Study On The Interaction Between DC-SIGN And Tollip And Study On The Biological Activities Of The Polysaccharide From The Spores Of Ganoderma Lucidum

PartⅠStudy on the interaction between DC-SIGN and TollipDC-SIGN(Dendritic cell-specific ICAM-3 grabbing nonintegrin) is a typeⅡmembrane protein mainly expressed on DCs(dendritic cells).Based on its structure, DC-SIGN belongs to the C-type lectin superfamily and it contains a short, cytoplasmic N-terminal tail,transmembrane region and an extracellular carbohydrate recognition domain(CRD).Via its extracellular CRD,DC-SIGN could recognize various kinds of pathogens or tissue protein containing high-mannose moieties or nonsialylated Lewis^x/y,such as HIV-1,Mycobacterium tuberculosis and carcinoembryonic antigen(CEA) on colorectal tumor cells.DC-SIGN plays an important role in mediating DCs adhesion,migration,and primary T cell activation. However,many studies have showed that DC-SIGN may contribute to the pathogenesis and the immune escape of some pathogens or tumors.For instance,upon binding HIV,DC-SIGN internalizes it rapidly and enhances its infection in trans of the target cells.ManLAM,a component of the Mycobacterium tuberculosis,targets DC-SIGN to induce IL-10 and inhibit the maturation of DCs.The interactions between DC-SIGN and colorectal tumor-associated Lewis glycans on CEA impair the function and differentiation of DC and may induce generalized failure of a host to mount an effective antitumor response.Although the ligands of DC-SIGN have been well defined,few studies have been done on its cytoplasmic tail.In the present study,we identified Tollip(Toll interacting protein) as a binding partner of the cytoplasmic tail of DC-SIGN using yeast two-hybrid screening,in human thymus cDNA library.Tollip is a integral part of the IL-1RI/TLR signaling cascade.It is also required for the sorting of the IL-1RI at late endosomes suggesting its involvement in protein sorting.GST pull-down assays showed that Tollip interacted with the cytoplasmic tail of DC-SIGN in vitro. Furthermore,the amino acid residues 57-164 of Tollip,which is part of the C2 domain was crucial for this interaction.The interaction was further confirmed in vivo by co-immunoprecipitation assay and confocal microscopic analysis.After internalization,DC-SIGN co-localized with Tollip on punctate structures throughout the cytoplasm.Knockdown of Tollip by siRNA impaired the lysosomal trafficking of DC-SIGN.Additionally,Tollip could be tyrosine phosphorylated upon DC-SIGN ligation by its specific antibody.Our results suggested that Tollip plays an important role in DC-SIGN sorting and might be an effector molecule in DC-SIGN signaling cascade.These results may be helpful in elucidating the immune escape by DC-SIGN and deserves further investigation. PartⅡStudy on the biological activities of the polysaccharide from the spores of Ganoderma lucidumGanoderma lucidum(G.lucidum),a species of basidiomycetes,has been widely used as a tonic in promoting longevity and health in China and other Asian countries. G.lucidum has been studied extensively in recent years because of its intrinsic immunomodulatory and anti-tumor properties.Though the fruit bodies of G.lucidum have been widely utilized,the spores of G.lucidum were realized and utilized only in the 20th century.The spores of G.lucidum also contain a large amount of bioactive substances like the fruit bodies of G.lucidum.The bioactivity of the spores may be higher than that of the fruit bodies of G.lucidum.However,the bioactive components in the spores of G.lucidum have been rarely studied due to the difficulties in collecting and sporoderm-breaking of the spores.In recent years,with the successful cultivation of G.lucidum indoors on a large scale and a breakthrough in sporoderm-breaking technology,much attention has been paid to chemical components of G.lucidum spores and their versatile biological activities.Recently,a number of triterpenes from the spores were isolated and characterized.However,only a few glucans from the spores were isolated and their functions remain to be investigated.GSG(G.lucidum spores glucan),a water-soluble polysaccharide from the spores of G.lucidum was extracted with the water extract and sequential alcohol precipitating method.The molecular weight of GSG ranges from 5000 Da to 11000 Da as determined by HPGPC(High performance gel permeation chromatography).IR, NMR and GC-MS confirmed the structure of GSG:it contains a backbone chain ofβ-1-3-linked D-glucopyranosyl residues and two 1-6-1inkedβ-D-glucose side chains that are further elaborated with additionalα(1,6) orα(1,4)-linked glucose regions.In vitro,GSG was an effective inducer of ROS production and MAPKs-dependent TNF-αand IL-6 secretion in murine resident peritoneal macrophages.Dectin-1 could recognize GSG and partially mediate its immunostimulatory activities.Additionally, in vivo administration of GSG potentiated the ConA-induced proliferative response of splenocytes and induced anti-tumor activity against Lewis lung cancer in mice.Our study demonstrated the biological activities of GSG both in vitro and in vivo. Due to the low molecular weight,great water-solubility,high purity and immunoenhancing effects,it is hopeful that GSG might be utilized as an intravenous injection for anti-tumor therapy and immunomodulation.The ability of Dectin-1 to mediate the activities of GSG would help to provide insights into the immunomodulatory mechanism by GSG.