Structural characterization of a lectin from Canavalia virosa seeds with inflammatory and cytotoxic activities

Abstract

A lectin from Canavalia virosa, Diocleinae subtribe, was purified by affinity chromatography with Sephadex G-50 matrix and named ConV. The primary structure of ConV was obtained by mass spectrometry and crystals were obtained by the vapor diffusion method at 293K and belonged to orthorhombic space group P222 with two molecules in its asymmetric unit. The structure obtained presented R and R of 18.91% and 24.92% respectively, with no residues in nonallowed regions of Ramachandran plot. The crystal structure was solved at 2.53Å and was demonstrated to be very similar to other lectins from the same subtribe. In inflammatory tests, ConV elicited paw edema, but incubation of lectin with glucose beforehand was able to reduce the edematogenic effect, indicating the involvement of the carbohydrate recognition domain in this process. The lectin also showed toxicity to rat C6 glioma cells, disrupting the mitochondrial membrane potential (ΔYm) and decreasing cell viability, indicating an anticancer potential for ConV. In silico studies confirmed that ConV interacts strongly with carbohydrates that comprise the N-glycans of glycoproteins. This finding corroborates the hypothesis which holds that the lectin domain interacts with glycans in molecular targets and that this contributes to the effects observed in biological activities.

ConA-Like Lectins: High Similarity Proteins as Models to Study Structure/Biological Activities Relationships

“…Edematogenic activity has been studied in ConA [31], ConGF [35], ConBr [29], Canavalia gladiata lectin (CGL) [29], C. maritima lectin (ConM) [29], C. virosa lectin (ConV) [36], C. oxyphylla lectin (CoxyL) [19], C. villosa lectin (CvilL) [20], Dioclea rostrata lectin (DRL) [37], D. wilsonii lectin (DWL) [38,39], D. virgata lectin (DvirL) [40], D. lasiophylla lectin (DlyL) [41], D. reflexa lectin (DrfL) [41] and Cymbosema roseum lectin (CRL1) [42] (Table 1). Anti-inflammatory effect has been studied in ConGF [43], DvirL [33], CRL1 [42], D. guianensis lectin (DguiL), D. violacea lectin (DVL) and Cratylia floribunda lectin (CFL) [28].…”

“…Some of these lectins affected mitochondrial membrane potential or cell migration, and all of them induced morphological changes on cells from a polygonal form to a spherical one, followed by reduction of cell adhesion. Staining methods indicated cell death by autophagic, necrotic and apoptotic processes induced by mitochondrial pathways and modulation of metalloproteinases [36,59,60,61]. Table 3 summarizes the antiglioma activities of the lectins cited above.…”

“…ConA primary structure was determined in 1975 by Cunningham and colleagues [94] by Edman degradation, and, since then, 22 ConA-like lectins from the Diocleinae subtribe have had their primary structures determined using Edman degradation, as well as mass spectrometry and DNA sequencing: ConV [36] (Uniprot id: C0HJY1), CGL (Uniprot id: P14894), ConM [95] (Uniprot id: P81364), ConBr [96] (Uniprot id: P55915), CboL [97] (Uniprot id: A0A023GPI8), CaBo [98] (Uniprot id: P58906), ConGF [99] (Uniprot id: A0A067XG71), DvirL (Uniprot id: P58907), CRL1 [42] (P86184), DLL [63] (PDB id: 5UUY), DlyL [44] (Uniprot id: C0HK27), DRL [100] (Uniprot id: P58908), DguiL [101] (Uniprot id: P81637), DGL [101] (Uniprot id: P08902), Cramoll [102] (Uniprot id: P83721), CFL [101] (Uniprot id: P81517), DVL (Uniprot id: I1SB09), Camptosema pedicaellatum lectin (CPL) [103] (Uniprot id: J9PBR3), DSL [104] (Uniprot id: B3EWJ2), DWL [38] (Uniprot id: P86624), DrfL [52] (Uniprot id: C0HK81) and Dioclea lehmanni lectin (DlehL) [95].…”

“…These proteins usually have Ca 2+ and Mn 2+ ions in a metal-binding site (MBS) located in the vicinity of the carbohydrate-recognition domain (CRD). These ions are important for CRD stabilization, thus allowing lectin binding to carbohydrates [36,113]. More details on the MBS can be found in Section 5.2.…”

Exploring the carbohydrate‐binding ability of Canavalia bonariensis lectin in inflammation models

“…In the paw edema test, the α-methyl-mannoside reversal assay ( Figure 4C The results presented in this paper are consistent with those shown in other works described for ConA-like lectins, in particular those describing the proinflammatory effect of lectins isolated from the Diocleineae subtribe, as reported also for the lectins from Dioclea lasiophylla and Canavalia virosa. 23,34,35 Thus, it is notorious that, even working with a group of lectins of high structural similarity and of the same specificity, the results of biological activity vary in terms of the potency of the biological effect, which justifies all the more acute tests regarding the fine specificity of these proteins.…”