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*

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-(α-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. grandifloralectin. 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. grandifloralectin. 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, whileC. 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.

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-(␣-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), 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.
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-␥ 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.
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  (15). A 1 cm 1% 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) 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 ⌬H (enthalpy change in kcal mol Ϫ1 ), K a (association constant in M Ϫ1 ), and n (number of binding sites/monomer), as adjustable parameters. The quantity c ϭ K a M t (0), where M t (0) is the initial macromolecule concentration, is of importance in titration microcalorimetry (28). All experiments were performed with c values 1 Ͻ c Ͻ 200. The instrument was calibrated using the calibration kit containing ribonuclease A (RNase A) and cytidine 2Ј-monophosphate supplied by the manufacturer. Thermodynamic parameters were calculated from the equation, where ⌬G, ⌬H, and ⌬S are the changes in free energy, enthalpy, and entropy of binding. T is the absolute temperature, and r ϭ 1.98 cal mol Ϫ1 K Ϫ1 .

RESULTS AND DISCUSSION
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 ␣-pyranosides of Man and Glc. The seven other Diocleinae lectins in Table I generally show similar preferential binding to the ␣-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-␣-D-mannopyranoside is as potent as Me␣Man. Methyl 2-deoxy-␣-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).
The thermodynamic binding parameters of the seven new Diocleinae lectins to Me␣Man determined by ITC measure-ments are listed in Table III. The thermodynamic parameters for ConA (5) and D. grandiflora lectin (3) binding to Me␣Man have previously been reported and are listed in Table III  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 Man␣(1-2)Man, a disaccharide moiety found in Nlinked oligomannose carbohydrates, is 5-fold greater than Me␣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 Man␣ (1)(2)Man. D. virgata shows 16-fold greater affinity for the disaccharide relative to Me␣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(␤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(␤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.
Binding to Trimannoside 1 and Its Deoxy Analogs-ConA is known to possess high affinity for the trisaccharide, 3,6-di-O-(␣-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 ␣-anomer (1) with a nearly Ϫ6 kcal mol Ϫ1 greater Ϫ⌬H and a 60-fold greater K a than Me␣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 ␣(1-6)-Man residue, the 2-and 4-hydroxyls of the central Man residue, and the 3-and 4-hydroxyls of the ␣(1-3)-Man residue (Fig. 2).
Chervenak and Toone (3) reported similar enhanced Ϫ⌬H and K a values for D. grandiflora lectin binding to 1 relative to Me␣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.
Hemagglutination inhibition data in the present study (Table II) show that the seven new Diocleinae lectins exhibit similar enhanced affinities for 1 relative to Me␣Man, as observed for ConA and D. grandiflora lectin. ITC data shown in Table III indicate that all seven new Diocleinae lectins show enhanced K a and Ϫ⌬H values for 1 relative to Me␣Man. The enhanced K a values of the lectins for 1 relative to Me␣Man are shown in Fig. 4. The Ϫ⌬H values for all seven lectins binding to 1 are Ϫ5 to Ϫ7 kcal mol Ϫ1 greater than that for Me␣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.
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) Table II. As expected, tetradeoxy analog 12 shows very little inhibition potency relative to 1 and is comparable with that of Me␣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.
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 ␣(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 GlcNAc␤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 GlcNAc␤1-2Man (Table I). In addition, ITC data in Table III    1) with respect to Me␣Man are shown in Fig. 4. Table III also shows that C. brasiliensis, D. guianensis, and D. virgata possess greater Ϫ⌬H values for 14 of the seven new lectins, and that ConA possesses the greatest Ϫ⌬H value of the nine lectins. Importantly, an enthalpy-entropy compensation plot (Ϫ⌬H versus ϪT⌬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 Ϫ⌬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   Table III.