In depth analysis on the carbohydrate-binding properties of a vasorelaxant lectin from Dioclea lasiophylla Mart Ex. Benth seeds.

Lectins are a class of proteins or glycoproteins capable of recognizing and interacting with carbohydrates in a specific and reversible manner. Owing to this property, these proteins can interact with glycoconjugates present on the cell surface, making it possible to decipher the glycocode, as well as elicit biological effects, such as inflammation and vasorelaxation. Here, we report a structural and biological study of the mannose/glucose-specific lectin from Dioclea lasiophylla seeds, DlyL. The study aimed to evaluate in detail the interaction of DlyL with Xman and high-mannose N-glycans (MAN3, MAN5 and MAN9) by molecular dynamics (MD) and the resultant in vitro effect on vasorelaxation using rat aortic rings. In silico analysis of molecular docking was performed to obtain the initial coordinates of the DlyL complexes with the carbohydrates to apply as inputs in MD simulations. The MD trajectories demonstrated the stability of DlyL over time as well as different profiles of interaction with Xman and N-glycans. Furthermore, aortic rings assays demonstrated that the lectin could relax pre-contracted aortic rings with the participation of the carbohydrate recognition domain (CRD) and nitric oxide (NO) when endothelial tissue is preserved. These results confirm the ability of DlyL to interact with high-mannose N-glycans with its expanded CRD, supporting the hypothesis that DlyL vasorelaxant activity occurs primarily through its interaction with cell surface glycosylated receptors.Communicated by Ramaswamy H. Sarma.

Vatairea guianensis lectin stimulates changes in gene expression and release of TNF-α from rat peritoneal macrophages via glycoconjugate binding.

Using a rat model of peritonitis, we herein report the inflammatory effect induced by the lectin isolated from Vatairea guianensis (VGL) seeds in the context of interactions between VGL and both toll-like receptor 4 (TLR4) and tumor necrosis factor receptor 1 (TNFR1). Peritoneal macrophages were stimulated with VGL for dose-dependent gene expression and release of TNF-α. In vivo results showed that VGL (1 mg/kg; intraperitoneal) induced peritonitis in female Wistar rats. Leukocyte migration, macrophage activation, and protein leakage were measured 3 and 6 hours after induction. In vitro, peritoneal macrophages were stimulated with VGL for gene expression and TNF-α dosage (mean ± SEM (n = 6), analysis of variance, and Bonferroni's test (P < .05)). In silico, VGL structure was applied in molecular docking with representative glycans. It was found that (a) VGL increases vascular permeability and stimulates leukocyte migration, both rolling and adhesion; (b) lectin-induced neutrophil migration occurs via macrophage stimulation, both in vitro and in vivo; (c) lectin interacts with TLR4 and TNFR1; and (d) stimulates TNF-α gene expression (RT-PCR) and release from peritoneal macrophages. Thus, upon lectin-glycan binding on the cell surface, our results suggest that VGL induces an acute inflammatory response, in turn activating the release of peritoneal macrophages via TNF-α and TLR and/or TNFR receptor pathways.

Antinociceptive effect of Lonchocarpus araripensis lectin: activation of L-arginine/NO/cGMP/K+ATP signaling pathway.

OBJECTIVE AND DESIGN: The involvement of nitric oxide pathway in the antinociceptive activity of Lonchocarpus araripensis lectin (LAL) was investigated in the model of carragenan-induced hypernociception. METHODS: Swiss mice received LAL (0.01-10 mg/kg; i.v.) 30 min before s.c. injection of carragenan in the paws. For the involvement of nociceptive pathways, animals were previously treated with the blockers: NOS (L-NAME, aminoguanidine, 7-nitroindazole); soluble guanylyl cyclase (ODQ); channels of ATP-dependent K+ (glibenclamide); L-type Ca2+ (nifedipine), or Ca2+-dependent Cl- (niflumic acid). Participation of lectin domain was evaluated by injection of LAL associated with N-acetyl-glucosamine (GlcNAc). nNOS gene relative expression was evaluated in the paw tissues and nNOS immunostaining in dorsal root ganglia. RESULTS: LAL at all doses inhibited carrageenan-induced hypernociception (4.12 ± 0.58 g), being maximal at 10 mg/kg (3 h: 59%), and reversed by GlcNAc. At this time, LAL effect was reversed by nifedipine (39%), niflumic acid (59%), L-NAME (59%), 7-nitroindazole (44%), ODQ (45%), and glibenclamide (34%), but was unaltered by aminoguanidine. LAL increased (95%) nNOS gene expression in mice paw tissues, but not its immunoexpression in the dorsal root ganglia. CONCLUSION: The antinociceptive effect of Lonchocarpus araripensis lectin involves activation of the L-arginine/NO/GMPc/K+ATP pathway.

Structural studies and nociceptive activity of a native lectin from Platypodium elegans seeds (nPELa).

A native lectin (nPELa), purified from seeds of the species Platypodium elegans, Dalbergieae tribe, was crystallized and structurally characterized by X-ray diffraction crystallography and bioinformatics tools. The obtained crystals diffracted to 1.6Å resolution, and nPELa structure were solved through molecular substitution. In addition, nPELa has a metal binding site and a conserved carbohydrate recognition domain (CRD) similar to other Dalbergieae tribe lectins, such as PAL (Pterocarpus angolensis) and CTL (Centrolobium tomentosum). Molecular docking analysis indicated high affinity of this lectin for different mannosides, mainly trimannosides, formed by α-1,3 or α-1,6 glycosidic bond, as evidenced by the obtained scores. In addition, molecular dynamics simulations were performed to demonstrate the structural behavior of nPELa in aqueous solution. In solution, nPELa was highly stable, and structural modifications in its carbohydrate recognition site allowed interaction between the lectin and the different ligands. Different modifications were observed during simulations for each one of the glycans, which included different hydrogen bonds and hydrophobic interactions through changes in the relevant residues. In addition, nPELa was evaluated for its nociceptive activity in mice and was reported to be the first lectin of the Dalbergieae tribe to show CRD-dependent hypernociceptive activity.

Partial characterization and immobilization in CNBr-activated Sepharose of a native lectin from Platypodium elegans seeds (PELa) and comparative study of edematogenic effect with the recombinant form.

The lectin from Platypodium elegans seeds (PELa) was purified by affinity chromatography in a mannose-agarose column. The lectin agglutinated rabbit erythrocytes and the agglutinating effect was inhibited by previous incubation with the glycoprotein fetuin, along with N-acetyl-d-glucosamine, D-mannose and its derivatives. The lectin maintained complete activity in temperatures ranging from 40 to 60°C and pH values ranging from 9 to 10. As a glycoprotein, PELa has a carbohydrate content of 2.2%, and its activity requires divalent cations such as Ca2+ and Mn2+. Based on SDS-PAGE, PELa displays a profile similar to that of other Dalbergieae lectins with the main chain of molecular mass around 30kDa and two subunits of 19kDa and 10 kDa each. Two-dimensional (2D) electrophoresis revealed the presence of isoforms with different isoelectric points, and high-performance size exclusion chromatography (HPSEC) was performed to confirm the purity of the sample. The lectin was immobilized in CNBr-activated Sepharose 4B and successfully captured fetuin in solution, demonstrating that this lectin remains active and capable of binding carbohydrates. PELa showed effects different from those of its recombinant form in both pro- and anti-inflammatory tests.

The leguminous lectin of Lonchocarpus araripensis promotes antinociception via mechanisms that include neuronal inhibition of Na(+) currents.

OBJECTIVE AND DESIGN: Sodium channels are highly expressed in nociceptive sensory neurons during hypernociceptive conditions. Based on the presence of a glycosidic portion in the sodium channel ß subunit associated to the antinociceptive effect of leguminous lectins via lectin domain, this study investigated the antinociceptive activity of the lectin isolated from Lonchocarpus araripensis seeds (LAL) in mice behavioral models and in NaV current in the nociceptor of rat dorsal root ganglion (DRG). MATERIAL/METHODS: LAL antinociceptive activity and the participation of opioid system, lectin domain and sodium channels were evaluated in Swiss mice models of nociception (formalin, capsaicin, hot plate, tail flick, von Frey) and in primary cultures of Wistar rats neurons of DRG (patch clamp). RESULTS: LAL presented inhibitory effects in the nociception induced by chemical and mechanical, but not by thermal stimuli and reduced total Na(+) current. LAL activity was inhibited by the lectin association with its binding sugar N-acethyl-glucosamine. CONCLUSION: LAL inhibits peripheral hypernociception by mechanisms that involve the lectin domain, inflammatory mediators and Na(+) channels. The innovative inhibitory action of leguminous lectins on NaV current brings new insights for the investigation of sodium channels role in nociception.