Construction Of A Knockout Cell Library For Genes Involved In GPI Biosynthesis And Functional Analysis Of The Genes

Glycosylphosphatidylinositol(GPI)anchor modification is a posttranslational modification of proteins that has been conserved in eukaryotes.The core structure of GPI is conserved and consists of Et NP-6-Man-?1,2-Man-?1,6-Man-?1,4-Glc N-?1,6-PI(Et NP,ethanolamine-phosphate;Man,mannose;Glc N,glucosamine;PI,phosphatidylinositol)among eukaryotic species.GPI-anchored proteins(GPI-APs)are synthesized in the endoplasmic reticulum(ER),and finally expressed on the cell surface via the Golgi apparatus.After GPI is transferred to proteins,the lipid and glycan parts of GPI moieties are processed at the ER and the Golgi during GPI-APs transport.Some GPI-APs are cleaved at the GPI moiety by GPI-cleaving enzymes on the cell surface.There are at least 23 steps in which 33 genes(including 21 phosphatidylinositol-glycan(PIG)genes and 6 post-GPI attachment to proteins(PGAP)genes)are involved for correct GPI-APs biogenesis.The most important biosynthetic step of GPI-APs: the connection of GPI and protein is mediated by the GPI transamidase complex.It recognizes and cleaves the C-terminal GPI attachment signal of precursor proteins.Then,GPI is transferred to the newly exposed C-terminus of the proteins.GPI transamidase consists of five subunits: PIGK,GAA1,PIGT,PIGS and PIGU,and the absence of any subunit leads to the loss of activity.Although the genes responsible for the reaction in most of the steps in GPI-APs biosynthesis have been identified,it is still unclear how GPI biosynthesis is regulated.In order to explore the biosynthesis and related mechanisms of GPI-APs,the main results of this study are summarized as follows:(1)Establishment of GPI-KO cell library based on CRISPR-Cas9 technology: knock out32 genes involved in GPI biosynthesis in human embryonic kidney 293(HEK293)cells.Analyzing the expression of GPI-APs(CD55,CD59,prion)in the GPI-KO cell library,it was found that the deletion of some genes involved in the biosynthesis of GPI-APs would affect the cell surface expression of GPI-APs.(2)Analysis of the sensitivity of KO cells in the GPI-KO cell library to phosphatidylinositol phospholipase C(PI-PLC).PI-PLC hydrolyzes PI at the site between the phosphate and glycerol backbone.Via treatment with PI-PLC,GPI-APs on the cell surface are cleaved and released.Approximately 50% of prion proteins were cleaved by PI-PLC in WT cells,whereas in PGAP2-KO cells,85% of prion proteins on the cell surface showed resistance against PI-PLC,suggesting that prion proteins have inositol-acylated GPI anchors.The sensitivity of prion proteins to PI-PLC increased when PGAP1 was overexpressed,and at the same time,prion expression was significantly reduced in PGAP2-KO cells.This confirms that PI-PLC resistance of prion proteins is caused by inefficient inositol deacylation.In addition,through the verification of prion chimeric protein,the mature part of the prion protein determines unique PI-PLC resistance.(3)Analysis of the sensitivity of KO cells in the GPI-KO cell library to aerolysin(a kind of toxin binding with GPI-APs to target cells).The results indicate that the presence of Et NP on Man2 negatively affects the binding of aerolysin to GPI-APs.The slightly high sensitivity of PGAP4-and B3GALT4-deficient cells to aerolysin means that the glycan side chain modifications of GPI weaken aerolysin binding.(4)Using the KO cell lines of the five subunits of GPI transamidase in the GPI-KO cell library,analysis of functionally important residues by comparing conserved sequences among homologous proteins.In addition to catalytic active sites on PIGK(His164 and Cys206)and Cys residues that form an intermolecular disulfide bond between PIGK(Cys92)and PIGT(Cys182),we also found some other important residues for GPI transamidase activity,including GAA1(Asp250),PIGT(Glu429),and PIGU(Leu375,Phe376).(5)Exploration of the best purification method for analyzing the structure of GPI transamidase.In K562K(K562 PIGK-KO)cells,using the GDN(glyco-diosgenin)-purified GPI transamidase complex containing h PIGK-Strep II,we performed a preliminary cryo-EM on the GPI transamidase complex to evaluate the sample quality.Single particle analysis of the GPI transamidase complex showed this method can effectively purify high-purity GPI transamidase complex,and the sample quality of GDN-purified GPI transamidase,which would be suitable for structural studies.