2 Departments of Molecular Pharmacology, and Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461; 3 Institut für Physiologische Chemie, Ludwig-Maximilians Universität München, 80539 München, Germany; 4 Du Pont Company Wilmington, DE 19880; and 5 Department of Organic Chemistry, Stockholm University, Stockholm, S-106 91, Sweden
Received on March 31, 2004; revised on May 4, 2004; accepted on May 10, 2004
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Abstract |
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Key words: binding thermodynamics / galectin-1 / galectin-3 / multivalent carbohydrates
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Introduction |
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Galectin-1 and -3 have been among the most widely studied members of the galectins because they have been reported to have different biological activities. For example, galectin-1 induces apoptosis in a variety of cells, including immature T cells (Pace et al., 2000), whereas galectin-3 is associated with antiapoptotic activities (Yang et al., 1996
). Interestingly, galectin-3 blocks the negative growth regulation of galectin-1 on neuroblastoma cells in culture (Kopitz et al., 2001
). Hence it is important to understand the structural and functional basis for their different biological activities.
Galectin-1 has been shown by analytical sedimentation data and mass spectrometry to be a dimer in solution, and galectin-3 to be a monomer in solution (Kopitz et al., 2003; Morris et al., 2004
). Galectin-1 crystallizes as a dimer in one-dimensional cross-linked complexes with a divalent complex oligosaccharide (Bourne et al., 1994
) and forms homogeneous cross-linked complexes with asialofetuin (Gupta and Brewer, 1994
). Galectin-3 precipitates as a pentamer with certain multivalent carbohydrates and glycoproteins and forms heterogeneous cross-linked complexes (Ahmad et al., 2004
). Hence a small amount of galectin-3 pentamer is in equilibrium with the monomer in solution, and the monomer is converted to pentamer by mass-action equilibria during precipitation with multivalent oligosaccharides (Ahmad et al., 2004
). It is also known that the N-terminal domain of galectin-3 is responsible for the oligomerization of the molecule (Birdsall et al., 2001
; Massa et al., 1993
; Yang et al., 1998
).
The carbohydrate binding specificities of galectins-1 and -3 are similar in that they both bind to LacNAc and lactose residues (Barondes et al., 1994; Gabius, 1997
; Kasai and Hirabayashi, 1996
). However, differences exist, including the observation that galectin-3 binds to internal and terminal LacNAc residues in polylactosamine chains, whereas galectin-1 prefers nonreducing terminal LacNAc residues (Ahmad et al., 2002
).
Because a large number of cell surface carbohydrates are branched chain structures and often multivalent in their interactions with lectins (Brewer, 1996), it is important to characterize the binding of multivalent carbohydrates with galectins-1 and -3. Hughes and co-workers (Sato and Hughes, 1992
), using hapten inhibition, reported binding of bi- and trivalent oligosaccharides to galectin-3. In addition, galectin-1 and galectin-3 from human lung show differential binding toward multivalent mucin type oligosaccharides by hapten inhibition (Sparrow et al., 1987
). Galectin-3 has recently been shown to precipitate with certain biantennary branched chain carbohydrates (the 2,4-, 2,6-, 3,6-, and 4,6-pentasaccharides in Figure 1) (Ahmad et al., 2004
). To date, however, their has been little thermodynamic data reported for the binding of galectins-1 and -3 to multivalent carbohydrates. Such data are also important in terms of designing carbohydrate inhibitors, such as cluster glycosides of the two galectins.
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Results |
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Thermodynamics of binding of divalent LacNAc oligosaccharides to galectin-3
Table II shows ITC data for the binding of galectin-3 to the oligosaccharides in Figure 1. There were little differences in the Ka values at 300 K of C2- to C4-DiLacNAc, and all were similar to that of LacNAc II. H showed an increase from 7.5 kcal mol1 for LacNAc II to 12.8 for C2-DiLacNAc, 17.2 kcal mol1 for C3-DiLacNAc, and 16.2 kcal mol1 for C4-DiLacNAc. The n values for the three analogs were close to 0.5.
Ka values at 300 K for the pentasaccharides in Figure 1 were similar to that of LacNAc II. The pentasaccharides possessed larger H and T
S values relative to LacNAc II. The highest affinity branched chain carbohydrate was lacto-N-hexaose, which showed a threefold enhancement relative to LacNAc II. ITC data was also obtained at 287 K for lacto-N-hexaose and the pentasaccharides to compare data with that for galectin-1. The highest affinity ligand was lacto-N-hexaose (14.7 x 104 M1), which was nearly fourfold greater than LacNAc II. The Ka values of galectins-1 and -3 for the 4,6-pentasaccharide at 287 K were similar, relative to LacNAc II, but the remaining three pentasaccharides showed slightly enhanced affinity at 287 K for galectin-3 relative to LacNAc II.
Thermodynamics of binding of divalent oligosaccharides to galectin-3 CRD
ITC thermodynamic binding data at 287 K for the binding of a variety of carbohydrates to the galectin-3 CRD are shown in Table III. Ka, H, and T
S values for the binding of LacNAc I and II and dithiogalactoside were similar to the intact galectin-3 molecule (Ahmad et al., 2002
). The thermodynamic binding parameters of 2,6-sialyl diLacNAc were similar to that of LacNAc II, indicating that galectin-3 CRD can recognize internal binding epitopes as well as the nonreducing terminal ones. The binding data for lacto-N-tetraose and Di-LacNAc did not show much change compared to that of LacNAc II.
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Discussion |
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Binding of linear and branched chain diLacNAc analogs to galectins-1 and -3
To determine the effects of varying the distance between two LacNAc residues in a molecule on their binding interactions with galectins-1 and -3, C2-, C3- and C4-DiLacNAc were synthesized that possess 2-, 3-, and 4-methylene groups, respectively, between two nonreducing LacNAc residues. Likewise, to determine the effects of varying the structures of biantennary LacNAc analogs on their binding interactions with the two galectins, the 2,3-, 2,4-, 3,6-, and 4,6-pentasaccharides in Figure 1 were tested. Hemagglutination inhibition data in Table I, however, show little difference in the inhibition potencies of the linear chain analogs for the two galectins relative to LacNAc II. Hemagglutination inhibition data for the biantennary analogs also show no enhanced inhibition potencies for galectin-1 but suggest some smaller inhibition enhancements for certain biantennary analogs for galectin-3, as was noted for wedgelike glycodendrimers (André et al., 2001). To determine if both types of analogs bind as bivalent carbohydrates as well as their thermodynamics of binding to the two galectins, ITC experiments were performed.
It has been previously shown that the ITC-derived n value, the number of binding sites per monomer of protein, is inversely proportional to the functional valency of carbohydrates (Dam et al., 2000). For example, bivalent carbohydrates binding to the lectins Concanavalin A and Dioclea grandiflora show n values close to 0.5. In these cases, the X-ray crystal structures of the lectins show a single binding site per monomer in both cases (Naismith and Field, 1996
; Rozwarski et al., 1998
), and therefore the reduced n value reflects doubling of the number of binding epitopes per molecule of the ligands. The X-ray crystal structures of galectin-1 (Liao et al., 1994
) and the CRD domain of galectin-3 (Seetharaman et al., 1998
) show that only one molecule of LacNAc II is associated with each monomer of the two proteins. Table II shows that the n values obtained by ITC for LacNAc II is close to 1.0 for galectins-1 and -3, as previously reported (Ahmad et al., 2002
), in agreement with the X-ray crystal data. Table II also shows that the n values for the linear and branched chain diLacNAc analogs are close to 0.5 for both galectins. Thus the analogs are functionally bivalent, which agrees with their structural valency, and therefore both LacNAc residues in each analog are involved in binding to the two galectins. Importantly, there are cases in which the functional valency of multivalent carbohydrates are less than their structural valency (Dam et al., 2000
).
The ITC-derived Ka values at 287 K and 300 K in Table II for C2-, C3-, and C4-DiLacNAc were similar to that of LacNAc II for both galectins, respectively. These results agree with the hemagglutination inhibition data in Table I. The binding enthalpies, H, of the two galectins to the linear spacer analogs increased by nearly a factor of two relative to LacNAc II. These increases, however, were offset by increases in the entropy of binding, T
S, such that no increases occurred in their Ka values. These results contrast with the two- to fourfold and 420-fold increases in affinities of the plant lectins Concanavalin A and D. grandiflora lectin, respectively, for a series of linear spacer analogs containing two nonreducing mannopyranoside residues, relative to methyl
-D-mannopyranoside (Dam et al., 2000
).
The Ka values at 287 K in Table II for lacto-N-hexaose and the 2,4-, 2,6-, 3,6-, and 4,6-pentasaccharides in Figure 1 for galectin-1 are very similar to that of LacNAc II, which agrees with the hemagglutination inhibition data in Table I. The binding enthalpies, H, of the branched chain analogs were close to those of the linear spacer analogs. The n values for these analogs were close to 0.5 indicating their functional bivalency.
The Ka values of galectin-3 at 300 K in Table II for the four pentasaccharides were similar to that of LacNAc II; however, the Ka value of lacto-N-hexaose was nearly threefold greater than LacNAc II. The Ka values at 287 K for the pentasaccharides were slightly greater than that of LacNAc II; however, the value for lacto-N-hexaose was nearly fourfold greater than that of LacNAc II. The H values for the pentasaccharides at 287 K (the ITC c value is more optimal than at 300 K) shows a near doubling of that for LacNAc II, as expected for divalent oligosaccharides binding to a lectin (Dam et al., 2000
). The observation that galectin-3 precipitates as a pentamer to a greater extent with the 3,6-pentasaccharide, followed by the 4,6-, 2,6-, and 2,4-pentasaccharides (Ahmad et al., 2004
), is thus not due to differences in their Ka values but rather to the stability and solubility of their respective cross-linked complexes. It is also interesting to note that although the Ka values for the four pentasaccharides are similar for both galectin-1 and -3, the four pentasaccharides show differential inhibition activities toward five Gal-specific plant lectins (Gupta et al., 1993
). Thus the architectures of the binding sites for galectins-1 and -3 appear to be more open toward binding these biantennary branch chain oligosaccharides as compared with certain Gal-specific plant lectins. However, the sixfold enhanced affinity of the plant lectin Erythrina cristagalli for the four pentasaccharides relative to LacNAc II (Gupta et al., 1993
) is not present in the binding of galectins-1 and -3 to the four pentasaccharides. These results are important in terms of designing carbohydrate inhibitors to the two galectins.
The data in Table II show that lacto-N-hexaose binds with three- to fourfold higher affinity to galectin-3 relative to LacNAc II. No similar increase in affinity occurs with galectin-1. Sato and Hughes (1992) reported that binding of lacto-N-hexaose to hamster kidney galectin-3 is weaker than that of straight chain lacto-N-tetraose having the same binding epitopes. However, the data in Table II show that lacto-N-hexaose (Ka = 6.5 x 104 M1) possesses 2.5-fold higher affinity than lacto-N-tetraose (Ka = 2.6 x 104 M1) for galectin-3 (Ahmad et al., 2002
), which confirms ITC studies with human galectin-3 (Bachhawat-Sikder et al., 2001
). The latter group, however, reported an n value for lacto-N-hexaose of one as compared to n values between 0.60.7 in the present study with galectins-1 and -3, consistent with the structural bivalency of the oligosaccharide.
The sugar binding sites of galectin-1 and galectin-3 show some differences that may help explain the higher affinity of lacto-N-hexaose for the latter. Galectin-3 has been shown to bind to internal LacNAc residues in polylactosamine oligomers, whereas galectin-1 is constrained primarily to nonreducing terminal LacNAc residues (Ahmad et al., 2002). The fact that lacto-N-hexaose possesses three potential binding epitopes for galectin-3, one on each branch chain arm, and the core lactose epitope may explain some of the enhanced affinity of galectin-3 for the lacto-N-hexaose relative to galectin-1.
Thermodynamics of binding of oligosaccharides to the CRD of galectin-3
Galectin-3 is the only chimera type galectin and possesses a nonlectin N-terminal region linked to a C-terminal CRD (Barondes et al., 1994; Gabius, 1997
).The C-terminal CRD domain of galectin-3 is homologous to that of galectin-1 and other members of the galectin family (Cooper, 2002
; Jia and Wang, 1988
). Indeed, the CRD alone of galectin-3 binds lactose but lacks the hemagglutination activity and cooperative binding associated with intact galectin-3 (Hsu et al., 1992
). This suggests that the N-terminal domain is important for aggregation of the protein. Indeed purified N-terminal fragments have been found to self-associate (Mehul et al., 1994
).
Recently, the quaternary structure of recombinant murine galectin-3 in solution was determined by sedimentation velocity and equilibrium measurements, and the results show that the lectin is predominantly a monomer (Morris et al., 2004). However, recent studies also show that recombinant murine galectin-3 precipitates as a pentamer with the 2,4-, 2,6-, 3,6-, and 4,6-pentasaccharides in Figure 1 (Ahmad et al., 2004
). This indicates that the galectin-3 monomer in solution is in equilibrium with a small percent of pentamer that undergoes cross-linking and precipitation with certain multivalent carbohydrates. Hence it is important to determine the effects of the N-terminal region on the C-terminal CRD domain of galectin-3 because large changes occur in the quaternary structure of galectin-3 on binding and precipitating with multivalent carbohydrates.
Table III shows ITC data at 287 K for the binding of the CRD domain of recombinant murine galectin-3 to the listed carbohydrates. The thermodynamic data is not significantly different from that of the intact molecule (Table II) and recently reported ITC data for the intact molecule (Ahmad et al., 2002). Little difference in Ka values is observed for the linear spacer DiLacNAc analogs as well as the 2,4-, 2,6-, 3,6-, and 4,6-pentasaccharides relative to LacNAc II. The other thermodynamic binding parameters are also similar to those of the intact molecule. A slight enhancement in affinity is observed for DiLacNAc, the linear dimeric analog of LacNAc II, relative to LacNAc II, which was observed for the intact galectin-3 molecule (Ahmad et al., 2002
). There is also a nearly threefold enhanced affinity toward lacto-N-hexaose, which is present for the intact molecule.
Interestingly, Figure 2 shows that the H versus T
S plot for the CRD of galectin-3 binding to the carbohydrates in Table III is similar to similar plots for galectin-1 and galectin-3 binding to the carbohydrates in Table II. This also suggest little change in the carbohydrate binding activity of the CRD domain of galectin-3 relative to intact galectin-3 as well as galectin-1.
The present results indicate that the N-terminal domain has little effect on the carbohydrate binding properties of the CRD domain of intact, monomeric galectin-3 in solution.
Summary
The observations that galectins-1 and -3 do not possess substantial enhanced affinities for straight chain and branch chain bivalent LacNAc analogs has important implications for the design of carbohydrate inhibitors of these proteins. So-called cluster glycosides have been synthesized and shown to possess enhanced affinities for certain lectins. For example, the mannose-specific D. grandiflora shows 20-fold enhanced affinity for a synthetic divalent mannose carbohydrate and nearly 60-fold enhanced affinity for a tetravalent trimannoside analog (Dam et al., 2000). The absence of substantial enhanced affinities of galectins-1 and -3 for the divalent LacNAc analogs suggest that carbohydrate analogs possessing higher valencies will not exhibit much greater affinities than LacNAc. Multivalent analogs of lacto-N-hexaose may also not possess much greater affinities than the parent molecule. Higher-affinity interactions of these two galectins and possible other members of the galectin family may depend on cross-linking interactions with separate carbohydrate epitopes on the surface of a matrix (extracellular matrix) or cell surface. Synthetic analogs that incorporate these design principles may give rise to higher affinities for galectins, including those in the present study. Alternatively, sugar analogs, which possess synthetic side chains, have been recently shown to possess enhanced affinity of the parent sugars for specific galectins, as in the case of galectin-3 (Sorme et al., 2003
).
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Materials and methods |
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Purification and expression of the proteins
Fetuin was desialylated and purified as described earlier (Gupta and Brewer, 1994). Asialofetuin-Sepharose was prepared by a published procedure (Parikh et al., 1974
). Galectin-1 from bovine heart was purified using the procedure for the isolation of calf spleen galectin-1 in our laboratory (Gupta and Brewer, 1994
). Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) of the affinity-purified lectin was found to contain one additional band. Affinity-purified lectin was fractionated into two clear peaks, I and II, by gel chromatography using Sephadex G-50 column in the presence of 0.05 mM lactose. The second peak corresponded to galectin-1 and was found to be pure by SDSPAGE. Fractions containing the second peak were pooled and dialyzed extensively against phosphate buffered saline containing 10 mM ß-mercaptoethanol, and stored at 4°C.
Recombinant murine galectin-3 was purified after recombinant expression, using the plasmid prCBP35 kindly provided by J. L. Wang (East Lansing, MI) and Escherichia coli JA221 cells (Agrwal et al., 1993). Galectin-3 thus produced was found to be pure by SDSPAGE. Galectin-3 batches were found to have different n values by ITC using LacNAc II as a ligand. Therefore each batch of galectin-3 was subjected to several affinity chromatography cycles on ASF-Sepharose until an n value of 1 was achieved by ITC. Galectin-3 samples were tested with LacNAc II before and after an ITC experiment with a new saccharide. Galectin-3's CRD was produced by collagenase treatment of the intact molecule and purified on a asialofetuin-Sepharose column. The purified galectin-3 CRD moved as a single band on SDSPAGE.
Monomeric lectin concentrations were determined spectrophotometrically at 280 nm using the specific extinction coefficient of the three proteins (). A value of 5.4 (
) was used for galectin-1, 6.1 (
) for galectin-3, and 5.5 (
) for galectin-3 CRD as determined from ITC experiments. Molecular masses of the galectins were determined by matrix-assisted laser desorption ionization and electrospray ionization mass spectrometry and were found to be 14,500 Da for galectin-1, 27,500 Da for galectin-3, and 14,500 Da for galectin-3 CRD. The carbohydrate concentration was measured by the phenolsulfuric acid method (Dubois et al., 1956
; Saha and Brewer, 1994
).
Hemagglutination inhibition assays
Hemagglutination assays were performed by serial dilution technique (Osawa and Matsumoto, 1972). Rabbit erythrocytes 3% (v/v) were prepared in 20 mM sodium phosphate buffer containing 0.15 M NaCl, pH 7.2, whereas the sugar and protein solutions were prepared in 20 mM sodium phosphate buffer containing 0.15 M NaCl and 10 mM ß-mercaptoethanol, pH 7.2. The final concentration of ß-mercaptoethanol in the assay was 5 mM.
Isothermal titration microcalorimetry
ITC experiments were performed using an MCS Microcalorimeter from Microcal (Northampton, MA). A typical titration profile is shown in Figure 3. Protein and carbohydrate samples were prepared in 20 mM sodium phosphate buffer containing 0.15 M NaCl and 10 mM ß-mercaptoethanol, pH 7.2. Protein solution (2.5 ml) was taken in a syringe and loaded into the ITC sample cell (cell volume = 1.34 ml). Reducing agents in the buffer were found to increase time for the baseline stabilization which varied from 3 to 6 h. The effect of dithiothreitol on baseline stabilization was greater than ß-mercaptoethanol. Degassing of protein solutions and using fresh buffers (maximum 12 h storage time) was found to decrease the time for baseline stabilization from 4 fto 1 h. After the baseline had stabilized, 4-µl injections of the carbohydrate solution were added from the computer-controlled 100-µl rotating syringe (400 rpm) into the protein solution, and exothermic heat changes accompanying the additions were recorded. The time period between the two consecutive injections was fixed at 4 min to allow the exothermic peak to return to the baseline. Control experiments were carried out by making identical injections into the cell containing buffer with no protein.
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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