminidase (Anthrobacter ureafaciens) was a gift from Drs. Tsukada and Ohta (Marukin Shoyu, Uji, Kyoto, Japan). Taka-amylase (an α-amylase from Aspergillus oryzae 3× crystallized), known (7Minobe S. Nakajima H. Itoh N. Funakoshi I. Yamashina I. J. Biochem. (Tokyo). 1979; 86: 1851-1854Crossref PubMed Scopus (34) Google Scholar) to have only oneN-glycosylation site of Man5GlcNAc2chain (8A in Table I) was a generous gift from Prof. T. Ikenaka (University of Osaka, Japan). Other reagents used were of reagent grade or higher. Microtiter plates (FluroNunc, MaxiSorp surface) were from Wallac (Gaithersburg, MD). Unit definitions of enzymes were those of the manufacturers.Table IList of oligosaccharidesPA, pyridyl-2-amino; Pep, peptide. Open table in a new tab PA, pyridyl-2-amino; Pep, peptide. CSL and TGL were purified from bulbs of C. sativus (1Oda Y. Tatsumi Y. Biol. Pharm. Bull. 1993; 16: 978-981Crossref PubMed Scopus (16) Google Scholar) andT. gesneriana (2Oda Y. Minami K. Eur. J. Biochem. 1986; 159: 239-245Crossref PubMed Scopus (62) Google Scholar), respectively. C. sativus is an autumn-flowering crocus distinct from Crocus vernus, a spring flowering plant (8Misaki A. Kakuta M. Meah Y. Goldstein I.J. J. Biol. Chem. 1997; 272: 25455-25461Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Concanavalin A (ConA) was from E-Y Laboratories (San Mateo, CA). HCC-labeled lectins were prepared using HCC succinimidyl ester (9Oda Y. Kinoshita M. Nakayama K. Ikeda S. Kakehi K. Anal. Biochem. 1999; 269: 230-235Crossref PubMed Scopus (4) Google Scholar). Briefly, a solution (50 μl) of HCC-succinimidyl ester (5 mg/ml in dimethyl sulfoxide) was added to one ml of lectin solution (1 mg/ml) in PBS and mixed gently. After 20 min at room temperature, solid glycine (1 mg) was added, and the mixture was kept for a further 10 min to quench the remaining HCC-succinimidyl ester. Each of the HCC-labeled lectins was purified by affinity chromatography as described previously (9Oda Y. Kinoshita M. Nakayama K. Ikeda S. Kakehi K. Anal. Biochem. 1999; 269: 230-235Crossref PubMed Scopus (4) Google Scholar). The purified HCC-labeled lectins were kept in PBS at −20 °C until use. For general carbohydrate analysis, the phenol-sulfuric acid method (10Dubois M. Gilles K.A. Hamilton J.K. Rebers P.A. Smith F. Anal. Chem. 1956; 28: 350-356Crossref Scopus (40620) Google Scholar) was used. For amino group determination, the 2,4,6-trinitrobenzenesulfonic acid method was used (11Qi X.-Y. Keyhani N.O. Lee Y.C. Anal. Biochem. 1988; 175: 139-144Crossref PubMed Scopus (30) Google Scholar, 12Lee Y.C. Scocca J.R. Muir L. Anal. Biochem. 1969; 27: 559-566Crossref PubMed Scopus (26) Google Scholar). MALDI-TOF of CSL was performed with a Voyager System (PE Biosystems, Foster City, CA) using cinapinic acid as the matrix at 25 kV. Ovomucoids from hen and Japanese quail egg white were prepared by the method of Fredericq and Deutsch (13Fredericq E. Deutsch H.F. J. Biol. Chem. 1949; 181: 499-510Abstract Full Text PDF PubMed Google Scholar). The glycopeptides were prepared from the ovomucoids as follows. Ovomucoid (1 g) was dissolved in 0.5 m Tris/HCl buffer (pH 8.5) containing 50 mm dithiothreitol, 6 m guanidine hydrochloride, and 5 mm EDTA, and the mixture was incubated at 37 °C for 1 h. Iodoacetamide was added, and the solution was kept in the dark at 37 °C for 30 min. The reaction mixture was dialyzed against water and freeze-dried. The freeze-dried material was dissolved in 50 mm Tris/HCl buffer (pH 7.5) and digested with Pronase (10 mg) at 37 °C for 24 h, at which time another portion of Pronase (5 mg) was added and incubation was continued for further 24 h. The digest was applied to a column (2.5 × 150 cm) of Sephadex G-50 equilibrated with 50 mm ammonium hydrogen carbonate. The carbohydrate-containing fractions detected by the phenol-sulfuric acid method (10Dubois M. Gilles K.A. Hamilton J.K. Rebers P.A. Smith F. Anal. Chem. 1956; 28: 350-356Crossref Scopus (40620) Google Scholar) were collected and freeze-dried to yield a mixture of glycopeptides (330 mg). The oligosaccharides were released from the glycopeptides by hydrazinolysis (14Parente J.P. Strecker G. Leroy Y. Montreuil J. Fournet B. J. Chromatogr. 1982; 249: 199-204Crossref Scopus (23) Google Scholar) and separated by HPLC (15Parente J.P. Wieruszeski J.-M. Strecker G. Montreuil J. Fournet B. van Halbeek H. Dorland L. Vliegenthardt J.F.G. J. Biol. Chem. 1982; 275: 13173-13176Abstract Full Text PDF Google Scholar) on a Cosmosil 5NH2 column (4.6 × 250 mm, Nakarai Tesque, Kyoto, Japan). From the quail ovomucoid, 10was isolated, and from hen ovomucoid, 21–23 were obtained in pure state. Their structures were confirmed by mass spectrometry and NMR (16Rozwarski D.A. Swami B.M. Brewer C.F. Sacchettini J.C. J. Biol. Chem. 1998; 273: 32818-32825Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 17Alonso J.M. Boulenguer P. Wieruszeski J.-M. Leroy Y. Montreuil J. Fournet B. Eur. J. Biol. Chem. 1988; 177: 187-197Crossref Scopus (9) Google Scholar). Stock saturated matrix ATT (6-aza-2-thio-thymine) solution was made in 50% (v/v) acetonitrile. The acting ATT solution was made fresh daily by mixing the stock ATT solution and 10% tribasic ammonium citrate solution in a 9:1 (v/v) ratio. The glycopeptide was dissolved in water to a 1 nmol/μl concentration, of which 0.3 μl was deposited on the sample slide followed immediately by 0.3 μl of saturated ammonium sulfate and 0.3 μl of ATT matrix solution. This mixture was left to dry at room temperature. Mass spectra were acquired on a Kompact MALDI IV (Kratos, Manchester, United Kingdom) time of flight mass spectrometer in the negative ion linear mode, equipped with a 337-nm nitrogen laser and a 20-kV extraction voltage with time-delayed extraction. Each spectrum was the average of 50 laser shots. The laser fluency used was23 of the maximum intensity (a laser power of 110). Glycopeptides (about 180 mg) were prepared from apotransferrin (1 g) in the same manner as described for ovomucoids, except that trypsin instead of Pronase was used for proteolysis. The oligosaccharide (about 53 mg) was obtained from the glycopeptides by hydrazinolysis. The oligosaccharide (10 mg) in 1 ml of 50 mm sodium acetate buffer, pH 5.5, was incubated with neuraminidase (1 unit) at 37 °C for 24 h and then heated in a boiling water bath for 3 min. The supernatant obtained from centrifugation was subjected to HPLC on a Cosmosil 5NH2column, and the asialo-oligosaccharide (about 8 mg) was obtained. The asialo-oligosaccharide (5 mg) was dissolved in 200 μl of 50 mm sodium acetate buffer, pH 3.5, and digested with β-galactosidase (1 unit) at 37 °C for 24 h. The product, GlcNAc2Man3GlcNAc2 (17) (about 4 mg) was purified by HPLC. Then the oligosaccharide17 (2 mg) was digested with β-N-acetylhexosaminidase (2 units) in 100 μl of 50 mm sodium citrate buffer, pH 5.0, at 37 °C for 24 h to produce oligosaccharide Man3GlcNAc2(10). Glycopeptides (about 335 mg) were obtained from bovine γ−globulins (3 g) in the same manner as described for ovomucoids, except pepsin was used in 0.2 m citrate/HCl buffer, pH 2.0. A mixture of the oligosaccharides (about 37 mg) was released from the glycopeptide mixture by hydrazinolysis. HPLC fractionation of this mixture (20 mg) yielded the oligosaccharide 25 (about 2 mg). The oligosaccharide 25 was digested with β-galactosidase followed by β-N-acetylhexosaminidase to produce oligosaccharide Man3GlcNAc2Fuc (15) (about 0.3 mg), which was purified by HPLC. One portion (50 μg) of15 obtained was digested with l-fucosidase (200 milliunits) in 60 μl of 50 mm sodium citrate buffer, pH 5.0, at 37 °C for 24 h, and an aliquot thereof was subjected to HPLC to confirm that the product was the oligosaccharide Man3GlcNAc2 (10). The Japanese quail ovomucoid (1.58 g) was denatured and digested with Pronase (1%, w/w) for 4 days at 55 °C, with daily addition of Pronase as described above. The digest was freeze-dried and dissolved in 15-ml 1% (v/v) pyridine-acetic acid in water (pH 5.5) and separated on a Sephadex G50 column (5 × 190 cm) equilibrated and eluted with the same buffer, collecting 18-ml fractions. The fractions containing glycopeptides were determined by analyzing carbohydrate with phenol-sulfuric acid and amino group with the 2,4,6-trinitrobenzenesulfonic acid method. The glycopeptide-containing fractions were freeze-dried, dissolved in 5 ml of 0.1 macetic acid, and further fractionated on a BioGel-P4 column (138 × 2 cm) in 0.1 m acetic acid, collecting 5-ml fractions. The fractions containing trimannosyl core glycopeptide were pooled, and its identity was confirmed by sugar composition analysis by high performance anion exchange chromatography (18Fan J.-Q. Namiki Y. Matsuoka K. Lee Y.C. Anal. Biochem. 1994; 219: 375-378Crossref PubMed Scopus (49) Google Scholar). Upon further fractionation with RP-HPLC (Shandon Hypercarb GCC column, using a gradient of acetonitrile in 10 mm NH4OH buffer), three glycopeptides were isolated (data not shown). By MALDI-TOF analysis and from the known primary structure of the ovomucoid (19Kato I. Shrode J. Wilson K.A. Laskowski M.J. Peeters H. Protides of the Biological Fluids: Proceedings of the 23rd Colloquium. Pergamon Press, New York1976: 235-243Google Scholar), they were determined to have the same sugar composition and different peptide length of PNTT (QOM-GP1), FPNTT (QOM-GP2), and FPNTTN (QOM-GP3), respectively. QOM-GP2 (1.3 μmol) prepared as above was dissolved in 250 μl of 10% (w/v) sodium bicarbonate. Solid diethylenetriaminepentaacetic acid dianhydride (7.8 μmol) was added, and the mixture was gently stirred overnight at room temperature. Dry Eu(NO3)3 (16 μmol) was added to the mixture and allowed to react for 1 h, and then the pH was adjusted to pH 7.2 with acetic acid. The mixture was vacuum-dried (SpeedVac, Holbrook, NY) and dissolved in 200 μl of water before applying to a Sephadex G-10 column (1.5 × 92 cm) and eluted with 50 mm ammonium acetate buffer, collecting 1-ml fractions. Man3GlcNAc2-glycopeptide (700 nmol) prepared as above was lyophilized in a screw-capped vial and mixed with 20 μl of 0.2 m sodium borate, pH 7.3, and 30 μl of 45 mm 6-carboxyfluorescein succinimidyl ester in Me2SO. The mixture was allowed to react overnight at room temperature, the progress of the reaction being monitored by TLC. The unreacted fluorescein reagent was separated from the labeled glycopeptide by a silica gel column (1 × 20 cm) eluted with ethyl acetate, acetic acid, and water at 3:2:1 (v/v), and 100-drop fractions were collected. Fluorescein-labeled glycopeptide fractions were pooled and further purified by HPLC using an ODS column with a linear gradient of 20–70% acetonitrile in 0.05% trifluoroacetic acid. Fluorescence signal was monitored at 520 nm using 490 nm for excitation. Man3GlcNAc2 (10, 300 μg) and endoglycosidase D (0.1 units) in 300 μl of 50 mm phosphate buffer, pH 6.0, was incubated at 37 °C for 24 h. The reaction mixture was filtered through an ultrafree-MC (10,000 nominal molecular weight limit filter; Millipore, Tokyo, Japan) to remove the enzyme, and the filtrate was used for inhibition studies. The purity of the digested product was confirmed by HPLC using a Cosmosil 5NH2 column under the conditions used for separation of quail oligosaccharides (see above). All pyridylaminated (PA) oligosaccharides were derived fromN-glycans of either human IgG or Japanese quail ovomucoid. PA derivatization was essentially as described (21Tomiya N. Awaya J. Kurono M. Endo S. Arata Y. Takahashi N. Anal. Biochem. 1988; 171: 73-90Crossref PubMed Scopus (390) Google Scholar). Briefly, to 200 nmol oligosaccharide (dried) in a screw-capped vial, 40 μl of 2-aminopyridine solution (1.0 g in 0.76 ml of concentrated HCl), pH 6.2, was added and heated at 90 °C for 20 min on a heating block. Four μl of freshly prepared NaCNBH3 (20 mg/12 μl of water) was then added to each vial, and heating was continued for 1.5 h. Derivatized oligosaccharides were separated from excess 2-aminopyridine by Sephadex G15 (1 × 40 cm) using 10 mm NH4HCO3 as eluent. Individual PA oligosaccharide was isolated, and its identity was confirmed with the two-dimensional HPLC technique as described (21Tomiya N. Awaya J. Kurono M. Endo S. Arata Y. Takahashi N. Anal. Biochem. 1988; 171: 73-90Crossref PubMed Scopus (390) Google Scholar), with the combination of an ODS and an amide silica gel column. In some cases, sequential digestion with fucosidase, β-galactosidase, andN-acetylglucosaminidase was used. Compound 20 was obtained from compound 11 by the action of GlcNAc-transferase 1 (gift from Dr. Harry Schachter, Toronto, Canada) and UDP-GlcNAc. Compound 12 was generated from 11by α-mannosidase (jack bean) digestion. Oligosaccharide (10) was labeled with AMF via reductive amination (22Camilleri P. Harland G.B. Okafo G. Anal. Biochem. 1995; 230: 115-122Crossref PubMed Scopus (64) Google Scholar) with minor modifications as follows. A dried sample of oligosaccharide 10 (200 μg, 2.3 nmol) was dissolved in 60 μl of 7:3 (v/v) dimethyl sulfoxide-acetic acid, containing AMF (1 mg, 2.5 μmol) and 1 m sodium cyanoborohydride, and was kept at 50 °C for 2 h. The mixture was applied onto a column (0.5 × 30 cm) of Sephadex G-10. Elution was carried out with PBS, and the effluent was fluorometrically monitored (e ex = 490 nm,e em = 520 nm). Fluorescent fractions were pooled and stored at −20 °C until use. The equipment was composed of a RF-535 fluorescence HPLC monitor (Shimadzu Corp., Kyoto, Japan) with a microflow cell (cell volume, 12 μl), an injector (type 7125; Rheodyne) equipped with a 20-μl sample loop, a Hitachi liquid chromatography pump (type 655, Hitachi-Naka, Japan) and a Chromatopack C-R6A data processor (Shimadzu). A Teflon tubing (60 cm × 0.25 mm inner diameter) was placed between the injector and the detector. The operation was performed at ambient temperature at a flow rate of 1.0 ml/min. Serially diluted inhibitor solutions in PBS (100 μl) were mixed in microcentrifuge tubes with an equal volume of HCC-labeled lectin (1 μm) in PBS. After incubation at 30 °C for 5 min, 100 μl of aSaccharomyces cerevisiae suspension in PBS (1 × 107 cells/ml) was added, and incubation was continued for further 10 min. The cells were washed three times with PBS and suspended in 200 μl of 50 mm glycine/NaOH, pH 9.0, containing 0.05% Tween 20. A 20-μl aliquot of the suspension was directly injected onto the flow injection system to determine the fluorescent intensity (e ex = 405 nm ande em = 449 nm). All binding assays were performed in triplicate, and the averaged values are presented. FP was measured in a quartz cell (volume, 300 μl) with a fluorescence spectrophotometer F 4010 equipped with a FP apparatus. The excitation and emission wavelengths were set at 490 and 520 nm, respectively. FP of the solution (200 μl) of the AMF-labeled oligosaccharide in PBS was measured first, and then 10 μl of a lectin solution of different concentrations in PBS was added. The solution was mixed by repeated pipetting, and FP was measured after 2 min. Fluorescence polarization assay was carried out with a Panvera BEACON 2000 (Madison, WI) using fluorescein-labeled glycopeptide in PBS with a final volume of 100 μl at 25 °C using 492-nm (excitation) and 520-nm (emission) wavelengths. For the inhibition assay, final concentrations of 1 μm in CSL, 100 pm in the fluorescent ligand, and various concentrations of each inhibitor were used. The mixture was vortex-mixed and allowed to reach equilibrium for 10 min before the measurement was made. When inhibition was carried out with Man3GlcNAc2 and its PA derivative, the results were indistinguishable. This PA group appears to have no influence in the FP measurement under the present set of conditions. BIACore 2000 (Amersham Pharmacia Biotech, Uppsala, Sweden) was used to measure the interaction between lectins (CSL, TGL, and ConA) and immobilized Taka-amylase (TA). TA (250 and 500 ng) was coated onto a CM5 sensor chip by activation of the sensor surface withN-hydroxysuccinimide/carbodiimide in 10 mmNaOAc, pH 3.6. Estimation of the immobilized protein from resonance unit values (427 and 1517) showed attachment of 10 and 36 fmol when 250 and 500 ng of TA were used, respectively. CSL was dissolved in HBS buffer (10 mm Hepes buffer, pH 7.4, containing 150 mm NaCl, 3 mm EDTA, and 0.005% polysorbate 20), and a 100-μl aliquot of the solution was introduced onto the surface chip at a flow rate of 20 μl/min. The change in the SPR was monitored for 5 min, and then HBS buffer was introduced onto the surface to initiate dissociation. Regeneration was carried out with 40 mm glycine/HCl, pH 1.5, containing 0.5m NaCl. The affinity constant was calculated by the ratios of off-rate/on-rate (K D =k d /k a) using BIA evaluation software version 2.1, assuming a single type of binding. A CSL solution in PBS (100 μl of 50 μg/ml) was coated onto microtiter plate wells overnight at 4 °C. The wells were then washed three times with 150 μl of PBST (PBS with 0.5% Triton X-100) and incubated with 100 nm europium-labeled trimannosyl core glycopeptide (50 μl) with a series of different inhibitors in different concentrations. After 1 h at 4 °C to allow for the binding, the excess inhibitor and the unbound ligand were removed by washing six times with 150 μl of PBST, and 200 μl of the enhancement solution (24Hemmilä I.A. Dakubu S. Mukkala V.-M. Siitarl H. Lövgren T. Anal. Biochem. 1984; 137: 335-343Crossref PubMed Scopus (761) Google Scholar) per well was added and gently shaken on a rotary shaker for 1 h before fluorescent measurement with a multilabel counter (Victor, model 1420, Wallac). Graph-Pad PRISM version 3.0 (San Diego, CA) was used for analysis of binding assays (nonlinear regression analysis using a hyperbolic equation) and Scatchard analysis (linear regression) as well as determination of 50% inhibition point (nonlinear regression using a logistic equation). Although CSL was previously estimated to be a hexamer of 8-kDa subunits by SDS-polyacrylamide gel electrophoresis (1Oda Y. Tatsumi Y. Biol. Pharm. Bull. 1993; 16: 978-981Crossref PubMed Scopus (16) Google Scholar), re-examination by MALDI-TOF revealed the subunit mass to be 12,635, and thus CSL is now considered to be a tetramer like many other plant lectins. CSL binding to the immobilized TA gave K D = 1.5 ± 0.22 × 10−7m(average of quadruplicates). This is somewhat lower than the values obtained by other methods (see Table IV).Table IVComparison of K D values obtained by different methodsMethodsLigandK DμmFIYeast cells0.30 ± 0.20FPMan3GlcNAc2-AMF1.55 ± 0.22FPMan3GlcNAc2-GP-fluorescein0.74 ± 0.17MPM3GlcNAc2-GP-Eu0.54 ± 0.04SPRTA0.15 ± 0.024-a Nonlinear regression analysis using a one-site model. Open table in a new tab 4-a Nonlinear regression analysis using a one-site model. Binding of CSL to S. cerevisiae cells showed K D (dissociation constant) of 0.34 μm by nonlinear regression and 0.56 μm by Scatchard plot (Fig. 2), values that are comparable with those reported for ConA (0.22 μm) and TGL (0.26 μm) (9Oda Y. Kinoshita M. Nakayama K. Ikeda S. Kakehi K. Anal. Biochem. 1999; 269: 230-235Crossref PubMed Scopus (4) Google Scholar). Interactions of AMF-labeled Man3GlcNAc2 with CSL, TGL, or ConA were analyzed using FP. Both CSL and ConA showed significant binding of AMF-labeled Man3GlcNAc2, but no detectable binding was shown by TGL (Fig. 3). Nonlinear regression analysis using a one-site model gaveK D for CSL and ConA as 1.93 ± 0.20 μm and 1.55 ± 0.22 μm, respectively. More detailed studies of structural requirements for binding by CSL were carried out by inhibition of HCC-CSL binding by oligosaccharides measured by FI. The results of inhibition of the lectin binding by oligosaccharides 1–6 and8 are shown in Table II. In general, these manno-oligosaccharides inhibited CSL and ConA more than TGL. Among the manno-oligosaccharides, 3, 4,6, and 8 were very potent inhibitors of CSL. For TGL, however, 5 was most inhibitory, followed by8, 6, and 2 in descending order, and3 and 4 were not inhibitory. In contrast, all of the tested manno-oligosaccharides were very inhibitory for ConA and almost to the same extent (51–57%), except for 8, which was the most effective (72%).Table IIInhibition of HCC-lectin binding to S. cerevisiae cellsLigands (10 μm)Inhibition2-aHCC-lectin (0.5 μM) was used for FI analysis.CSLTGLConA%%%10056271457370953438257504051666175487627722-a HCC-lectin (0.5 μM) was used for FI analysis. Open table in a new tab In addition to the above oligomannoses, 11 other oligosaccharides were used to obtain inhibition curves by FI (Fig.4). The inhibition curve of the branched mannotriose, 6, was included for comparison. The most potent inhibitors were 9, 10, and 15, all of which contain the tetrasaccharide unit depicted in Fig. 1. The two poorest ligands are 16 and 24. In 16, both branching mannose residues are covered with GlcNAc and, in24, by Galβ(1–4)GlcNAc. The three oligosaccharides of medium potency, 21–23, possess the bisecting GlcNAc and are substituted on at least one of the terminal Man residues in the core structure. Manβ(1–4)GlcNAc and chitobiose (GlcNAcβ(1–4)GlcNAc) showed no inhibition up to a concentration of 10 mm (data not shown). The results (Table III) are similar to those obtained by FI (Table II). Among the mannobioses, Manα(1–3)Man, 3, was the most potent inhibitor of CSL, whereas all of the manno-oligosaccharides showed potent inhibition of ConA.Table IIIInhibition of binding of AMF-oligosaccharide by CSL and ConALigands (20 mm)Inhibition3-aBinding of AMF-ligand (2 or 5 μm) measured by FP.CSLConA%%1051.42030.5351.658.447.554.455.474.63-a Binding of AMF-ligand (2 or 5 μm) measured by FP. Open table in a new tab As shown in Fig. 5, the best inhibitors are those containing structural elements shown in Fig. 1. PA oligosaccharides manifest the same inhibitory potency as their parent oligosaccharides (compare 10 and 11), confirming the noninvolvement of the (reducing) terminal GlcNAc in the binding. A dramatic difference is observed by the position of GlcNAc placed on Manα residues. When GlcNAc is on the Manα(1–6) residue (19), it remains as potent as unmodified parent compound (11). However, when GlcNAc is on the Manα(1–3) residue (20), the loss of potency is dramatic (10-fold in I50 values). Even more drastic is the loss of the Manα(1–3) residue, as shown by comparison of 10 and12. The influence of bisecting GlcNAc, judging from the comparison of inhibition by 13 and 14, seems negligible. Manβ(1–4)GlcNAc was totally ineffective in this assay. The results of binding of europium-labeled Man3GlcNAc2-glycopeptide to the microplate-coated CSL produced K D of 0.54 μm (estimated by nonlinear regression, data not shown)." @default.
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• W4251113918 title "Crocus sativus Lectin Recognizes Man3GlcNAc in the N-Glycan Core Structure" @default.
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