RESUMO
The biomolecular recognition of D-mannose-binding lectin from Artocarpus heterophyllus (ArtinM) by Horseradish Peroxidase (HRP) mediated by glycosylation allows their application in a multitude of biological systems. The present work describes the use of molecular dynamics (MD) to assess the Gibbs free energy associated with the formation of a ArtinM-HRP conjugate mediated by a glycosylation molecule. For the enthalpy term, we applied the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method and for the vibrational entropy term, we use the quasi-harmonic approximation. Our results show that, even without glycosylation, the binding free energy between ArtinM and HRP is - 196.154 kJmol- 1, an extremely high affinity with low selectivity, originated mainly through the van der Waals energy terms. The binding free energy between ArtinM and the glycosylated HRP (gHRP) was calculated at - 66.156 kJmol- 1, an absolute and considerably lower value, however, originated from electrostatic energy terms, which increases the selectivity of molecular recognition. Our work has shown that the HRP active site region has a high affinity and low selectivity for other biomolecules. The presence of glycosylation plays a role in increasing this selectivity for this region. Thus, we conclude that performing mutagenesis of amino acid residues near the entrance of the catalytic site, can improve the activity of non-glycosylated HRPs. This illustrates new insights that can be applied to carbohydrate-based immunochemistry.
Assuntos
Artocarpus/metabolismo , Lectina de Ligação a Manose/metabolismo , Simulação de Dinâmica Molecular , Glicosilação , Peroxidase do Rábano Silvestre/química , Peroxidase do Rábano Silvestre/metabolismo , Lectina de Ligação a Manose/química , Lectinas de Plantas , TermodinâmicaRESUMO
The immunomodulatory activity of plant lectins has been evaluated because of their high selectivity for glycans linked to receptors on innate and adaptative immune cells. ArtinM is a mannosyl-binding lectin, obtained from the seeds of Artocarpus heterophyllus, that induces the differentiation of CD4+ T cells and macrophages by interacting with CD3 and TLR2/CD14, respectively. This ArtinM property ultimately favors the combat of intracellular pathogens, opening new perspectives on the lectins application as immunomodulatory agents. The current section describes protocols for purification and evaluation of ArtinM biological activity. The purification is based on the ArtinM-D-mannose affinity. The effect of inducing IL-12 production by murine macrophages cell line is adopted to evaluate the ArtinM biological activity.
Assuntos
Artocarpus/metabolismo , Linfócitos T CD4-Positivos/citologia , Fatores Imunológicos/farmacologia , Macrófagos/citologia , Lectinas de Plantas/farmacologia , Animais , Artocarpus/química , Complexo CD3/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Fatores Imunológicos/isolamento & purificação , Interleucina-12/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Manose/metabolismo , Camundongos , Lectinas de Plantas/isolamento & purificação , Células RAW 264.7 , Sementes/química , Sementes/metabolismo , Receptor 2 Toll-Like/metabolismoRESUMO
BACKGROUND: As an alternative to the use of widely investigated agro-industrial residues, the present study aimed to promote the valorization of two selected residues, yellow mombin seed (YS) and jackfruit seed (JS), as a result of their enhanced performance. RESULTS: YS was applied as a solid state substrate for Penicillium roqueforti ATCC 101110 cultivation (25 °C, Aw = 0.963, 107 spores g-1 and 142 h) to produce a crude multi-enzymatic extract (CE-YS) containing activities of CMCase = 31.95 U g-1 , xylanase = 56.85 U g-1 , exoglucanase = 5.55 U g-1 and FPase = 24.60 U g-1 . CE-YS was then applied to six different residues saccharification and the best performance was obtained with jackfruit seed residue (JS), which was selected for enzymatic saccharification. The highest productivity of reducing sugars expressed as glucose (6.26 mg g-1 h-1 ) was obtained under the conditions: 40.7 g L-1 JS, 5 mmol L-1 MgCl2 , 65 °C, 120 rpm, pH 3.0 (citrate buffer 50 mmol L-1 ) and 18 h. CONCLUSION: The residues, YS and JS, can be used satisfactorily for the production of bioproducts of great industrial applicability, such as crude extracts (containing cellulolytic enzymes) and RS (which can be converted, for example, into bioethanol). © 2020 Society of Chemical Industry.
Assuntos
Anacardiaceae/microbiologia , Artocarpus/microbiologia , Penicillium/metabolismo , Açúcares/metabolismo , Anacardiaceae/metabolismo , Artocarpus/metabolismo , Biocatálise , Celulase/química , Meios de Cultura/metabolismo , Fermentação , Concentração de Íons de Hidrogênio , Hidrólise , Sementes/metabolismo , Resíduos/análiseRESUMO
Mast cells are connective tissue resident cells with morphological and functional characteristics that contribute to their role in allergic and inflammatory processes, host defense and maintenance of tissue homeostasis. Mast cell activation results in the release of pro-inflammatory mediators which are largely responsible for the physiological functions of mast cells. The lectin ArtinM, extracted from Artocarpus heterophyllus (jackfruit), binds to D-manose, thus inducing degranulation of mast cells. ArtinM has several immunomodulatory properties including acceleration of wound healing, and induction of cytokine release. The aim of the present study was to investigate the role of ArtinM in the activation and proliferation of mast cells. The rat mast cell line RBL-2H3 was used throughout this study. At a low concentration (0.25µg/mL), ArtinM induced mast cell activation and the release of IL-6 without stimulating the release of pre-formed or newly formed mediators. Additionally, when the cells were activated by ArtinM protein tyrosine phosphorylation was stimulated. The low concentration of ArtinM also activated the transcription factor NFkB, but not NFAT. ArtinM also affected the cell cycle and stimulated cell proliferation. Therefore, ArtinM may have therapeutic applications by modulating immune responses due to its ability to activate mast cells and promote the release of newly synthesized mediators. Additionally, ArtinM could have beneficial effects at low concentrations without degranulating mast cells and inducing allergic reactions.
Assuntos
Degranulação Celular/efeitos dos fármacos , Lectinas/farmacologia , Proteínas de Plantas/farmacologia , Animais , Artocarpus/metabolismo , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Proliferação de Células/efeitos dos fármacos , Interleucina-6/metabolismo , Mastócitos/citologia , Mastócitos/metabolismo , Mitose/efeitos dos fármacos , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , RatosRESUMO
ArtinM, a d-mannose-binding lectin from Artocarpus heterophyllus, activates antigen-presenting cells by recognizing Toll-like receptor (TLR)2 and cluster of differentiation (CD)14 N-glycans, induces cytokine production, and promotes type 1 T helper (Th1) immunity, a process that plays an assisting role in the combat against fungal infections. We recently demonstrated that ArtinM stimulates CD4⺠T cells to produce interleukin (IL)-17 through direct interaction with CD3. Here, we further investigated the effects of ArtinM on the production of IL-17 by B cell activation. We showed that ArtinM activates murine B cells, increasing IL-17 and IL-12p40 production. The direct effect of ArtinM was sufficient to induce IL-17 production in B cells, and we did not find differences in the levels of IL-17 between the B cells purified from the wild-type (WT) and knockout (KO) mice for TLR2 or CD14 in the presence of ArtinM. Thus, the effects of ArtinM on splenic B cells through carbohydrate recognition may contribute to Th17 immunity; however, the mechanism involved is not associated with the interaction of ArtinM with TLR2 and CD14. The current work represents a pioneering effort in the understanding of the induction of IL-17 by lectins in B cells.
Assuntos
Linfócitos B/efeitos dos fármacos , Interleucina-17/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Lectinas de Plantas/farmacologia , Receptor 2 Toll-Like/metabolismo , Animais , Artocarpus/metabolismo , Linfócitos B/citologia , Linfócitos B/metabolismo , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Receptores de Lipopolissacarídeos/genética , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Receptor 2 Toll-Like/genéticaRESUMO
Different parts of plant foods are generally discarded by consumers such as peel, stalk and leaves, which could however possess a nutritional value. However, few studies have analysed the composition of these marginal foods. The phenolic compound, flavonoid, polyamine, nitrate and pesticide contents of parts of vegetables that are usually discarded--but which were cultivated according to conventional and non-conventional procedures--were analysed to provide suggestions on how to improve the consumption of these parts and to reduce the production of urban solid waste. Few, but significant, differences between the two manuring procedures were observed. Higher nitrate content and the presence of organochlorine pesticides were found in conventional cultivated papaya peel, lemon balm leaves, jack fruit pulp, and beet stalk and peel. Discarded parts of plant foods such as stalk, leaves and peels can be used as a source of antioxidant compounds, such as phenolic compounds.