Structural studies of an anti-inflammatory lectin from Canavalia boliviana seeds in complex with dimannosides

Abstract

Plant lectins, especially those purified from species of the Leguminosae family, represent the best-studied group of carbohydrate-binding proteins. Lectins purified from seeds of the Diocleinae subtribe exhibit a high degree of sequence identity notwithstanding that they show very distinct biological activities. Two main factors have been related to this feature: variance in key residues influencing the carbohydrate-binding site geometry and differences in the pH-dependent oligomeric state profile. In this work, we have isolated a lectin from Canavalia boliviana (Cbol) and solved its x-ray crystal structure in the unbound form and in complex with the carbohydrates Man(α1-3)Man(α1-O)Me, Man(α1-4)Man(α1-O)Me and 5-bromo-4-chloro-3-indolyl-α-D-mannose. We evaluated its oligomerization profile at different pH values using Small Angle X-ray Scattering and compared it to that of Concanavalin A. Based on predicted pKa-shifts of amino acids in the subunit interfaces we devised a model for the dimer-tetramer equilibrium phenomena of these proteins. Additionally, we demonstrated Cbol anti-inflammatory properties and further characterized them using in vivo and in vitro models.

Figure 1. Proposed amino acid sequence of Cbol.

Assemble from sequences of degradation products generated by cleavages with trypsin (T-), chymotrypsin (Q-) and pepsin (P-). The letter X in Cbol sequence represents residues of leucine or isoleucine, which cannot be distinguished by mass.

Figure 2. Overall structure of Cbol.

Subunits displayed in cartoon representation and colored in grey. (A) One molecule in the asymmetric unit of Cbol in complex with Xman (shown as blue sticks). (B) Unbound Cbol with two molecules in the asymmetric unit. (C) Cbol in complex with M13M (shown as green sticks). (D) Cbol in complex with M14M (shown as yellow sticks).

Figure 3. 2F_obs - F_calc electron density maps from the carbohydrates contoured at 1.0 σ.

(A) Xman. (B) Man(α1-3)Man(α1-O)Me, (C) Man(α1-4)Man(α1-O)Me.

Figure 4. Hydrophobic subsite of Cbol.

M14M (shown as yellow sticks) performs one more hydrogen bond with Tyr12 (shown as blue sticks) when compared to M13M (shown as green sticks).

Figure 5. The pH-dependent oligomerization of ConA and Cbol determined with SAXS.

Figure 6. Proposed model for the oligomerization process in Canavalia lectins.

Figure 7. Inhibitory effect of C. boliviana lectin (Cbol) upon the neutrophil migration induced by carrageenan.

Neutrophil migration was induced by i.p. injection of carrageenan (Cg; 500 µg) and evaluated 4 h later. Animals were treated i.v. (0.1 ml), 30 min before stimuli with: Saline and CboL (0.5, 1 and 5 mg/Kg). Values represent mean ± S.E.M. (n = 6); *indicate significant differences from Cg alone. (p<0.05) ANOVA-Bonferroni.

Figure 8. Inhibitory effect of carbohydrate and heat-treated on anti-inflamatory effect induced by Cbol.

(A) CboL (1 mg/Kg i.v), native or heat-treated (100°C) for 30 min; (B) CboL (1 mg/Kg i.v) alone or combined with α-methyl-D-Glucose (0.5 M). The black bar represents neutrophil migration induced by the injection of the carbohydrate alone. Values are reported as means ± S.E.M. for six animals. *p<0.05 compared with carrageenan (Cg) alone (analysis of variance-Bonferroni).

Figure 9. Inhibitory effect of C. boliviana lectin (Cbol) upon the rat paw oedema induced by carrageenan: reversion by glucose.

Paw oedema was induced by s.c. injection of carrageenan (Cg; 500 µg). Animals were treated i.v. (0.1 ml), 30 min before stimuli, with: Saline or CboL (1 mg/Kg; i.v) alone or associated to glucose (0.5 M). Control groups received saline i.v. and Cg s.c. or only saline s.c. (A) Oedema was measured at 1, 2, 3 and 4 h after stimuli and expressed as the increase in paw volume (ml). (B) The area under the time-course curve was determined using a trapezoidal rule. Values represent mean ± S.E.M. (n = 6). *indicate significant differences from Cg alone (p<0.05) ANOVA-Bonferroni.

Figure 10. Chemotaxis assay of human neutrophils incubated in the presence of Cbol.

Cells were allowed to migrate in the Boyden chamber toward the medium alone or IL-8. Cells were pre incubated with different concentrations of CboL. Data show means ± SD from three independent experiments performed in triplicate.