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• W2023147995 abstract "The jack bean lectin concanavalin A (ConA) and the Dioclea grandiflora lectin (DGL) are highly homologous Man/Glc-specific members of the Diocleinae subtribe. Both lectins bind, cross-link, and precipitate with carbohydrates possessing multiple terminal nonreducing Man residues. The present study investigates the binding and cross-linking interactions of ConA and DGL with a series of synthetic divalent carbohydrates that possess spacer groups with increasing flexibility and length between terminal α-mannopyranoside residues. Isothermal titration microcalorimetry was used to determine the thermodynamics of binding of the two lectins to the divalent analogs, and kinetic light scattering and electron microscopy studies were used to characterize the cross-linking interactions of the lectins with the carbohydrates. The results demonstrated that divalent analogs with flexible spacer groups between the two terminal Man residues possess higher affinities for the two lectins as compared with those with inflexible spacer groups. Furthermore, despite their high degree of homology, ConA and DGL exhibit differences in their kinetics of cross-linking and precipitation with the divalent analogs. Electron microscopy shows the loss of organized cross-linked lattices of the two lectins with analogs possessing increased distance between the terminal Man residues. The loss of lattice patterns with the analogs is distinct for each lectin. These results have important implications for the interactions of lectins with multivalent carbohydrate receptors in biological systems. The jack bean lectin concanavalin A (ConA) and the Dioclea grandiflora lectin (DGL) are highly homologous Man/Glc-specific members of the Diocleinae subtribe. Both lectins bind, cross-link, and precipitate with carbohydrates possessing multiple terminal nonreducing Man residues. The present study investigates the binding and cross-linking interactions of ConA and DGL with a series of synthetic divalent carbohydrates that possess spacer groups with increasing flexibility and length between terminal α-mannopyranoside residues. Isothermal titration microcalorimetry was used to determine the thermodynamics of binding of the two lectins to the divalent analogs, and kinetic light scattering and electron microscopy studies were used to characterize the cross-linking interactions of the lectins with the carbohydrates. The results demonstrated that divalent analogs with flexible spacer groups between the two terminal Man residues possess higher affinities for the two lectins as compared with those with inflexible spacer groups. Furthermore, despite their high degree of homology, ConA and DGL exhibit differences in their kinetics of cross-linking and precipitation with the divalent analogs. Electron microscopy shows the loss of organized cross-linked lattices of the two lectins with analogs possessing increased distance between the terminal Man residues. The loss of lattice patterns with the analogs is distinct for each lectin. These results have important implications for the interactions of lectins with multivalent carbohydrate receptors in biological systems. Lectins are carbohydrate-binding proteins that are widely conserved in nature, such as those in animals, plants, and microorganisms (1Varki A. Cummings R. Esko J. Freeze H. Hart G. Marth J. Essentials of Glycobiology. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York1999Google Scholar). The biological activities of many animal lectins have been determined, including receptor-mediated endocytosis of glycoproteins, cellular recognition and adhesion (cf. Ref. 2Drickamer K. Taylor M.E. Annu. Rev. Cell Biol. 1993; 9: 237-264Crossref PubMed Scopus (711) Google Scholar), regulation of inflammation (3Liu F.-T. Clin. Immunol. 2000; 97: 79-88Crossref PubMed Scopus (188) Google Scholar), and metastasis and control of cell growth (4Konstantinov K.N. Robbins B.A. Liu F.-T. Am. J. Pathol. 1996; 148: 25-30PubMed Google Scholar, 5Akahani S. Nangia-Makker P. Inohara H. Kim H.-R.C. Raz A. Cancer Res. 1997; 57 (R. C.): 5272-5276PubMed Google Scholar). A common feature of lectins is their multivalent binding properties (6Rini J.M. Annu. Rev. Biophys. Biomol. Struct. 1995; 24: 551-577Crossref PubMed Scopus (428) Google Scholar, 7Loris R. Hamelryck T. Bouckaert J. Wyns L. Biochim. Biophys. Acta. 1998; 1383: 9-36Crossref PubMed Scopus (478) Google Scholar). As a consequence, lectin binding to cells leads to cross-linking and aggregation of glycoprotein and glycolipid receptors. In many cases, these interactions are associated with signal transduction effects, including the arrest of bulk transport in ganglion cell axons (8Edmonds B.T. Koenig E. Cell Motil. Cytoskeleton. 1990; 17: 106-117Crossref PubMed Scopus (13) Google Scholar), molecular sorting of glycoproteins in the secretory pathways of cells (9Chung K.-N. Walter P. Aponte G.W. Moore H.-P. Science. 1989; 243: 192-197Crossref PubMed Scopus (117) Google Scholar), apoptosis of human T cells (10Perillo N.L. Pace K.E. Seilhamer J.J. Baum L.G. Nature. 1995; 378: 736-739Crossref PubMed Scopus (950) Google Scholar, 11Perillo N.L. Uittenbogaart C.H. Nguyen J.T. Baum L.G. J. Exp. Med. 1997; 185: 1851-1858Crossref PubMed Scopus (267) Google Scholar), regulation of the T cell receptor (12Vespa G.N.R. Lewis L.A. Kozak K.R. Moran M. Nguyen J.T. Baum L.G. Miceli M.C. J. Immunol. 1999; 162: 799-806PubMed Google Scholar, 13Demetriou M. Granovsky M. Quaggin S. Dennis J.W. Nature. 2001; 409: 733-739Crossref PubMed Scopus (745) Google Scholar), and growth regulation of neuroblastoma cells (14Kopitz J. von Reitzenstein C. Burchert M. Cantz M. Gabius H.-J. J. Biol. Chem. 1998; 273: 11205-11211Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). Thus, the carbohydrate cross-linking properties of lectins are a key feature of their biological activities. The cross-linking properties of a variety of plant and animal lectins with multivalent carbohydrates and glycoproteins have recently been reviewed (15Dam T.K. Brewer C.F. Methods Enzymol. 2003; 362: 455-486Crossref PubMed Scopus (22) Google Scholar). Studies show that a number of lectins form homogeneous cross-linked complexes with branched chain oligosaccharides and glycoproteins. For example, quantitative precipitation experiments with the Man/Glc-specific lectin concanavalin A (ConA) 1The abbreviations used are: ConA, concanavalin A; DGL, Dioclea grandiflora lectin; αMDM, methyl α-d-mannopyranoside; ITC, isothermal titration microcalorimetry; EM, electron microscopy. in the presence of binary mixtures of a series of bivalent N-linked oligomannose glycopeptides indicate that each glycopeptide forms its own unique cross-linked complex with the lectin (16Bhattacharyya L. Khan M.I. Brewer C.F. Biochemistry. 1988; 27: 8762-8767Crossref PubMed Scopus (33) Google Scholar). Subsequent x-ray crystallographic studies have demonstrated different lattice structures of crystalline cross-linked complexes of the soybean agglutinin with four different divalent carbohydrates (17Olsen L.R. Dessen A. Gupta D. Sabesan S. Sacchettini J.C. Brewer C.F. Biochemistry. 1997; 36: 15073-15080Crossref PubMed Scopus (100) Google Scholar). The different lattice structures are due to differences in the structures of the cross-linking carbohydrates (17Olsen L.R. Dessen A. Gupta D. Sabesan S. Sacchettini J.C. Brewer C.F. Biochemistry. 1997; 36: 15073-15080Crossref PubMed Scopus (100) Google Scholar). The ability to form unique cross-linked complexes with glycoconjugates and to separate different counter-receptors into homogeneous cross-linked aggregates has recently been implicated in the apoptotic activity of galectin-1, a member of the β-galactosidase-specific animal lectin family (18Pace K.E. Lee C. Stewart P.L. Baum L.G. J. Immunol. 1999; 163: 3801-3811Crossref PubMed Google Scholar). Recently, galectin-3, another member of the galectin family, has been shown to form disorganized, heterogeneous cross-linked complexes with multivalent carbohydrates (19Ahmad N. Gabius H.-J. André S. Kaltner H. Sabesan S. Roy R. Liu B. Macaluso F. Brewer C.F. J. Biol. Chem. 2004; 279: 10841-10847Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). The biological properties of galectin-3, including its anti-apoptotic activities (20Liu F.-T. Patterson R.J. Wang J.L. Biochim. Biophys. Acta. 2002; 1572: 263-273Crossref PubMed Scopus (562) Google Scholar) and ability to antagonize the growth inhibitory activity of galectin-1 in neuroblastoma cells (21Kopitz J. von Reitzenstein C. Andre S. Kaltner H. Uhl J. Ehemann V. Cantz M. Gabius H.-J. J. Biol. Chem. 2001; 276: 35917-35923Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar), may relate to its ability to randomly cross-link glycoconjugates and prevent separation of different receptors. Hence, the ability of lectins to form organized or disorganized cross-linked complexes with multivalent glycoconjugate receptors, such as galectin-1 and -3, respectively, may relate to their biological activities. In the present study, we investigated the effects of varying the flexibility and distance between terminal Man residues in divalent carbohydrate analogs on their binding thermodynamics and cross-linking interactions with ConA and the lectin from Dioclea grandiflora (DGL). Isothermal titration calorimetry (ITC), kinetic light scattering, and electron microscopy (EM) studies were used to characterize these interactions. The results demonstrated that the thermodynamics of binding and cross-linking properties of the two lectins are sensitive to the flexibility and spacing between the carbohydrate epitopes of the analogs. ConA was purchased from Sigma and/or prepared from jack bean (Canavalia ensiformis) seeds (Sigma) according to the method of Agrawal and Goldstein (22Agrawal B.B.L. Goldstein I.J. Biochim. Biophys. Acta. 1967; 147: 262-271Crossref PubMed Scopus (449) Google Scholar). The concentration of ConA was determined spectrophotometrically at 280 nm using A1%,1 cm = 13.7 and 12.4 at pH 7.2 and 5.2, respectively (23Goldstein I.J. Poretz R.D. Liener I.E. Sharon N. Goldstein I.J. The Lectins. Academic Press, Inc., New York1986: 35-244Google Scholar), and expressed in terms of monomer (Mr = 25,600). DGL was isolated from D. grandiflora seeds obtained from northeastern Brazil (Albano Ferreira Martin Ltd., São Paulo, Brazil) as described previously (24Moreira R.A. Barros A.C.H. Stewart J.C. Pusztai A. Planta. 1983; 158: 63-69Crossref PubMed Scopus (59) Google Scholar). The concentration of DGL was determined spectrophotometrically at 280 nm using A1%,1 cm = 12.0 at pH 7.2 and expressed in terms of monomer (Mr = 25,000) (24Moreira R.A. Barros A.C.H. Stewart J.C. Pusztai A. Planta. 1983; 158: 63-69Crossref PubMed Scopus (59) Google Scholar). αMDM was purchased from Sigma. The synthesis of carbohydrate analogs 1, 2, 3, and 4 has been reported previously (25Roy R. Das S.K. Dominique R. Trono M.C. Mateo F.H. Gonzalez F.S. Pure Appl. Chem. 1999; 71: 565-571Crossref Scopus (59) Google Scholar), as have analogs 9–13 (26Page D. Roy R. Glycoconj. J. 1997; 14: 345-356Crossref PubMed Scopus (51) Google Scholar). Synthesis of 5–8 will be reported elsewhere. The concentrations of carbohydrates were determined by modification of the Dubois phenolsulfuric acid method (27Dubois M. Gilles K.A. Hamilton J.K. Rebers P.A. Smith F. Anal. Chem. 1956; 28: 350-356Crossref Scopus (41132) Google Scholar, 28Saha S.K. Brewer C.F. Carbohydr. Res. 1994; 254: 157-167Crossref PubMed Scopus (271) Google Scholar) using appropriate monosaccharides as standards. Structures of these analogs are shown in Figs. 1 and 2.Fig. 2Structures of bivalent mannosides 5–13.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Isothermal Titration Microcalorimetry—ITC experiments were performed using an MCS ITC instrument from Microcal, Inc. (Northampton, MA). Injections of 4 ml of carbohydrate solution were added from a computer-controlled 250- or 100-μl microsyringe at an interval of 4 min into the sample solution of lectin (cell volume = 1.34 ml) with stirring at 350 revolutions/min. An example of an ITC experiment is shown in Fig. 3 for bivalent analog 11 with ConA at 27 °C. Control experiments performed by making identical injections of saccharide into a cell containing buffer without protein showed insignificant heats of dilution. The concentrations of lectins were 0.1–0.19 mm, and the sugars were 1.0–6.0 mm, respectively. Titrations were done at pH 5.0–5.2 and at NaCl concentrations from 0.05 to 0.15 m. The experimental data were fitted to a theoretical titration curve using software supplied by Microcal, with ΔH (enthalpy change in kcal/mol), Ka (association constant in m–1), and n (number of binding sites/monomer) as adjustable parameters. The quantity c = Ka Mt(0), where Mt(0) is the initial macromolecule concentration, is of importance in titration microcalorimetry (29Wiseman T. Williston S. Brandt J.F. Lin L.-N. Anal. Biochem. 1989; 179: 131-137Crossref PubMed Scopus (2446) Google Scholar). All experiments were performed with c values 1 < c < 200. The instrument was calibrated using the calibration kit containing ribonuclease A (RNase A) and cytidine 2′-monophosphate (2′-CMP) supplied by the manufacturer microcal. Thermodynamic parameters were calculated from the equation ΔG =ΔH – TΔS =–RT ln Ka, where ΔG, ΔH, and ΔS are the changes in free energy, enthalpy, and entropy of binding, respectively, T is the absolute temperature, and R = 1.98 cal mol–1 K–1. Kinetics of Precipitation—Measured volumes of lectin and saccharide solution at stoichiometric concentration (2:1) were mixed in a 1-ml quartz cuvette, and the time-dependent development of turbidity was measured at 420 nm (30Bhattacharyya L. Fant J. Lonn H. Brewer C.F. Biochemistry. 1990; 29: 7523-7530Crossref PubMed Scopus (43) Google Scholar). The buffer was Hepes (0.1 m Hepes, 0.15 m NaCl, 1 mm CaCl2, and 1 mm MnCl2, pH 7.2). All experiments were done at room temperature. Absorbances were monitored continuously until they remained constant for 30 min. After each experiment, a portion of the precipitate was treated with 100 mm αMDM to check whether or not the precipitation was due to the binding of the saccharides. Electron Microscopy—Negative stain electron microscopy of the precipitates was performed by placing the samples on 300-mesh carboncoated Parlodion grids that had been freshly glow-discharged for 2 min, touched to filter paper and floated on a drop of 1% phosphotungstic acid, pH 7.0, and blotted immediately. The samples were observed at 80 kV in a JEOL 1200EX electron microscope. Thermodynamics of Binding of ConA and DGL to Analogs 1–13—At pH 5.0 and low salt concentrations, both ConA and DGL are dimers and do not precipitate with analogs 1–13. Hence, ITC studies were performed under these conditions. ITC data for ConA binding to 1–13 at 300 K are shown in Table I. Ka values for 1–4 are very similar and nearly twice as great as that of the monosaccharide αMDM. Ka values for 5–13 are nearly 4–6 times greater than that of αMDM. The –ΔH values for ConA binding to 1–13 are greater than that of αMDM, with values for 1–4 generally lower than those of 6–13. The n values for ConA binding to 1–13 are between 0.68 and 0.52, as compared with 1.0 for αMDM.Table IThermodynamic binding parameters for ConA with bivalent sugars at pH 5.0, 27 °CKacErrors in Ka range from 1 to 7%.-ΔGdErrors in ΔG are < 1%.-ΔHeErrors in ΔH are 1-4%.-TΔSfErrors in TΔS are 1-7%.ngErrors in n are <2%.M-1 × 10-4kcal / molkcal / molkcal / molsites / monomerαMDMaαMDM, methyl α-d-mannopyranoside.1.25.68.42.81.012.36.011.25.20.6222.56.014.18.10.523bData taken from Ref. 32.2.25.912.76.70.594bData taken from Ref. 32.2.56.011.45.40.6754.36.310.34.00.6864.76.413.47.00.6174.96.414.78.30.578bData taken from Ref. 32.5.36.515.28.70.549bData taken from Ref. 32.4.76.417.010.60.5210bData taken from Ref. 32.5.46.516.610.10.52116.76.615.48.80.5812bData taken from Ref. 32.6.86.614.37.70.60135.36.515.99.40.57a αMDM, methyl α-d-mannopyranoside.b Data taken from Ref. 32Dam T.K. Roy R. Das S.K. Oscarson S. Brewer C.F. J. Biol. Chem. 2000; 275: 14223-14230Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar.c Errors in Ka range from 1 to 7%.d Errors in ΔG are < 1%.e Errors in ΔH are 1-4%.f Errors in TΔS are 1-7%.g Errors in n are <2%. Open table in a new tab The ITC data for DGL binding to 1–13 are shown in Table II. Ka values for DGL binding to 1–4 are similar and nearly four times as great as that of αMDM. Ka values for 5–8 are 8–20-fold greater than that of αMDM, whereas the Ka values of 9–13 are 3–9-fold greater than that of αMDM. The –ΔH values for DGL binding to 1–13 are generally greater than that of αMDM (Table II). The n values for DGL binding to 1–13 are between 0.76 and 0.53, as compared with 1.0 for αMDM.Table IIThermodynamic binding parameters for DGL with bivalent sugars at pH 5.0, 27 °CKacErrors in Ka are 7-10%.-ΔGdErrors in ΔG are 1%.-ΔHeErrors in ΔH are 1-7%.-TΔSfErrors in TΔS are 2-13%.ngErrors in n are 1-7%.M-1 × 10-4kcal / molkcal / molkcal / molsites / monomerαMDMaαMDM, methyl α-D-mannopyranoside.0.464.98.23.31.011.95.810.14.30.7622.25.910.84.90.683bData taken from Ref. 32.2.05.911.25.30.614bData taken from Ref. 32.1.65.711.05.30.7054.16.310.44.10.7566.36.511.65.10.6877.66.712.96.20.618bData taken from Ref. 32.10.66.914.87.90.569bData taken from Ref. 32.1.65.714.38.60.6010bData taken from Ref. 32.2.56.014.88.80.57114.56.415.69.20.5212bData taken from Ref. 32.3.76.212.05.80.70132.66.015.39.30.53a αMDM, methyl α-D-mannopyranoside.b Data taken from Ref. 32Dam T.K. Roy R. Das S.K. Oscarson S. Brewer C.F. J. Biol. Chem. 2000; 275: 14223-14230Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar.c Errors in Ka are 7-10%.d Errors in ΔG are 1%.e Errors in ΔH are 1-7%.f Errors in TΔS are 2-13%.g Errors in n are 1-7%. Open table in a new tab Time-dependent Light Scattering of ConA and DGL in the Presence of 1–13—Fig. 4a shows the time-dependent light-scattering profiles of 90 μm ConA with 45 μm of 1–4, respectively, at pH 7.2, room temperature. The kinetic profile is slowest and lowest for 1, fastest for 2, and greatest for 3, with an intermediate profile for 4. Fig. 4b shows the time-dependent light scattering of 90 μm DGL with 1–4 (45 μm each) at pH 7.2 at room temperature. The profiles for 1 and 2 appear the slowest of the four, with 3 nearly as fast as 4. Fig. 5a shows the time-dependent light-scattering profiles of 90 μm ConA with 45 μm each of 9–12, respectively, at pH 7.2, room temperature. Analog 11 showed the slowest precipitation kinetics, with 9, 12, and 10 showing increasing rates of precipitation. Fig. 5b shows the time-dependent light-scattering profile of 90 μm DGL with 45 μm each of 9–12 at pH 7.2, room temperature. The kinetic precipitation profile of 10 is slowest, with 9 somewhat faster. Analogs 11 and 12 precipitate much faster with DGL as compared with 9 and 10. Analogs 5–8 also showed unique kinetics of precipitation with the two lectins under the above conditions (data not shown). Electron Micrographs of the Precipitates of ConA and DGL with 1–13—Negative stain electron micrographs of the precipitates of ConA and DGL with 1, 2, and 3 are shown in Fig. 6. Patterns are observed for all three precipitates of ConA, whereas patterns are observed for the precipitates of DGL with 1 and 3 but not for 2. ConA and DGL precipitates with 4 failed to show patterns. Fig. 7 shows negative stain electron micrographs of the precipitates of ConA with 5, 6, and 7 and DGL with 5 and 6. The precipitates of ConA with 5–7 all show observable lattice patterns, whereas the precipitates of DGL with 5 and 6 also show lattice patterns. The precipitates of DGL with 7 failed to show a pattern. The precipitates of ConA and DGL with 8 also failed to show a pattern. Fig. 8 shows negative stain electron micrographs of the precipitates of ConA with 9, 10, and 11, and the precipitates of DGL with 9–13. Although the precipitates of ConA with 9, 10, and 11 showed lattice patterns, the precipitates of ConA with 12 and 13 failed to show patterns. All of the precipitates of DGL with 9–13 showed lattice patterns. ConA and DGL are highly homologous lectins with 81% similarities in amino acid sequences and identical quaternary structures and subunit composition (cf. Ref. 31Rozwarski 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). Both are tetramers at pH 7.0 and dimers at pH 5.0 having low salt concentration (cf. Ref. 32Dam T.K. Roy R. Das S.K. Oscarson S. Brewer C.F. J. Biol. Chem. 2000; 275: 14223-14230Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Both lectins possess similar carbohydrate binding specificities for Man containing oligosaccharides (cf. Ref. 33Dam T.K. Cavada B.S. Grangeiro T.B. Santos C.F. Ceccatto V.M. de Sousa F.A.M. Oscarson S. Brewer C.F. J. Biol. Chem. 2000; 275: 16119-16126Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). X-ray crystallographic data show that amino acid residues of the binding sites of ConA (34Naismith J.H. Field R.A. J. Biol. Chem. 1996; 271: 972-976Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar) and DGL (31Rozwarski 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) are conserved and that there is one binding site/subunit in both lectins. The tetrameric forms of ConA and DGL are known to bind and precipitate with multivalent carbohydrates and glycoproteins (35Brewer C.F. Chemtracts Biochem. Mol. Biol. 1996; 6: 165-179Google Scholar, 36Gupta D. Oscarson S. Raju T.S. Stanley P. Toone E.J. Brewer C.F. Eur. J. Biochem. 1996; 242: 320-326Crossref PubMed Scopus (46) Google Scholar). ConA has also been shown to form homogeneous cross-linked lattices with individual glycopeptides (16Bhattacharyya L. Khan M.I. Brewer C.F. Biochemistry. 1988; 27: 8762-8767Crossref PubMed Scopus (33) Google Scholar) and glycoproteins (37Mandal D.K. Brewer C.F. Biochemistry. 1992; 31: 12602-12609Crossref PubMed Scopus (62) Google Scholar). ITC studies have shown that ConA and DGL bind with higher affinities to multivalent carbohydrates containing 2–4 mannopyranoside residues/molecule, relative to the monosaccharide αMDM (32Dam T.K. Roy R. Das S.K. Oscarson S. Brewer C.F. J. Biol. Chem. 2000; 275: 14223-14230Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). The effects of varying the flexibility and spacing between binding epitopes in multivalent carbohydrates on their thermodynamics of binding, kinetics of precipitation, and structures of their cross-linked complexes with lectins has not been investigated. The present study investigated these interactions of ConA and DGL with bivalent Man analogs 1–13 in Figs. 1 and 2. Thermodynamics of ConA and DGL Binding to 1–13—For comparison, the thermodynamics of ConA and DGL binding to αMDM as well as to 3, 4, 8, 9, 10, and 12 are shown in Tables I and II, respectively (32Dam T.K. Roy R. Das S.K. Oscarson S. Brewer C.F. J. Biol. Chem. 2000; 275: 14223-14230Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). The results for αMDM show n values for both lectins close to 1.0, demonstrating that the monosaccharide binds as a monovalent ligand to both proteins. The ΔH values of –8.4 and –8.2 kcal/mol for ConA and DGL, respectively, are similar, as are the Ka values of 1.2 × 104m–1 and 0.46 × 104m–1, respectively. Analogs 1–13 show enhanced affinities relative to αMDM for ConA and DGL and different ΔH and n values. Previous ITC studies have shown that the value of n is inversely proportional to the functional valency of carbohydrate ligands for lectins (32Dam T.K. Roy R. Das S.K. Oscarson S. Brewer C.F. J. Biol. Chem. 2000; 275: 14223-14230Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Values of n of 0.5 have been observed for higher affinity carbohydrate ligands binding to ConA and DGL, with values between 0.5 and 0.8 for lower affinity bivalent ligands due to incomplete binding of the second epitope (32Dam T.K. Roy R. Das S.K. Oscarson S. Brewer C.F. J. Biol. Chem. 2000; 275: 14223-14230Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). The n values for ConA and DGL in Tables I and II, respectively, are consistent with lower affinity divalent carbohydrates binding to both lectins. The greater –ΔH values for 1–13 binding to both lectins, relative to αMDM, are also consistent with divalent binding of the analogs (32Dam T.K. Roy R. Das S.K. Oscarson S. Brewer C.F. J. Biol. Chem. 2000; 275: 14223-14230Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Interestingly, the enhanced Ka values of both lectins for 1–13 appear to cluster into two groups for each lectin. Analogs 1–4 show approximate 2-fold enhanced affinities for ConA, and 3–4-fold enhanced affinities for DGL, relative to αMDM. On the other hand, analogs 5–13 show enhanced affinities of 4–6-fold for ConA, and 5–20-fold for DGL, relative to αMDM (the exception is 9, which possesses ∼3-fold higher affinity). Thus, 1–4 possess smaller enhanced affinities for both lectins as compared with 5–13. The most obvious structural differences between these two groups of bivalent analogs is the flexibility of the linker regions between the outer two Man residues in each molecule. Analogs 1–4 possess relatively ridged linkers consisting of one or two acetylenic groups with or without a phenyl group, whereas 5–8 and 10–13 possess relatively flexible methylene groups. 9 is absent a methylene group between the two aryl glycoside moieties, which may be part of the reason for its relatively modest enhanced affinity for DGL in that group. Thus, the degree of flexibility of the spacer groups of the analogs appears to modulate their enhanced affinities for ConA and DGL. Kinetics of Precipitation of ConA and DGL with Analogs 1–13—At pH 7.0 and high salt concentration, ConA and DGL are tetramers and precipitate with analogs 1–13. The kinetics of precipitation of both lectins with the analogs is shown in the time-dependent light-scattering profiles in Figs. 4 and 5. Because the concentrations of the two lectins and the concentrations of the analogs are the same, a comparison of the precipitation rate profiles of different analogs with the two lectins can be made. Differences in the precipitation rates are due to several factors, including the affinities of the analogs, the rates of formation of soluble cross-linked complexes, and the solubility constants of their cross-linked lattices. Fig. 4a shows the kinetics of cross-linking and precipitation of ConA with analogs 1–4. Analog 1 shows the slowest rate of precipitation with ConA followed by 4, 3, and 2, respectively. Analog 3, however, shows a greater degree of precipitation with ConA than 2. Thus, the effects of different spacer groups of the analogs are observed in their kinetics and extent of precipitation with ConA. Fig. 4b shows the time-dependent light-scattering profiles of DGL with 1–4. Although analog 1 is the slowest to precipitate with DGL, similar to ConA, the order of kinetics and extent of precipitation of DGL with 2–4 is different from that with ConA. Analogs 3 and 4 show the fastest and largest degree of precipitation of DGL, whereas 2 and 3 are fastest with ConA. Thus, the different spacer groups of analogs 1–4 exhibit different kinetics and extents of precipitation with ConA and DGL, even though the structures of the two proteins are very similar (31Rozwarski 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). Fig. 5 shows the time-dependent light-scattering profiles of ConA and DGL with analogs 9–12, respectively. Similar to 1–4, analogs 9–12 show differential kinetics and the extent of precipitation with ConA and DGL. Furthermore, the relative kinetics and extent of precipitation of the analogs differs for the two lectins. These results are similar to those of analogs 1–4 with the two lectins. These results demonstrate that differences in the structures of the two lectins affect their kinetics of cross-linking interactions with 1–4 and 9–12 (Figs. 4 and 5). Electron Microscopy of the Cross-linked Lattices of ConA and DGL with 1–13—We have previously used negative stain EM to observe the presence of organized lattices in cross-linked complexes of lectins with multivalent carbohydrates (cf. Ref. 15Dam T.K. Brewer C.F. Methods Enzymol. 2003; 362: 455-486Crossref PubMed Scopus (22) Google Scholar). In the present study, differences in the spacer groups in 1–13 are observed to affect the structures of their cross-linked complexes with ConA and DGL (15Dam T.K. Brewer C.F. Methods Enzymol. 2003; 362: 455-486Crossref PubMed Scopus (22) Google Scholar). For example, ConA shows EM patterns for precipitates with 1–3, 5–7, and 9–11. No patterns are observed for the precipitates of 4, 8, 12, and 13. The lack of patterns observed for these precipitates correlates with the increased distance and flexibility separating the Man residues in the analogs, which, in turn, prevents the formation of organized cross-linked complexes. DGL also shows a pattern of structures for its precipitates with 1–13. EM patterns for precipitates are observed with 1, 3, 5, 6, and 9–13. No patterns are observed for 2, 4, 7, and 8. With the exception of 2, the lack of patterns observed with 4, 7, and 8 correlate with the increased distance and flexibility between the Man residues in the molecules, a finding similar to that for ConA. Although the structures and binding specificities of ConA and DGL are very similar, both lectins show differences in their patterns of lattice structures with 1–13. DGL shows patterns with 12 and 13, unlike ConA, which shows no patterns with these two analogs. ConA shows a pattern for precipitates with 2, whereas DGL shows no pattern. ConA also shows a pattern for precipitates with 7, whereas DGL shows no pattern. Thus, two highly homologous lectins show differences in the observed patterns of their precipitates with 1–13. The detailed lattice structures of the cross-linked complexes of the two lectins with the analogs in Figs. 6 and 7 will await x-ray fiber diffraction and image reconstruction of the EMs (cf. Ref. 38Cheng W. Bullitt E. Bhattacharrya L. Brewer C.F. Makowski L. J. Biol. Chem. 1998; 273: 35016-35022Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) or x-crystallographic analysis of crystals of the respective cross-linked complexes (cf. Ref. 17Olsen L.R. Dessen A. Gupta D. Sabesan S. Sacchettini J.C. Brewer C.F. Biochemistry. 1997; 36: 15073-15080Crossref PubMed Scopus (100) Google Scholar) The results demonstrated that bivalent Man analogs with flexible spacer groups exhibit higher affinities for ConA and DGL than analogs with rigid spacer groups. ConA and DGL also showed differences in their kinetics of precipitation with the bivalent analogs and differences in the EM patterns of their precipitates with 1–13. The present findings indicated that the spacing and flexibility of carbohydrate epitopes in divalent carbohydrates affects their thermodynamics of binding, kinetics of precipitation, and structures of their cross-linked complexes with different lectins. These results have important implications for the interaction of lectins with multivalent carbohydrate receptors in biological systems (39Brewer C.F. Miceli M.C. Baum L.G. Curr. Opin. Struct. Biol. 2002; 12: 616-623Crossref PubMed Scopus (380) Google Scholar)." @default.
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• W2023147995 title "Thermodynamic, Kinetic, and Electron Microscopy Studies of Concanavalin A and Dioclea grandiflora Lectin Cross-linked with Synthetic Divalent Carbohydrates" @default.
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