| Progranulin(PGRN)is a highly glycosylated secreted 593 amino acid precursor protein with a signal peptide(N terminal 17 amino acides)and 7.5 cysteine-rich granulin peptide domains which were named as paragranulin、granulin G、F、B、A、C、D and E respectively.These different domains were isolated by short linker peptides.The mature PGRN can be proteolytically cleaved to generate seven～6kDa granulin/Grn or epithelin/Epi peptides,and GmA,GrnB,GrnC,GrnD and GrnE peptide fragments of PGRN corresponding to the individual Grn/Epi domains have been isolated from human body.Though both PGRN and the constituent Grn peptides have biological activities,now,most research was focused on the functional properties of the intact precursor PGRN.PGRN as a multifunctional growth factor widely expressed in a variety of tissues is involved in many important physiological events such as early embryogenesis,wound repair and inflammatory.Furthermore,PGRN plays an important role in the tumorigenesis,which is involved in the cell division,invasion,and angiogenesis.Recent studies indicated that PGRN haploinsufficiency resulted from a mutation in PGRN gene is a major reason for leading to the development of frontotemporal lobar degeneration with ubiquitin-positive inclusions(FTLD-U),which was therefore paid a great attention by scientists.There is growing evidence in vitro and in vivo that PGRN and GrnE have neurotrophic effects and function as a neuronal survival factor,but the mechanism of FTLD-U resulted from PGRN mution is not very clear.The aim of this project was to study in vitro the effect of mutated PGRN on the biological function of PGRN by constructing a series of PGRN mutants using molecular biology methods and screen the proteins interacting with PGRN using PGRN as bait by yeast two-hybrid system.All the findings will lay a basis for elucidating the biological functions of PGRN and its molecular mechanisms in FTLD-U.In this study,firstly,PGRN-6His fusion protein was purified by eukaryotic expression system,and then used as an antigen to prepare McAbs against PGRN which could help us obtain domain-specific anti-PGRN McAbs.These antibodies will be an essential tool for investigating the the biological functions of PGRN and Grns and their roles in central nervous system(CNS).Next,a series of PGRN mutants including PGRN missense mutations and PGRN N-glycosylation mutations were constructed and then we analyzed the effect of these mutations on the expression and function of PGRN.Finally,PGRN was used as bait protein to screen the interacting proteins with PGRN from a human leukocyte cDNA library by yeast two-hybrid system followed by further analysis and validation.The contents of the research project are as follows:(1)Expression and purification of PGRN-6His and PGRN-Flag in eukaryotic expression system.First of all,human PGRN gene was cloned by molecular biology methods,then identified by restriction enzyme digestion and sequencing.The PGRN gene was cloned into the adenovirus shuttle vector pAd5-El-CMV-6His and pAd5-E1-CMV-Flag respectively.By homologous recombination in eukaryotic cells,the non-replicative adenovirus Ad5-PGRN-6His and Ad5-PGRN-Flag were prepared.The supernatant were harvested from Hela cells infected by the adenovirus for 72h,then concentrated by ammonium sulfate.Finally,the PGRN-6His and PGRN-Flag protein were purified from the concentrated proteins by Ni-NTA resin and anti-Flag immobilized affinity chromatography.(2)Preparation and identification of monoclonal antibodies against human PGRN.Splenocytes from the Balb/c mice immunized by human PGRN-6His fusion protein were fused with myeloma cells SP2/0 for producing hybridoma.Hybridoma cell lines secreting antibodies against human PGRN was determined by indirect ELISA using PGRN-Flag as coating antigen.The hybridoma cells of positive wells were cloned by limiting dilution in aminopterin-free selection medium and were used for producing antibody-rich fluid called ascites fluid by inoculating enlarged hybridoma cell lines into the peritoneal cavity of Balb/c mice.Seven strains of McAbs which were named as 2A5,4F10,5B7,6C1,6E2,3E7 and 8H9 specific for human PGRN were obtained and characterized using Western blot and immunocytochemistry(ICH).The results showed that the particular domain recognized by 5B7,6C1 and 3E7 was GrnB,the particular domain recognized by 4F10 was GrnA,and that recognized by 2A5,6E2 and 8H9 was GrnC.(3)Construct of PGRN single-point mutants and its function study.Site-directed mutagenesis was used to generate 10 PGRN single-point mutations which contained the five single N-glycosylation site mutations(N118Q,N236Q,N265Q,N368Q,and N530Q),four missense mutations in the mature PGRN(C139R,P248L,R432C,and S258N)and one mutation in the signal peptide(A9D).All the PGRN mutants cDNA were directionally cloned into pAd5-E1-CMV-MCS-TAA and pAd5-E1-CMV-MCS-Flag respectively,then the plasmids were transfected into HEK 293 cells.The expression of PGRN mutants were analyzed by RT-PCR and Western blotting respectively.Moreover,the effects of PGRN mutants on the cell proliferation of SH-SY5Y were analyzed.The results indicated that PGRN with single-point mutations did not show decreased expression of PGRN mRNA and the expression and secretion level of PGRN protein were not significantly affected except A9D with a mutation in signal peptide.Furthermore,the PGRN mutants have little effect on the SH-SY5Y cell proliferation.(4)Screening of PGRN interacting proteins using the yeast two-hybrid system.The pGBKT7/PGRN containing the bait was constructed.The bait Gal4-BD-PGRN was then tested for its autoactivation and toxicity to ensure that it does not activate the reporter genes HIS3 and ADE and has no toxicity to AH 109.Then,the pGBKT7/PGRN used as a bait to screen PGRN interacting proteins from human leukocyte cDNA library using the yeast two-hybrid system.Through sequencing and an intial identification of screened genes,a novel nuclear protein of unknown function FAM76B was obtained(5)Validation of the interaction between PGRN and FAM76B.Full length cDNA of FAM76B was cloned by PCR from human cDNA library followed by the construction of the prey vector pGADT7/FAM76B.Then the interaction of FAM76B with PGRN was tested with the yeast two-hybrid system on a one-to-one basis.Next,the interaction between FAM76B and PGRN was strengthened by the co-immunoprecipitated in vivo and GST pull-down assay in vitro.Further,the precise domains of PGRN interacting with FAM76B were analyzed by constructing the deletions of PGRN and FAM76B followed testing with yeast two-hybrid system.Finally,the colocalization of PGRN and FAM76B in CHO cells was examined by using confocal microscopy.The results showed that PGRN has a direct interaction with FAM76B.The precise domain of PGRN interacting with FAM76B resides within GrnB and GmC,and the precise site of FAM76B interacting with PGRN resides within the region containing the His-repeat tract.Confocal laser microscopy showed that co-expression of GrnBACD with FAM76B in the CHO cells results in a good amount of GrnBACD translocation from cytoplasm into the nuclei,and was colocalized with FAM76B there.In summary,some results were obtained from the the project as follows:(1)monoclonal antibodies against the particular domains GmB,GrnA and GrnC of PGRN were prepared successfully;(2)a series of PGRN single-point mutants were constructed,then the effect of these mutants on the expression of PGRN and the cell proliferation of SH-SY5Y was analyzed;(3)a novel PGRN interacting protein FAM76B with a unknown function of nuclear protein was screened using the yeast two-hybrid system;(4)the direct interaction between FAM76B and PGRN was validated by the co-immunoprecipitated,GST pull-down assay and confocal laser microscopy;(5)by using gene deletion technique and the yeast two-hybrid system,the precise domain of PGRN interacting with FAM76B was found to reside within GrnB and GrnC,and the precise site of FAM76B interacting with PGRN was found to be present in the region containing the His-repeat tract.All the findings above will lay a basis for the elucidation of PGRN biological functions and its molecular mechanisms in FTLD-U. |
