Biological Activity Research And Application Of Macrofungi Derived Lectins

Lectins are proteins of non-immune origin,which have at least one carbohydrate recognition domain and can reversibly bind to specific monosaccharides or oligosac-charides.Lectins are widely distributed in the nature and have a variety of biological activities,such as immuneregulatory activity,antitumor activity,anti-bacterial activity and so on.At the same time,the glycan binding specificity of lectins makes them as useful tools in the study of glycobiology.So it is particularly necessary to find novel lectins to facilitate the study of drugs discavery and glycobiology.In this thesis,on the one hand we isolate a novel lectin?CMIP?from the fruiting body of Cordyceps militaris;study its structure composition,glycan-binding speci-ficity,immuneregulatory activity and antitumor activity.At the same time,we cloned and prokaryoticly expressed two other Cordyceps militaris derived proteins ConA-like lectin?CMCL?and Recin-B related lectin?CMRBL?,studied their haemaggluti-nation,antitumor and mitogen activity.On the other hand,based on the fact that Agrocybe aegerita derived lectin Dimer AAL2 specifically binds to GlcNAc terminated glycan,we use Di-AAL2 weak lectin chromatography or CTD 110.6 coupled with HCD/ETD-LC-MS/MS in the identification of O-GlcNAcylated proteins from the tissue of diabetic rats.1.The purification,clone and activity investigation of CMIP:A novel lectin CMIP was isolated through multistage molecular sieve chromato-graphy form the fruiting body of Cordyceps militaris.Through LC-MS/MS identi-fication we obtained the 8 tryptic peptides of CMIP.And after blasted to the transcrip-tome of Cordyceps militaris,the CMIP coding gene?CCM₀1955?was identified.Then we cloned the gene of CMIP and prokaryoticly expressed it.Circular dichroism spectrum analyze was performed to understand the secondary structure of CMIP and the results showed that it is mainly composed of ?-sheet.The hemagglutination test showed that CMIP specifically agglutinated erythrocyte of rat,but not the erythrocyte of mice or human beings.To further analyze the glycan biding specificity of CMIP,a glycan microarray?CFG,version 5.2?composed of 609 glycans were employed and the result showed that CMIP specifically bound to glycans that contain core pentasaccharides and polylactosamine structure.In the cell binding assay,CMIP can specially bind to the immortalized immune cells,such as Jurkat,RAW264.7 and Raji,but not the tested tumor cells?4T1,HeLa<5%,MCF-7,H22<10%and HepG2<16%?.Moreover,the binding between CMIP and Jurkat cell can't be blocked by other lectins?ConA,WGA,AAL,AAL2,LCA and Galectin-1?tested,this indicated that the cell surface ligand of Jurkat cell for CMIP is totally different form the other lectins.For the human peripheral blood lymphocytes,CMIP can specifically bind to CD8T lymphocytes?binding ratio 68.5%?,but less bind to the CD4T lymphocytes?binding ratio 10.3%?.For binding property of CMIP on immune cells,we focused on the immuneregulatory activity of CMIP.We fund that:1)CMIP significantly promoted the proliferation of spleen lymphocytes;2)CMIP showed to induce the M1 polariza-tion of macrophage RAW264.7 cells.And we further studied the mechanism of CMIP induced macrophage M1 polarization.The results showed that CMIP treatment increased CD86 expression of macrophages,and significantly induce the expression of M1 cytokines,such as IL-1?,iNOS,CXCL11,CCL2 and so on.Moreover,CMIP synergized with IFN-y to stimulate macrophage M1 polarization.And the phosphory-lation activation of STAT1 and p38 was involved in this process.In the antitumor assay,CMIP can't bind to any tumor cells,and showed no cytotoxicity to any of the tumor cells tested.But on the mouse model of 4T1 breast cancer lung metastasis,CMIP significantly reduced the number of tumor nodules in the lung of tumor bearing mice and prolonged their survival time.2.Clone,purification and activity investigation of two Cordyceps militaris derived proteins CMCL and CMRBLFrom the transcriptome database of Cordyceps militaris,we selected two fruiting body derived genes CCM₀3227 and CCM₀3832,these two genes contain ConA-like carbonhydrate recognize domain and Ricin B carbonhydrate recognize domain respectively.We designed primers,cloned and prokaryoticly expressed them.We named them CMCL?Cordyceps militaris ConA-like lecin?and CMRBL?Cordyceps militaris Recin B-related lectin?.CMRBL specifically agglutinated erythrocyte of rat which indicated that CMRBL was a lectin candidate,and it was worthy of further in-depth study.In the cytotoxicity test,neither of these two proteins showed any cytotoxicity to the tumor cell tested.But both of them can promote the proliferation of spleen lymphocytes.3.The application of Agrocybe aegerita derived lectin AAL2 dimerization in the identification of O-GlcNAcylationWe use lectin Di-AAL2 low affinity chromatography coupled with HCD/ETD-LC-MS/MS for the identification of O-GlcNAc modified protein from the tissue of diabetic rats.In the insulin sensitive muscles cell model?L6 myoblast cell differen-tiated myotubes?,insulin stimulation increased the protein O-GlcNAc modification levels,which were detected by CTD110.6 and RL2 antibody detection.But in the palmitic acid induced insulin resistant cell model,the increasing of O-GlcNAc levels was slightly inhibited.In vivo assay,we use STZ?streptozocin?to establish the rat model of type I diabetes,and the heart tissue and liver tissue were used for the identi-fication of O-GlcNAcylated proteins.In the assay,after trypsin enzymolysis,the peptides were incubated with other lectins?ConA,AAL and WGA?to remove the N-link Glycosylated peptides.And then use PNGase F treatment to remove the remaining N-link glycans of N-glycan modified peptides.Finally,the sample was applied to the POROS AL-Di-AAL2 column in the HPLC system or for the CTD110.6 antibody enrichment;the indicated fraction was collected and used for the HCD/ETD-LC-MS/MS O-GlcNAc identification.In total,we identified 44 modified proteins,50 peptides and 65 modified sites from the liver tissue of diabetic rats;32 midified proteins,35 peptides,and 40 modified sites form the heart of diabetic rats.We chose two MS identified proteins ERP44 and PPP1R12A to verify their O-GlcNAc modify cation.The immuneprecipitation and western blot assay showed that these two proteins were detected by CTD 110.6 and their modification lever was higher in the diabetic tissue than in the normal tissue.