RESUMO
S-layer (glyco)-proteins (SLPs) form a nanostructured envelope that covers the surface of different prokaryotes and show immunomodulatory activity. Previously, we have demonstrated that the S-layer glycoprotein from probiotic Lactobacillus kefiri CIDCA 8348 (SLP-8348) is recognized by Mincle (macrophage inducible C-type lectin receptor), and its adjuvanticity depends on the integrity of its glycans. However, the glycan's structure has not been described so far. Herein, we analyze the glycosylation pattern of three SLPs, SLP-8348, SLP-8321, and SLP-5818, and explore how these patterns impact their recognition by C-type lectin receptors and the immunomodulatory effect of the L. kefiri SLPs on antigen-presenting cells. High-performance anion-exchange chromatography-pulse amperometric detector performed after ß-elimination showed glucose as the major component in the O-glycans of the three SLPs; however, some differences in the length of hexose chains were observed. No N-glycosylation signals were detected in SLP-8348 and SLP-8321, but SLP-5818 was observed to have two sites carrying complex N-glycans based on a site-specific analysis and a glycomic workflow of the permethylated glycans. SLP-8348 was previously shown to enhance LPS-induced activation on both RAW264.7 macrophages and murine bone marrow-derived dendritic cells; we now show that SLP-8321 and SLP-5818 have a similar effect regardless of the differences in their glycosylation patterns. Studies performed with bone marrow-derived dendritic cells from C-type lectin receptor-deficient mice revealed that the immunostimulatory activity of SLP-8321 depends on its recognition by Mincle, whereas SLP-5818's effects are dependent on SignR3 (murine ortholog of human DC-SIGN). These findings encourage further investigation of both the potential application of these SLPs as new adjuvants and the protein glycosylation mechanisms in these bacteria.
Assuntos
Antígenos CD/metabolismo , Lactobacillus/metabolismo , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Antígenos CD/genética , Cromatografia Líquida de Alta Pressão , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Glicopeptídeos/análise , Glicopeptídeos/química , Glicosilação , Imunização , Interferon gama/metabolismo , Lectinas Tipo C/deficiência , Lectinas Tipo C/genética , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/química , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Polissacarídeos/análise , Polissacarídeos/química , Células RAW 264.7 , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
C-type lectins are multifunctional sugar-binding molecules expressed on dendritic cells (DCs) and macrophages that internalize antigens for processing and presentation. Macrophage galactose-type lectin 1 (MGL1) recognizes glycoconjugates expressing Lewis X structures which contain galactose residues, and it is selectively expressed on immature DCs and macrophages. Helminth parasites contain large amounts of glycosylated components, which play a role in the immune regulation induced by such infections. Macrophages from MGL1(-/-) mice showed less binding ability toward parasite antigens than their wild-type (WT) counterparts. Exposure of WT macrophages to T. crassiceps antigens triggered tyrosine phosphorylation signaling activity, which was diminished in MGL1(-/-) macrophages. Following T. crassiceps infection, MGL1(-/-) mice failed to produce significant levels of inflammatory cytokines early in the infection compared to WT mice. In contrast, MGL1(-/-) mice developed a Th2-dominant immune response that was associated with significantly higher parasite loads, whereas WT mice were resistant. Flow cytometry and RT-PCR analyses showed overexpression of the mannose receptors, IL-4Rα, PDL2, arginase-1, Ym1, and RELM-α on MGL1(-/-) macrophages. These studies indicate that MGL1 is involved in T. crassiceps recognition and subsequent innate immune activation and resistance.
Assuntos
Antígenos de Helmintos/imunologia , Assialoglicoproteínas/metabolismo , Resistência à Doença/imunologia , Lectinas Tipo C/metabolismo , Macrófagos Peritoneais/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais , Taenia/imunologia , Teníase/imunologia , Acetilgalactosamina/metabolismo , Animais , Assialoglicoproteínas/deficiência , Citocinas/biossíntese , Feminino , Galactose/metabolismo , Glicoconjugados/metabolismo , Imunidade , Espaço Intracelular/metabolismo , Cinética , Lectinas Tipo C/deficiência , Ativação de Macrófagos/imunologia , Proteínas de Membrana/deficiência , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fosforilação , Fosfotirosina/metabolismo , Ligação Proteica , Solubilidade , Teníase/parasitologiaRESUMO
Agaricus brasiliensis is a well-known medicinal mushroom. We have previously demonstrated that Agaricus-derived polysaccharides exhibit potent antitumor effects; however, the underlying mechanism(s) have not been elucidated yet. In this study, we examined the immunoenhancing activities of Agaricus extracts. Agaricus-derived polysaccharides were characterized as 1,6-ß-glucan with a small amount of 1,3-ß-glucan using anti-ß-glucan antibody and nuclear magnetic resonance analysis. These polysaccharides strongly induced the production of various cytokines from both murine splenocytes and bone marrow-derived dendritic cells in the presence of exogenous granulocyte-macrophage colony-stimulating factor. Polysaccharide-induced cytokine production was significantly reduced in bone marrow-derived dendritic cells derived from dectin-1-deficient mice. Furthermore, a binding assay revealed that the Agaricus-derived polysaccharides can be recognized by dectin-1, a pivotal receptor for 1,3-ß-glucan. Taken together, our results clearly indicate that the immunostimulation induced by Agaricus-derived polysaccharides is exerted, at least in part, via dectin-1 in combination with granulocyte-macrophage colony-stimulating factor.