Evidence for Conformational Mechanism on the Binding of TgMIC4 with ß-Galactose-Containing Carbohydrate Ligand.

A deeper understanding of the role of sialic/desialylated groups during TgMIC4-glycoproteins interactions has importance to better clarify the odd process of host cell invasion by members of the apicomplexan phylum. Within this context, we evaluated the interaction established by recombinant TgMIC4 (the whole molecule) with sialylated (bovine fetuin) and desialylated (asialofetuin) glycoproteins by using functionalized quartz crystal microbalance with dissipation monitoring (QCM-D). A suitable receptive surface containing recombinant TgMIC4 for monitoring ß-galactose-containing carbohydrate ligand (limit of quantification ∼ 40 µM) was designed and used as biomolecular recognition platform to study the binding and conformational mechanisms of TgMIC4 during the interaction with glycoprotein containing (fetuin), or not, terminal sialic group (asialofetuin). It was inferred that the binding/interaction monitoring depends on the presence/absence of sialic groups in target protein and is possible to be differentiated through a slower binding kinetic step using QCM-D approach (which we are inferring to be thus associated with ß-galactose ligand). This slower binding/interaction step is likely supposed (from mechanical energetic analysis obtained in QCM-D measurements) to be involved with Toxoplasma gondii (the causative agent of toxoplasmosis) parasitic invasion accompanied by ligand (galactose) induced binding conformational change (i.e., cell internalization process can be additionally dependent on structural conformational changes, controlled by the absence of sialic groups and to the specific binding with galactose), in addition to TgMIC4-glycoprotein solely recognition binding process.

Impedance-derived electrochemical capacitance spectroscopy for the evaluation of lectin-glycoprotein binding affinity.

Characterization of lectin-carbohydrate binding using label-free methods such as impedance-derived electrochemical capacitance spectroscopy (ECS) is desirable to evaluate specific interactions, for example, ArtinM lectin and horseradish peroxidase (HRP) glycoprotein, used here as a model for protein-carbohydrate binding affinity. An electroactive molecular film comprising alkyl ferrocene as a redox probe and ArtinM as a carbohydrate receptive center to target HRP was successfully used to determine the binding affinity between ArtinM and HRP. The redox capacitance, a transducer signal associated with the alkyl ferrocene centers, was obtained by ECS and used in the Langmuir adsorption model to obtain the affinity constant (1.6±0.6)×10(8) L mol(-1). The results shown herein suggest the feasibility of ECS application for lectin glycoarray characterization.

Evaluating the Equilibrium Association Constant between ArtinM Lectin and Myeloid Leukemia Cells by Impedimetric and Piezoelectric Label Free Approaches.

Label-free methods for evaluating lectin-cell binding have been developed to determine the lectin-carbohydrate interactions in the context of cell-surface oligosaccharides. In the present study, mass loading and electrochemical transducer signals were compared to characterize the interaction between lectin and cellular membranes by measuring the equilibrium association constant, Ka , between ArtinM lectin and the carbohydrate sites of NB4 leukemia cells. By functionalizing sensor interfaces with ArtinM, it was possible to determine Ka over a range of leukemia cell concentrations to construct analytical curves from impedimetric and/or mass-associated frequency shifts with analytical signals following a Langmuir pattern. Using the Langmuir isotherm-binding model, the Ka obtained were (8.9 ± 1.0) × 10(-5) mL/cell and (1.05 ± 0.09) × 10(-6) mL/cell with the electrochemical impedance spectroscopy (EIS) and quartz crystal microbalance (QCM) methods, respectively. The observed differences were attributed to the intrinsic characteristic sensitivity of each method in following Langmuir isotherm premises.

Topical application of the lectin Artin M accelerates wound healing in rat oral mucosa by enhancing TGF-ß and VEGF production.

The lectin Artin M has been shown to accelerate the wound-healing process. The aims of this study were to evaluate the effects of Artin M on wound healing in the palatal mucosa of rats and to investigate the effects of Artin M on transforming growth factor beta (TGF-ß) and vascular endothelial growth factor (VEGF) secretion by rat gingival fibroblasts. A surgical wound was created on the palatal mucosa of 72 rats divided into three groups according to treatment: C--Control (nontreated), A--Artin M gel, and V--Vehicle. Eight animals per group were sacrificed at 3, 5, and 7 days postsurgery for histology, immunohistochemistry and determination of the levels of cytokines, and growth factors. Gingival fibroblasts were incubated with 2.5 µg/mL of Artin M for 24, 48, and 72 hours. The expression of VEGF and TGF-ß was determined by enzyme-linked immunosorbent assay. Histologically, at day 7, the Artin M group showed earlier reepithelialization, milder inflammatory infiltration, and increased collagen fiber formation, resulting in faster maturation of granular tissue than in the other groups (p < 0.05). Artin M-induced cell proliferation in vivo and promoted a greater expression of TGF-ß and VEGF in both experiments (p < 0.05). Artin M was effective in healing oral mucosa wounds in rats and was associated with increased TGF-ß and VEGF release, cell proliferation, reepithelialization, and collagen deposition and arrangement of fibers.

The immunomodulatory effect of plant lectins: a review with emphasis on ArtinM properties.

Advances in the glycobiology and immunology fields have provided many insights into the role of carbohydrate-protein interactions in the immune system. We aim to present a comprehensive review of the effects that some plant lectins exert as immunomodulatory agents, showing that they are able to positively modify the immune response to certain pathological conditions, such as cancer and infections. The present review comprises four main themes: (1) an overview of plant lectins that exert immunomodulatory effects and the mechanisms accounting for these activities; (2) general characteristics of the immunomodulatory lectin ArtinM from the seeds of Artocarpus heterophyllus; (3) activation of innate immunity cells by ArtinM and consequent induction of Th1 immunity; (4) resistance conferred by ArtinM administration in infections with intracellular pathogens, such as Leishmania (Leishmania) major, Leishmania (Leishmania) amazonensis, and Paracoccidioides brasiliensis. We believe that this review will be a valuable resource for more studies in this relatively neglected area of research, which has the potential to reveal carbohydrate targets for novel prophylactic and therapeutic strategies.

ArtinM offers new perspectives in the development of antifungal therapy.

The thermally dimorphic fungus Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis (PCM), the most frequent systemic mycosis that affects the rural populations in Latin America. Despite significant developments in antifungal chemotherapy, its efficacy remains limited since drug therapy is prolonged and associated with toxic side effects and relapses. In response to these challenges, it is now recognized that several aspects of antifungal immunity can be modulated to better deal with fungal infections. A common idea for halting fungal infections has been the need to activate a cell-based, pro-inflammatory Th1 immune response to improve the fungal elimination. ArtinM, a D-mannose binding lectin from Artocarpus heterophyllus, has the property of modulating immunity against several intracellular pathogens. Here, we review the immunomodulatory activity of ArtinM during experimental PCM in mice. Both prophylactic and therapeutic protocols of ArtinM administration promotes a Th1 immune response balanced by IL-10, which outstandingly reduces the fungal load in organs of the treated mice while maintaining a controlled inflammation at the site of infection. A carbohydrate recognition-based interaction of ArtinM with Toll-like receptor 2 (TLR2) accounts for initiating the immunomodulatory effect of the lectin. The precise identification of the TLR2 N-glycan(s) targeted by ArtinM may support novel basis for the development of antifungal therapy.