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1.
PLoS Negl Trop Dis ; 10(4): e0004609, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27058234

RESUMO

ArtinM, a D-mannose binding lectin from Artocarpus heterophyllus, has immunomodulatory activities through its interaction with N-glycans of immune cells, culminating with the establishment of T helper type 1 (Th1) immunity. This interaction protects mice against intracellular pathogens, including Leishmania major and Leishmania amazonensis. ArtinM induces neutrophils activation, which is known to account for both resistance to pathogens and host tissue injury. Although exacerbated inflammation was not observed in ArtinM-treated animals, assessment of neutrophil responses to ArtinM is required to envisage its possible application to design a novel immunomodulatory agent based on carbohydrate recognition. Herein, we focus on the mechanisms through which neutrophils contribute to ArtinM-induced protection against Leishmania, without exacerbating inflammation. For this purpose, human neutrophils treated with ArtinM and infected with Leishmania major were analyzed together with untreated and uninfected controls, based on their ability to eliminate the parasite, release cytokines, degranulate, produce reactive oxygen species (ROS), form neutrophil extracellular traps (NETs) and change life span. We demonstrate that ArtinM-stimulated neutrophils enhanced L. major clearance and at least duplicated tumor necrosis factor (TNF) and interleukin-1beta (IL-1ß) release; otherwise, transforming growth factor-beta (TGF-ß) production was reduced by half. Furthermore, ROS production and cell degranulation were augmented. The life span of ArtinM-stimulated neutrophils decreased and they did not form NETs when infected with L. major. We postulate that the enhanced leishmanicidal ability of ArtinM-stimulated neutrophils is due to augmented release of inflammatory cytokines, ROS production, and cell degranulation, whereas host tissue integrity is favored by their shortened life span and the absence of NET formation. Our results reinforce the idea that ArtinM may be considered an appropriate molecular template for the construction of an efficient anti-infective agent.


Assuntos
Fatores Imunológicos/farmacologia , Lectinas/farmacologia , Leishmania major/imunologia , Neutrófilos/imunologia , Neutrófilos/parasitologia , Artocarpus/química , Degranulação Celular , Sobrevivência Celular , Células Cultivadas , Citocinas/metabolismo , Humanos , Fatores Imunológicos/isolamento & purificação , Lectinas/isolamento & purificação , Leishmania major/fisiologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/fisiologia , Espécies Reativas de Oxigênio/metabolismo
2.
Int J Biol Macromol ; 82: 22-30, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26433176

RESUMO

Recent advances in glycobiology have revealed the essential role of lectins in deciphering the glycocodes at the cell surface to generate important biological signaling responses. ArtinM, a d-mannose-binding lectin isolated from the seeds of jackfruit (Artocarpus heterophyllus), is composed of 16 kDa subunits that are associated to form a homotetramer. Native ArtinM (n-ArtinM) exerts immunomodulatory and regenerative effects, but the potential pharmaceutical applicability of the lectin is highly limited by the fact that its production is expensive, laborious, and impossible to be scaled up. This led us to characterize a recombinant form of the lectin obtained by expression in Saccharomyces cerevisiae (y-ArtinM). In the present study, we demonstrated that y-ArtinM is similar to n-ArtinM in subunit arrangement, oligomerization and carbohydrate binding specificity. We showed that y-ArtinM can exert n-ArtinM biological activities such as erythrocyte agglutination, stimulation of neutrophil migration and degranulation, mast cell degranulation, and induction of interleukin-12 and interleukin-10 production by macrophages. In summary, the expression of ArtinM in yeast resulted in successful production of an active, recombinant form of ArtinM that is potentially useful for pharmaceutical application.


Assuntos
Carboidratos/química , Lectinas de Ligação a Manose/química , Estrutura Molecular , Proteínas Recombinantes , Animais , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/imunologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Citocinas/biossíntese , Hemaglutinação , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Lectinas de Ligação a Manose/metabolismo , Lectinas de Ligação a Manose/farmacologia , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Polissacarídeos/química , Ligação Proteica , Receptor 2 Toll-Like , Leveduras/genética , Leveduras/metabolismo
3.
PLoS One ; 9(2): e88422, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24558388

RESUMO

Lectins are carbohydrate-binding proteins that recognize and modulate physiological activities and have been used as a toll for detection and identification of biomolecules, and therapy of diseases. In this study we have isolated a lectin present in the latex of Euphorbia tirucalli, and named it Eutirucallin. The latex protein extract was subjected to ion exchange chromatography and showed two peaks with haemagglutinating activity. Polypeptides of 32 kDa protein extract strongly interacted with immobilized galactose (α-lactose > D-N-acetylgalactosamine). The Eutirucallin was obtained with a yield of 5.6% using the α-lactose column. The lectin domain has 32 kDa subunits and at least two of which are joined by disulfide bridges. The agglutinating capacity for human erythrocytes A(+), B(+) and O(+) is inhibited by D-galactose. The haemagglutinating activity of Eutirucallin was independent of Ca(2+) and maintained until the temperature of 55°C. Eutirucallin presented biological activities such as neutrophils recruitment and cytokine prodution by macrophages. The analysis of the trypsin-digested Eutirucallin by ms/ms in ESI-Q-TOFF resulted in nine peptides similar to type 2 ribosome-inactivating protein (type-2 RIP). It's partial sequence showed a similarity of 67.4 - 83.1% for the lectin domain of type-2 RIP [Ricin and Abrin (83.1%), Viscumin, Ebulin, Pulchellin, Cinnamomin, Volkensin and type-2 RIP Iris hollandica]. Our data suggest that Eutirucallin is a new member of type 2 ribosome-inactivating protein and presents biotechnological potential.


Assuntos
Euphorbia/química , Inflamação/induzido quimicamente , Látex/química , Extratos Vegetais/química , Lectinas de Plantas/química , Acetilgalactosamina/química , Cálcio/química , Dissulfetos/química , Eritrócitos/efeitos dos fármacos , Galactose/química , Humanos , Lactose/química , Lectinas/química , Macrófagos/efeitos dos fármacos , Peptídeos/química , Estrutura Terciária de Proteína , Ribossomos/química , Espectrometria de Massas por Ionização por Electrospray , Temperatura
4.
Inflamm Allergy Drug Targets ; 11(6): 433-41, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22762379

RESUMO

Lectins are ubiquitous proteins that exhibit selective and reversible carbohydrate-binding activities, and have become increasingly known as cell recognition mediators in a wide range of biological systems. Besides being useful tools in the study of underlying mechanisms involved in inflammation, lectins have also emerged as suitable molecules for pharmaceutical applications. Since the discovery that mammalian lectins exert crucial roles in neutrophil adhesion, mobilization, and activation, the experimental use of lectins from exogenous sources, such as plants, as modulators of leukocyte functions has been considered. Indeed, specific mammalian cell responses triggered by different plant lectins have contributed to delineation of the signaling mechanisms underlying cell adhesion, intracellular activation, and modulation of cell responses. This review presents a comprehensive summary of research concerning the effects of plant lectins on the main physiological activities of neutrophils, such as migration, degranulation, release of inflammatory mediators, phagocytosis, and apoptosis. The reports included herein illustrate the modulation of inflammatory processes by plant lectins.


Assuntos
Inflamação/tratamento farmacológico , Ativação de Neutrófilo/efeitos dos fármacos , Lectinas de Plantas/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Humanos , Inflamação/fisiopatologia , Mediadores da Inflamação/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Fagocitose/efeitos dos fármacos
5.
PLoS One ; 6(11): e27892, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22132163

RESUMO

ArtinM, a D-mannose-binding lectin from Artocarpus heterophyllus (jackfruit), interacts with N-glycosylated receptors on the surface of several cells of hematopoietic origin, triggering cell migration, degranulation, and cytokine release. Because malignant transformation is often associated with altered expression of cell surface glycans, we evaluated the interaction of ArtinM with human myelocytic leukemia cells and investigated cellular responses to lectin binding. The intensity of ArtinM binding varied across 3 leukemia cell lines: NB4>K562>U937. The binding, which was directly related to cell growth suppression, was inhibited in the presence of Manα1-3(Manα1-6)Manß1, and was reverted in underglycosylated NB4 cells. ArtinM interaction with NB4 cells induced cell death (IC(50) = 10 µg/mL), as indicated by cell surface exposure of phosphatidylserine and disruption of mitochondrial membrane potential unassociated with caspase activation or DNA fragmentation. Moreover, ArtinM treatment of NB4 cells strongly induced reactive oxygen species generation and autophagy, as indicated by the detection of acidic vesicular organelles in the treated cells. NB4 cell death was attributed to ArtinM recognition of the trimannosyl core of N-glycans containing a ß1,6-GlcNAc branch linked to α1,6-mannose. This modification correlated with higher levels of N-acetylglucosaminyltransferase V transcripts in NB4 cells than in K562 or U937 cells. Our results provide new insights into the potential of N-glycans containing a ß1,6-GlcNAc branch linked to α1,6-mannose as a novel target for anti-leukemia treatment.


Assuntos
Leucemia Mieloide/patologia , Lectinas de Ligação a Manose/metabolismo , Polissacarídeos/metabolismo , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Leucemia Mieloide/enzimologia , Lectinas de Ligação a Manose/farmacologia , Modelos Biológicos , Espécies Reativas de Oxigênio/metabolismo
6.
PLoS One ; 6(12): e29216, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22216217

RESUMO

The fungus Paracoccidioides brasiliensis is a human pathogen that causes paracoccidioidomycosis, the most prevalent systemic mycosis in Latin America. The cell wall of P. brasiliensis is a network of glycoproteins and polysaccharides, such as chitin, that perform several functions. N-linked glycans are involved in glycoprotein folding, intracellular transport, secretion, and protection from proteolytic degradation. Here, we report the effects of tunicamycin (TM)-mediated inhibition of N-linked glycosylation on P. brasiliensis yeast cells. The underglycosylated yeasts were smaller than their fully glycosylated counterparts and exhibited a drastic reduction of cell budding, reflecting impairment of growth and morphogenesis by TM treatment. The intracellular distribution in TM-treated yeasts of the P. brasiliensis glycoprotein paracoccin was investigated using highly specific antibodies. Paracoccin was observed to accumulate at intracellular locations, far from the yeast wall. Paracoccin derived from TM-treated yeasts retained the ability to bind to laminin despite their underglycosylation. As paracoccin has N-acetyl-ß-d-glucosaminidase (NAGase) activity and induces the production of TNF-α and nitric oxide (NO) by macrophages, we compared these properties between glycosylated and underglycosylated yeast proteins. Paracoccin demonstrated lower NAGase activity when underglycosylated, although no difference was detected between the pH and temperature optimums of the two forms. Murine macrophages stimulated with underglycosylated yeast proteins produced significantly lower levels of TNF-α and NO. Taken together, the impaired growth and morphogenesis of tunicamycin-treated yeasts and the decreased biological activities of underglycosylated fungal components suggest that N-glycans play important roles in P. brasiliensis yeast biology.


Assuntos
Proteínas Fúngicas/fisiologia , Morfogênese , Paracoccidioides/crescimento & desenvolvimento , Acetilglucosaminidase/metabolismo , Proteínas Fúngicas/metabolismo , Glicosilação , Lectinas/metabolismo , Macrófagos/metabolismo , Paracoccidioides/efeitos dos fármacos , Paracoccidioides/enzimologia , Paracoccidioides/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Tunicamicina/farmacologia
7.
PLoS One ; 5(5): e10757, 2010 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-20505765

RESUMO

This study aimed to investigate the immunological mechanisms involved in the gender distinct incidence of paracoccidioidomycosis (pcm), an endemic systemic mycosis in Latin America, which is at least 10 times more frequent in men than in women. Then, we compared the immune response of male and female mice to Paracoccidioides brasiliensis infection, as well as the influence in the gender differences exerted by paracoccin, a P. brasiliensis component with carbohydrate recognition property. High production of Th1 cytokines and T-bet expression have been detected in the paracoccin stimulated cultures of spleen cells from infected female mice. In contrast, in similar experimental conditions, cells from infected males produced higher levels of the Th2 cytokines and expressed GATA-3. Macrophages from male and female mice when stimulated with paracoccin displayed similar phagocytic capability, while fungicidal activity was two times more efficiently performed by macrophages from female mice, a fact that was associated with 50% higher levels of nitric oxide production. In order to evaluate the role of sexual hormones in the observed gender distinction, we have utilized mice that have been submitted to gonadectomy followed by inverse hormonal reconstitution. Spleen cells derived from castrated males reconstituted with estradiol have produced higher levels of IFN-gamma (1291+/-15 pg/mL) and lower levels of IL-10 (494+/-38 pg/mL), than normal male in response to paracoccin stimulus. In contrast, spleen cells from castrated female mice that had been treated with testosterone produced more IL-10 (1284+/-36 pg/mL) and less IFN-gamma (587+/-14 pg/mL) than cells from normal female. In conclusion, our results reveal that the sexual hormones had a profound effect on the biology of immune cells, and estradiol favours protective responses to P. brasiliensis infection. In addition, fungal components, such as paracoccin, may provide additional support to the gender dimorphic immunity that marks P. brasiliensis infection.


Assuntos
Paracoccidioides/imunologia , Paracoccidioidomicose/imunologia , Paracoccidioidomicose/microbiologia , Caracteres Sexuais , Animais , Feminino , Proteínas Fúngicas/imunologia , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Regulação da Expressão Gênica , Hormônios Esteroides Gonadais/metabolismo , Interferon gama/biossíntese , Interleucina-10/biossíntese , Interleucina-4/genética , Interleucina-4/metabolismo , Lectinas/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Masculino , Camundongos , Viabilidade Microbiana , Modelos Imunológicos , Óxido Nítrico/biossíntese , Especificidade de Órgãos/imunologia , Baço/metabolismo , Baço/microbiologia , Baço/patologia , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo
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