Diocleinae Lectins Are a Group of Proteins with Conserved Binding Sites for the Core Trimannoside of Asparagine-linked Oligosaccharides and Differential Specificities for Complex Carbohydrates*

Tarun K. DamDagger , Benildo S. Cavada§, Thalles B. Grangeiro, Claudia F. Santos§, Flavia A. M. de Sousa§, Stefan Oscarsonparallel , and C. Fred BrewerDagger **

From the Dagger  Department of Molecular Pharmacology and Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461, § Laboratorio de Lectinas, Departamento de Bioquimica e Biologia Molecular, UFC, 60451-970 Fortaleza, Ceara, Brazil,  Departamento de Biologia, UFC, 60451-970 Fortaleza, Ceara, Brazil, and the parallel  Department of Organic Chemistry, Stockholm University, Stockholm, Sweden

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References

The seed lectin from Dioclea grandiflora and jack bean lectin concanavalin A (ConA) are both members of the Diocleinae subtribe of Leguminosae lectins. Both lectins have recently been shown to possess enhanced affinities and extended binding sites for the trisaccharide, 3,6-di-O-(alpha -D-mannopyranosyl)-D-mannose, which is present in the core region of all asparagine-linked carbohydrates (Gupta, D., Oscarson, S., Raju, S., Stanley, P. Toone, E. J. and Brewer, C. F. (1996) Eur. J. Biochem. 242, 320-326). In the present study, the binding specificities of seven other lectins from the Diocleinae subtribe have been investigated by hemagglutination inhibition and isothermal titration microcalorimetry (ITC). The lectins are from Canavalia brasiliensis, Canavalia bonariensis, Cratylia floribunda, Dioclea rostrata, Dioclea virgata, Dioclea violacea, and Dioclea guianensis. Hemagglutination inhibition and ITC experiments show that all seven lectins are Man/Glc-specific and have high affinities for the core trimannoside, like ConA and D. grandiflora lectin. All seven lectins also exhibit the same pattern of binding to a series of monodeoxy analogs and a tetradeoxy analog of the trimannoside, similar to that of ConA and D. grandiflora lectin. However, C. bonariensis, C. floribunda, D. rostrata, and D. violacea, like D. grandiflora, show substantially reduced affinities for a biantennary complex carbohydrate with terminal GlcNAc residues, while C. brasiliensis, D. guianensis, and D. virgata, like ConA, exhibit affinities for the oligosaccharide comparable with that of the trimannoside. Thermodynamic data obtained by ITC indicate different energetic mechanisms of binding of the above two groups of lectins to the complex carbohydrate. The ability of the lectins to induce histamine release from rat peritoneal mast cells is shown to correlate with the relative affinities of the proteins for the biantennary carbohydrate.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References

Phytohemagglutinins from the Leguminosae family comprise one of the largest group of homologous proteins with carbohydrate binding properties (see Ref. 1). Despite similarities in their physicochemical properties and their relatively conserved primary sequences, Leguminosae lectins display considerable diversity in their carbohydrate binding properties (2). This diversity is present not only in terms of recognizing different monosaccharides but also in lectins with the same nominal monosaccharide specificity. For example, Man-specific Leguminosae lectins have been isolated from the Diocleinae subtribe, which include the jack bean lectin concanavalin A (ConA)1 and seed lectin from Dioclea grandiflora, and from the Vicieae tribe, which includes the sweet pea, garden pea, lentil, and fava bean lectins. However, ConA and D. grandiflora lectin have recently been shown to possess substantially enhanced affinities for the "core" trimannoside, 3,6-di-O-(alpha -D-mannopyranosyl)-D-mannose, which is present in all asparagine-linked (N-linked) carbohydrates (3, 4). In addition, recent hemagglutination inhibition studies have reported that ConA and D. grandiflora lectin have nearly the same pattern of binding to deoxy analogs 2-11 of the trimannoside (Fig. 1) (4). These studies indicate that two nominal Man/Glc-specific lectins from the Diocleinae subtribe (Scheme I

<AR><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C><OVL><UP>‖              </UP></OVL></C></R><R><C><UP>Genus 1</UP></C></R><R><C><IT>Canavalia</IT></C></R><R><C><UNL><UP>Species</UP></UNL></C></R><R><C><UP>C. ensiformis</UP></C></R><R><C>(<UP>Con A</UP>)</C></R><R><C><IT>C. brasiliensis</IT></C></R><R><C><IT>C. bonariensis</IT></C></R><R><C><IT> </IT></C></R></AR><AR><R><C><IT>Family:</IT></C></R><R><C><IT>Leguminosae</IT></C></R><R><C><IT>‖</IT></C></R><R><C><IT>‖</IT></C></R><R><C><IT>Tribe:</IT></C></R><R><C><IT>Phaseoleae</IT></C></R><R><C><IT>‖</IT></C></R><R><C><IT>‖</IT></C></R><R><C><IT>Subtribe:</IT></C></R><R><C><IT>Diocleinae</IT></C></R><R><C><IT>‖</IT></C></R><R><C><IT>‖</IT></C></R><R><C><OVL><IT>‖              </IT></OVL></C></R><R><C><IT>Genus 2</IT></C></R><R><C><IT>Cratylia</IT></C></R><R><C><UNL><UP>Species</UP></UNL></C></R><R><C><UP>C. floribunda</UP></C></R><R><C></C></R><R><C></C></R><R><C></C></R></AR><AR><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C></C></R><R><C><UP>‖</UP></C></R><R><C><UP>Genus 3</UP></C></R><R><C><IT>Dioclea</IT></C></R><R><C><UNL><UP>Species</UP></UNL></C></R><R><C><UP>D. grandiflora</UP></C></R><R><C><UP>D. rostrata</UP></C></R><R><C><UP>D. guianensis</UP></C></R><R><C><UP>D. violacea</UP></C></R><R><C><UP>D. virgata</UP></C></R></AR>
Scheme 1

), possess extended binding sites and high affinities for the trimannoside, unlike members of the Vicieae tribe (1). In addition, the hemagglutination inhibition study showed that, while ConA possesses high affinity for a biantennary complex carbohydrate (14, Fig. 1), D. grandiflora lectin shows much lower affinity for the oligosaccharide (4). These results indicate a further divergent specificity of these two Diocleinae subtribe lectins for complex type carbohydrates.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 1.   Structures of core trimannoside 1, deoxy analogs 2-12, Man5 oligomannose carbohydrate 13, and biantennary complex carbohydrate 14. Man, GlcNAc, 2-dMan, 3-dMan, 4-dMan, and 6-dMan represent mannose, N-acetylglucosamine, 2-deoxymannose, 3-deoxymannose, 4-deoxymannose, and 6-deoxymannose residues, respectively. 1, methyl 3,6-di-O-(alpha -D-mannopyranosyl)-alpha -D-mannopyranoside; 2, me- thyl 6-O-(alpha -D-mannopyranosyl)-3-O-(2-deoxy-alpha -D-mannopyranosyl)-alpha -D-manno-pyranoside; 3, methyl 6-O-(alpha -D-mannopyranosyl)-3-O-(3-deoxy-alpha -D-mannopyranosyl)-alpha -D-mannopyranoside; 4, methyl 6-O-(alpha -D-mannopyranosyl)-3-O-(4-deoxy-alpha -D-mannopyranosyl)-alpha -D-mannopyranoside; 5, methyl 6-O-(alpha -D-mannopyranosyl)-3-O-(6-deoxy-alpha -D-mannopyranosyl)-alpha -D-mannopyranoside; 6, methyl 6-O-(2-deoxy-alpha -D-mannopyranosyl)-3-O-(alpha -D-mannopyranosyl)-alpha -D-mannopyranoside; 7, methyl 6-O-(3-deoxy-alpha -D-mannopyranosyl)-3-O-(alpha -D-mannopyranosyl)-alpha -D-mannopyranoside; 8, me-thyl 6-O-(4-deoxy-alpha -D-mannopyranosyl)-3-O-(alpha -D-mannopyranosyl)-alpha -D-mannopyranoside; 9, methyl 6-O-(6-deoxy-alpha -D-mannopyranosyl)-3O-(alpha -D-mannopyranosyl)-alpha -D-mannopyranoside; 10, methyl 6-O(alpha -D-mannopyranosyl)-3-O-(alpha -D-mannopyranosyl)-2-deoxy-alpha -D-mannopyranoside; 11, methyl 6-O-(alpha -D-mannopyranosyl)-3-O-(alpha -D-mannopyranosyl)-4-deoxy-alpha -D-mannopyranoside; 12, methyl 6-O-(alpha -D-mannopyranosyl)-3-O-(3,4-dideoxy-alpha -D-mannopyranosyl)-2,4-dideoxy-alpha -Dmannopyranoside.

Isothermal titration microcalorimetry (ITC) has been used to determine the thermodynamics of carbohydrate binding to ConA (3, 5, 6), including binding of the methyl alpha -anomer of the core trimannoside (1) and its deoxy analogs (2-11) (7). The thermodynamic data (7) identified the 3-, 4- and 6-OH of the alpha (1-6)-Man, the 3- and 4-OH of the alpha (1-3)-Man, and the 2- and 4-hydroxyls of the central Man of 1 in binding, confirming the hemagglutination results (4). Importantly, both the hemagglutination inhibition (4) and ITC data (7) agree with the recently determined x-ray crystal structure of ConA complexed with the trimannoside (8), which shows binding of the above hydroxyls of the trisaccharide (Fig. 2).


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 2.   View of the x-ray crystal structure of trimannoside 1 (no anomeric methoxy group) bound to ConA. The trimannoside is shown with the central Man indicated by C, the alpha (1-6)-Man by 6, and the alpha (1-3)-Man by 3. Data are from Naismith and Field (8).

Seven other lectins of the subtribe Diocleinae from different genera and different species (Scheme I) have recently been described. The lectins are from Canavalia brasiliensis, Canavalia bonariensis, Cratylia floribunda, Dioclea rostrata, Dioclea virgata, Dioclea violacea, and Dioclea guianensis. The SDS-polyacrylamide gel electrophoresis patterns of the subunit structures of the lectins resemble ConA and D. grandiflora lectin, with molecular masses ranging from 26 to 30 kDa (9-15). The x-ray crystal structure of C. brasiliensis has recently been reported (16) and is similar to ConA (17, 18). Although the complete primary sequences of all of the Diocleinae lectins are not known, the high degree of sequence homologies of ConA, D. grandiflora, and C. brasiliensis suggests that other members of the Diocleinae subtribe possess relatively conserved sequences.

Despite their phylogenetic proximity and apparently conserved sequences, the above Diocleinae lectins possess different biological activities such as histamine release from rat peritoneal mast cells (19), lymphocyte proliferation and interferon-gamma production (20), peritoneal macrophage stimulation and inflammatory reaction (21), and induction of paw edema and peritoneal cell immigration in rats (22). Thus, it is important to determine the fine carbohydrate binding specificities of this group of lectins.

The present study reports hemagglutination inhibition and ITC studies of the binding of the above seven new Diocleinae lectins to a variety of mono- and oligosaccharides, trimannoside 1, deoxy analogs 2-12, Man5 oligomannose carbohydrate 13, and biantennary complex carbohydrate 14 (Fig. 1). Together with results for ConA and D. grandiflora lectin, the present findings indicate that nine members of the Diocleinae subtribe of Leguminosae lectins possess conserved binding specificities toward 1 but differential specificities for 14. Furthermore, the relative affinities of the lectins for 14 correlate with their abilities to stimulate histamine release from the rat peritoneal mast cells.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References

Mealpha Glc, Mealpha Man, methyl-beta -D-glucopyranoside, 2-deoxy-D-glucose, Gal, Fuc, Manalpha 1-2Man, Manalpha 1-3Man, Manalpha 1-6Man, maltose, trehalose, maltotriose, lactose, melibiose, sialic acid, and 1 were purchased from Sigma. Methyl-2-deoxy-alpha -D-mannopyranoside was a gift from Dr. S. Sabesan (DuPont). Biantennary complex carbohydrate 14, GlcNAcbeta 1-2Man, and Man5 oligosaccharide (13) were obtained from Dextra Laboratories Ltd. The synthesis of oligosaccharides 2-11 (Fig. 1) has been described elsewhere (23). Synthesis of 12 will be presented elsewhere. The concentrations of carbohydrates were measured by the phenol-sulfuric acid method using an appropriate mixture of Man, Glc, and Gal as the standard (24, 25). The purity of the oligosaccharides was checked by 500-MHz 1H NMR spectroscopy. Seeds of all of the species were obtained from the States of Ceara and Rio Grande do Sul, Brazil.

Purification of the Lectins-- Lectins were purified by affinity chromatography using Sephadex G-50, as described previously (see Ref. 26). Concentrations of the lectins were determined spectrophotometrically at 280 nm and expressed in terms of monomer. The Acm1% at pH 7.2 and the subunit molecular mass of the lectins used are as follows: 10.47 and 26 kDa (C. brasiliensis) (9), 11.15 and 30 kDa (C. bonariensis) (13), 11.36 and 29.5 kDa (C. floribunda) (10), 11.22 and 30 kDa (D. rostrata) (12), 10.50 and 30 kDa (D. guianensis) (11), 10.19 and 30 kDa (D. virgata) (14), 9.75 and 29.5 kDa (D. violacea) (15). Acm1% of C. floribunda, D. rostrata, and D. violacea were reported above, whereas that of the remaining lectins were determined in the present study.

Hemagglutination Inhibition Assay-- The assay was performed at room temperature using a 2-fold serial dilution technique (27) and 3% (v/v) rabbit erythrocytes in HEPES buffer (0.1 M HEPES, 0.15 M NaCl, 1 mM CaCl2, and 1 mM MnCl2, pH 7.2). The minimum concentration of saccharide required for complete inhibition of four hemagglutination doses was determined by visual inspection.

Isothermal Titration Microcalorimetry-- ITC experiments were performed using an OMEGA Microcalorimeter from Microcal, Inc. (Northampton, MA). In individual titrations, injections of 4 µl of carbohydrate were added from the computer-controlled 250-µl microsyringe at an interval of 4 min into the lectin solution (cell volume = 1.3424 ml) dissolved in the same buffer as the saccharide, while stirring at 350 rpm. An example of an ITC experiment is shown in Fig. 3 for binding of 1 to D. violacea at 27 °C. Control experiments performed by making identical injections of saccharide into a cell containing buffer with no protein showed insignificant heats of dilution. The experimental data were fitted to a theoretical titration curve using software supplied by Microcal, with Delta H (enthalpy change in kcal mol-1), 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 (28). 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 supplied by the manufacturer. Thermodynamic parameters were calculated from the equation,
&Dgr;G=&Dgr;H−T&Dgr;S=<UP>−</UP>RT <UP>ln</UP>K<SUB>a</SUB> (Eq. 1)
where Delta G, Delta H, and Delta S are the changes in free energy, enthalpy, and entropy of binding. T is the absolute temperature, and r = 1.98 cal mol-1 K-1.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 3.   ITC profile of D. violacea (0.18 mM) with trisaccharide 1 (9.7 mM) at 27 °C. Top, data obtained for 30 automatic injections, 4 µl each, of 1; bottom, the integrated curve showing experimental points (black-square) and the best fit (------). The buffer was 0.1 M HEPES with 1.0 M NaCl and 5 mM each CaCl2 and MnCl2 (pH 7.2). For details, see "Materials and Methods."

    RESULTS AND DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References

Monosaccharide Binding Specificities-- The monosaccharide binding properties of ConA (29) and D. grandiflora lectin (4, 30) are well defined, with both lectins showing preferential binding to the alpha -pyranosides of Man and Glc. The seven other Diocleinae lectins in Table I generally show similar preferential binding to the alpha -pyranosides of Man and Glc, and not to Gal, Fuc, lactose, melibiose, or sialic acid. The C-2 hydroxyl group of Man is not essential for binding to the Diocleinae lectins, since methyl 2-deoxy-alpha -D-mannopyranoside is as potent as Mealpha Man. Methyl 2-deoxy-alpha -D-mannopyranoside was previously reported not to inhibit D. grandiflora lectin (4); however, a reinvestigation shows that it does inhibit the lectin (Table I). 2-D-Glc inhibits C. floribunda, D. rostrata, and D. guianensis more poorly than Glc. GlcNAc at a relatively high concentration (150 mM) shows some inhibition of C. brasiliensis, C. bonariensis, and D. virgata but did not inhibit the other four lectins. 3-deoxymannose, 4-deoxymannose, and 6-deoxymannose show no inhibitory activity, suggesting that Diocleinae lectins recognize the 3-, 4-, and 6-hydroxyl groups of Man, as observed for ConA (29) and D. grandiflora lectin (4).

                              
View this table:
[in this window]
[in a new window]
 
Table I
Inhibitory potencies of monosaccharides and disaccharides for Diocleinae lectin-mediated hemagglutination of rabbit erythrocytes
Values in parenthesis are normalized with respect to Mealpha Man for each lectin.

The thermodynamic binding parameters of the seven new Diocleinae lectins to Mealpha Man determined by ITC measurements are listed in Table III. The thermodynamic parameters for ConA (5) and D. grandiflora lectin (3) binding to Mealpha Man have previously been reported and are listed in Table III for comparison. The values for D. grandiflora lectin binding to Mealpha Man have been reexamined and confirmed in the present study. C. brasiliensis displays the highest Ka value (1.3 × 104 M-1), with D. rostrata possessing the lowest Ka value (1.7 × 103 M-1). C. brasiliensis, D. guianensis, D. violacea, and D. virgata have Delta H values between -5.8 and -4.9 kcal mol-1, while C. bonariensis, C. floribunda, D. rostrata, ConA, and D. grandiflora possess -Delta H values between -6.9 and -8.9 kcal mol-1. However, the relative Ka values of the lectins for binding Mealpha Man do not correlate with their respective -Delta H values, indicating compensating entropic factors.

Disaccharide Binding Specificities-- Inhibitory potencies of most of the disaccharides for the seven Diocleinae lectins were comparable with that of ConA and D. grandiflora lectin (Table I). None of the lectins were inhibited by lactose and melibiose, as expected. It has previously been shown that the affinity of ConA for Manalpha (1-2)Man, a disaccharide moiety found in N-linked oligomannose carbohydrates, is 5-fold greater than Mealpha Man, as compared with weaker binding of D. grandiflora lectin to the disaccharide relative to the monosaccharide (4). The seven new Diocleinae lectins display a range of relative affinities for Manalpha (1-2)Man. D. virgata shows 16-fold greater affinity for the disaccharide relative to Mealpha Man, whereas D. guianensis and D. violacea show enhanced affinities for the disaccharide comparable with that of ConA. The remaining four lectins show lower relative affinities for the disaccharide.

Large differences in the binding specificity of ConA and D. grandiflora lectin toward GlcNAc(beta 1-2)Man, a disaccharide moiety found in a variety of N-linked carbohydrates, have been reported (4). While ConA binds to the disaccharide, no binding was detected for D. grandiflora lectin. This is consistent with the difference in relative affinities of ConA and D. grandiflora lectin for biantennary complex carbohydrate 14 (Fig. 1) (Table II), with ConA showing high affinity for the pentasaccharide but D. grandiflora lectin showing low affinity (4). The other Diocleinae lectins exhibit distinct patterns of binding to GlcNAc(beta 1-2)Man. While C. brasiliensis, D. guianensis, and D. virgata bind the disaccharide, the remaining lectins show little or no affinity for it, similar to D. grandiflora lectin. This observation is significant in light of the results for binding of the lectins to biantennary carbohydrate 14, discussed below.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Inhibitory potencies of trimannoside 1 and its deoxy analogues for Diocleinae lectin-mediated hemagglutination of rabbit erythrocytes
Inhibitory potencies in parentheses are relative to trimannoside 1 for each lectin.

Binding to Trimannoside 1 and Its Deoxy Analogs-- ConA is known to possess high affinity for the trisaccharide, 3,6-di-O-(alpha -D-mannopyranosyl)-D-mannose, which is present in the core region of all asparagine-linked carbohydrates (31). ITC data established that ConA binds to the trimannoside and its methyl alpha -anomer (1) with a nearly -6 kcal mol-1 greater -Delta H and a 60-fold greater Ka than Mealpha Man (5). These results suggested extended site binding of ConA to the trimannoside, which was confirmed by the x-ray crystal structure of the trimannoside-ConA complex (Fig. 3) (8). ITC studies of ConA binding to deoxy analogs 2-11 established the binding energetics of the various hydroxyl groups of trimannoside 1 to ConA (7). The results also demonstrated that the solution complex of the trimannoside involves binding of the same hydroxyl groups of 1 observed in the x-ray crystal complex. Thus, ConA binds to 1 via the 3-, 4-, and 6-hydroxyls on the alpha (1-6)-Man residue, the 2- and 4-hydroxyls of the central Man residue, and the 3- and 4-hydroxyls of the alpha (1-3)-Man residue (Fig. 2).

Chervenak and Toone (3) reported similar enhanced -Delta H and Ka values for D. grandiflora lectin binding to 1 relative to Mealpha Man, which were confirmed in the present study (Table III). In addition, hemagglutination inhibition experiments with deoxy analogs 2-11 established that the pattern of binding of the hydroxyl groups of 1 to D. grandiflora lectin is similar to that for ConA (Table II for comparison) (4). These findings suggest similar extended sites for both lectins to the trimannoside.

                              
View this table:
[in this window]
[in a new window]
 
Table III
Isothermal titration microcalorimetry data at 27 °C of lectins from the subtribe Diocleinae

Hemagglutination inhibition data in the present study (Table II) show that the seven new Diocleinae lectins exhibit similar enhanced affinities for 1 relative to Mealpha Man, as observed for ConA and D. grandiflora lectin. ITC data shown in Table III indicate that all seven new Diocleinae lectins show enhanced Ka and -Delta H values for 1 relative to Mealpha Man. The enhanced Ka values of the lectins for 1 relative to Mealpha Man are shown in Fig. 4. The -Delta H values for all seven lectins binding to 1 are -5 to -7 kcal mol-1 greater than that for Mealpha Man, similar to the differences observed for ConA and D. grandiflora lectin (Table III). These data strongly suggest similar extended binding sites for all nine Diocleinae lectins.


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 4.   Plot of the ratio of Ka values of the nine Diocleinae lectins for trimannoside 1 and complex carbohydrate 14, relative to Mealpha Man, derived from the ITC data in Table III.

In order to determine which hydroxyl groups of 1 are involved in binding to the Diocleinae lectins, hemagglutination inhibition experiments were performed using monodeoxy analogs 2-11 and tetradeoxy analog 12 (Table II). As an example, hemagglutination inhibition data for the C. floribunda lectin are shown in Fig. 5. The results indicate the involvements of the 3-, 4-, and 6-hydroxyls of the alpha (1-6)-Man, the 3- and 4-hydroxyls of the alpha (1-3)-Man, and the 2- and 4-hydroxyls of the central Man of trimannoside 1 in binding. There is also an indication of possible participation of the 2-hydroxyl of the alpha (1-6)-arm, as observed for a few of the lectins in Table II. As expected, tetradeoxy analog 12 shows very little inhibition potency relative to 1 and is comparable with that of Mealpha Man. The data in Table II show a similar pattern of inhibition by the analogs for the seven new Diocleinae lectins as observed for ConA and D. grandiflora lectin (4). These results indicate highly conserved binding sites for 1 in all nine Diocleinae lectins.


View larger version (43K):
[in this window]
[in a new window]
 
Fig. 5.   Plot of the hemagglutination inhibition potencies of deoxy analogs 2-12 (Fig. 1) in the presence of C. floribunda lectin.

Binding of Man5 Oligomannose Carbohydrate-- Hemagglutination inhibition data in Table II show that the Diocleinae lectins bind Man5 oligosaccharide 13 with almost the same inhibitory potency as 1. This indicates that the trimannoside moiety on the alpha (1-6)-arm is the primary epitope for interaction, as observed for ConA and D. grandiflora lectin (4).

Binding of Biantennary Complex Oligosaccharide 14-- The affinities of D. grandiflora lectin for biantennary complex oligosaccharide 14 and the longer chain analog with terminal Gal residues have been reported to be weak compared with that of ConA (4). These results are related to the lack of D. grandiflora lectin binding to the disaccharide GlcNAcbeta 1-2Man, which is present in 14 (Table I) (4). All of the Diocleinae lectins tested showed distinct correlated binding affinities toward this disaccharide and 14. Hemagglutination inhibition data in Table II indicates that 14 has much higher inhibition potencies with C. brasiliensis, D. guianensis, and D. virgata as compared with the other new lectins. Longer chain analogs of 14 also show a similar pattern (data not shown). This parallels the binding activities of the lectins toward GlcNAcbeta 1-2Man (Table I). In addition, ITC data in Table III show an order of magnitude greater Ka values of C. brasiliensis, D. guianensis, and D. virgata for 14 relative to the other four lectins. Among the nine Diocleinae lectins, ConA shows the highest Ka value for 14, with D. grandiflora lectin showing a relatively low Ka. The relative Ka values for all nine lectins binding to 14 (along with 1) with respect to Mealpha Man are shown in Fig. 4. Table III also shows that C. brasiliensis, D. guianensis, and D. virgata possess greater -Delta H values for 14 of the seven new lectins, and that ConA possesses the greatest -Delta H value of the nine lectins.

Importantly, an enthalpy-entropy compensation plot (-Delta H versus -TDelta S) of the data in Table III for 14 shows different slopes for the above two groups of the Diocleinae lectins (Fig. 6B). The lectins from C. brasiliensis, D. guianensis, D. virgata, and ConA fall on a line with a slope of 1.44 (correlation coefficient 0.85), while the lectins from C. bonariensis, C. floribunda, D. rostrata, D. violacea, and D. grandiflora fall on a line with a slope of 0.85 (correlation coefficient 0.98). Although the D. grandiflora data point appears to intersect both plots, it is associated with the latter group of lectins because of its relatively low affinity and -Delta H values for 14. By comparison, a similar plot of the lectins binding to 1 shows a single line with a slope of 1.21 (correlation coefficient 0.97) (Fig. 6A). These results indicate different energetic mechanisms of binding of the four relatively high affinity lectins to 14, as compared with the five lower affinity lectins. Thus, although all nine Diocleinae lectins show conserved high affinities binding for 1, four of the lectins show relatively high affinities for 14, with the other five lectins showing relatively low affinities. Therefore, binding discrimination among this group of lectins occurs toward biantennary complex carbohydrates. The structural basis for this discrimination is currently under investigation.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 6.   Enthalpy-entropy compensation plots for the binding of the Diocleinae lectins with trimannoside 1 (slope = 1.21) (A) and biantennary complex carbohydrate 14 at 27 °C (300 K) (B). In B, closed circles (bullet ) represent the values for ConA, C. brasiliensis, D. guianensis, and D. virgata (slope = 1.4) while the closed squares (black-square) represent the values for D. grandiflora, C. bonariensis, C. floribunda, D. rostrata, and D. violacea (slope = 0.85).

Histamine Release Activities of the Diocleinae Lectins Are Correlated with Relative Affinities for 14-- ConA has long been known for its ability to induce histamine release from cells (see Refs. 32 and 33). Recently, Gomes and co-workers (19) investigated the histamine release properties from rat peritoneal mast cells of several other lectins from the same subtribe. At the level of 10 µg/ml lectin concentration, ConA, C. brasiliensis, D. guianensis, and D. virgata induced a higher level of histamine release from rat peritoneal mast cells, whereas D. grandiflora, C. bonariensis, C. floribunda, D. rostrata, and D. violacea displayed lower abilities for induction. A significant correlation between the histamine releasing properties of these lectins and their affinity constants for 14 is apparent from the present study. Fig. 7 shows that the Ka values of the Diocleinae lectins for 14 and the amount of histamine released by the lectins at 10 µg/ml are correlated. The strong histamine-inducing lectins ConA, C. brasiliensis, D. guianensis, and D. virgata exhibit relatively high affinities (Ka) for 14. On the other hand, the remaining relatively inactive lectins possess lower affinities for the complex carbohydrate. It appears, therefore, that induction of histamine release from rat peritoneal mast cells by ConA, C. brasiliensis, D. guianensis, and D. virgata involves binding of the lectins to a biantennary complex carbohydrate and/or structurally homologous epitope present on the cell surface.


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 7.   Graph showing Ka values (gray bars) of the nine Diocleinae lectins for complex carbohydrate 14 and their histamine releasing properties (black bars). The data of histamine release are taken from Gomes et al. (19).

Conclusions-- The present study demonstrates that nine lectins from the Diocleinae subtribe are Man/Glc-binding proteins with conserved binding specificities for the core trimannoside of N-linked carbohydrates. Using deoxy analogs of the trimannoside, all nine lectins were shown to possess conserved binding sites that recognize the 3-, 4-, and 6-hydroxyl groups on the alpha (1-6)-Man, the 3- and 4-hydroxyl groups on the alpha (1-3)-Man, and the 2- and 4-hydroxyl groups of the central Man of the trimannoside. While the binding specificities of the lectins are conserved for the trimannoside, their specificities are different for biantennary complex carbohydrate 14 and longer chain analogs. Thermodynamic data from ITC experiments indicate different energetic mechanisms of binding of the Diocleinae lectins to 14. The relative affinities of the lectins for 14 correlate with their induced histamine release activities from rat peritoneal mast cells, suggesting that the lectin receptors on the cells involve a carbohydrate(s) structure similar to 14.

    FOOTNOTES

* This work was supported by NCI, National Institutes of Health, Grant CA-16054 and Core Grant P30 CA-13330 (to C. F. B.) and by grants from Conselho Nacional de Desenvolvimento Cientifico e Tecnológico, PADCT, Financiadora de Estudos e Projetos, and FUNCAP (to B. S. C., T. B. G., C. F. S., and F. A. M. de S.). The NMR facility at AECOM was supported by Instrumentation Grant I-S10-RR02309 from the National Institute of Health and DMB-8413723 from the National Science Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed. Tel.: 718-430-2227; Fax: 718-430-8922.

1 The abbreviations used are: ConA, concanavalin A, lectin from jack bean; ITC, isothermal titration microcalorimetry; Mealpha Man, methyl alpha -D-mannopyranoside; Mealpha Glc, methyl-alpha -D-glucopyranoside.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References

  1. Liener, I. E., Sharon, N., and Goldstein, I. J. (eds) (1986) The Lectins: Properties, Functions and Applications in Biology and Medicine, Academic Press, Inc., Orlando, FL
  2. Sharon, N., and Lis, H. (1990) FASEB J. 4, 3198-3208[Abstract]
  3. Chervenak, M. C., and Toone, E. J. (1995) Biochemistry. 34, 5685-5695[Medline] [Order article via Infotrieve]
  4. Gupta, D., Oscarson, S., Raju, T. S., Stanley, P., Toone, E. J., and Brewer, C. F. (1996) Eur. J. Biochem. 242, 320-326[Abstract]
  5. Mandal, D. K., Kishore, N., and Brewer, C. F. (1994) Biochemistry 33, 1149-1156[Medline] [Order article via Infotrieve]
  6. Mandal, D. K., Bhattacharyya, L., Koenig, S. H., Brown, R. D., III, Oscarson, S., and Brewer, C. F. (1994) Biochemistry 33, 1157-1162[Medline] [Order article via Infotrieve]
  7. Gupta, D., Dam, T. K., Oscarson, S., and Brewer, C. F. (1997) J. Biol. Chem. 272, 6388-6392[Abstract/Free Full Text]
  8. Naismith, J. H., and Field, R. A. (1996) J. Biol. Chem. 271, 972-976[Abstract/Free Full Text]
  9. Moreira, R. A., and Cavada, B. S. (1984) Biol. Plant. (Prague) 26, 113-120
  10. Oliveira, J. T. A., Cavada, B., S., and Moreira, R. D. A. (1991) Rev. Bras. Bot. 14, 61-66
  11. Vasconcelos, I. M., Cavada, B. S., Moreira, R. D. A., and Oliveira, J. T. A. (1991) J. Food Biochem. 15, 137-154
  12. Cavada, B. S., Grangeiro, T. B., Ramos, M. V., Cordeiro, E. F., Oliveria, J. T. A., and Moreira, R. A. (1996) Rev. Bras. Fisiol. Vegetal 8, 31-36
  13. Cavada, B. S., Moreira-Silva, L. I. M., Grangeiro, T. B., Santos, C. F., Pinto, V. P. T., Barral-Netto, M., Roque-Barreira, M. C., Gomes, J. C., Martins, J. L., Oliveira, J. T. A., and Moreira, R. A. (1996) in Lectins: Biology, Biochemistry, Clinical Biochemistry (Van Driessche, E., Rouge, P., Beeckmans, S., and Bog-Hansen, T. C., eds), Vol. 11, pp. 74-80, TEXTOP, Hellerup, Denmark
  14. Cavada, B. S., Ramos, M. V., Cordeiro, E. F., Grangeiro, T. B., Oliveira, J. T. A., Carvalho, A. F. F. U., and Moreira, R. A. (1996) Rev. Bras. Fisiol. Vegetal 8, 37-42
  15. Moreira, R. de A., Cordeiro, E. de F., Ramos, M. V., Grangeiro, T. B., Martins, J. L., Oliveria, J. T. A. de, and Cavada, B. S. (1996) Rev. Bras. Fisiol. Veg. 8, 23-29
  16. Sanz-Aparicio, J., Hermoso, J., Grangeiro, T. B., Calvete, J. J., and Cavada, B. S. (1997) FEBS Lett. 405, 114-118[CrossRef][Medline] [Order article via Infotrieve]
  17. Hardman, K. D., and Ainsworth, C. F. (1972) Biochemistry 11, 4910-4919[Medline] [Order article via Infotrieve]
  18. Derewenda, Z., Yariv, J., Helliwell, J. R., Kalb, A. J., Dodson, E. J., Papiz, M. Z., Wan, T., and Campbell, J. (1989) EMBO J. 8, 2189-2193[Abstract]
  19. Gomes, J. C., Rossi, R. R., Cavada, B. S., Moreira, R. A., and Oliveira, J. T. A. (1994) Agents Actions 41, 132-135[Medline] [Order article via Infotrieve]
  20. Barral-Netto, M., Santos, S. B., Barral, A., Moreira, L. I. M., Santos, C. F., Moreira, R. A., Oliveira, J. T. A., and Cavada, B. S. (1992) Immunol. Invest. 21, 297-303[Medline] [Order article via Infotrieve]
  21. Rodrigues, D., Cavada, B. S., Oliveira, J. T. A., Moreira, R. D. A., and Russo, M. (1992) Braz. J. Med. Biol. Res. 25, 823-826[Medline] [Order article via Infotrieve]
  22. Bento, C. A. M., Cavada, B. S., Oliveira, J. T. A., Moreira, R. A., and Barja Fidalgo, C. (1993) Agents Actions 38, 48-54[Medline] [Order article via Infotrieve]
  23. Oscarson, S., and Tedebark, U. (1995) Carbohydr. Res. 278, 271-287[CrossRef][Medline] [Order article via Infotrieve]
  24. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F. (1956) Anal. Chem. 28, 350-356
  25. Saha, S. K., and Brewer, C. F. (1994) Carbohydr. Res. 254, 157-167[CrossRef][Medline] [Order article via Infotrieve]
  26. Agrawal, B. B. L., and Goldstein, I. J. (1967) Biochim. Biophys. Acta 147, 262-271[Medline] [Order article via Infotrieve]
  27. Osawa, T., and Matsumoto, I. (1972) Methods Enzymol. 28B, 323-327
  28. Wiseman, T., Williston, S., Brandt, J. F., and Lin, L.-N. (1989) Anal. Biochem. 179, 131-135[Medline] [Order article via Infotrieve]
  29. Goldstein, I. J., and Poretz, R. D. (1986) in The Lectins (Liener, I. E., Sharon, N., and Goldstein, I. J., eds), pp. 35-244, Academic Press, Inc., New York
  30. Moreira, R. A., Barros, A. C. H., Stewart, J. C., and Pusztai, A. (1983) Planta 158, 63-69
  31. Brewer, C. F., and Bhattacharyya, L. (1986) J. Biol. Chem. 261, 7306-7310[Abstract/Free Full Text]
  32. Sugiyama, K., Sasaki, J., and Yamasaki, H. (1975) Jpn. J. Pharmacol 25, 485-487[Medline] [Order article via Infotrieve]
  33. Sullivan, T. J., Greene, W. C., and Parker, C. W. (1975) J. Immunol. 115, 278-282[Abstract]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.