| Ⅰ.ObjectiveThe incidence of pregnancy failure is consistently increased.It is estimated that 23 million miscarriages occur every year worldwide,and the population prevalence of women who have recurrent pregnancy loss is up to 2.5%,with high probability of infertility.One of the major reasons leading to infertility is embryo dysfunction.Although the molecular causes of pregnancy loss,such as genetic abnormality and pregnancy related gene expression unbalance have been reported,there still is an urgent need to better understand the mechanisms for the embryonic pathogenesis of miscarriage patients,and find the new biomarkers for pregnancy evaluation,diagnosis and therapy.Trophoblast is a thin layer of cells that helps a mature embryo to attach to the wall of the uterus.Trophoblast directly initiates the apposition,adhesion and attachment of embryo to the receptive uterus,and drives embryo to penetrate into deeper uterine tissue.It also transforms to vascularization which sets up the communication with maternal blood vessels.Furthermore,trophoblast develops into the functional placenta which supplies nutrients and mediates the gas exchange at fetal-maternal interface.Thus,trophoblast steers successful implantation,and dysfunction of trophoblast hampers embryo implantation,angiogenesis and placentation,even lead to pregnancy loss and infertility.However,the molecular mechanism associated with trophoblast invasion is not fully clear.Protein glycosylation is one of the important posttranslational modifications.Glycans carried by a glycoprotein molecule not only affect the biochemical properties of the molecule,but also regulate a myriad of critical physiological and pathological processes,including embryogenesis and implantation,immune response,host-pathogen recognition and tumorigenesis.Protein fucosylation is an important type of glycosylation,and generally serves as the final step in the biosynthesis of sugar chains.It includes two major forms:N-fucosylation and O-fucosylation.Glycotransferases are responsible for catalyzing the biosynthesis of protein glycosylation.Fucosyltransferases(FUTs)are the key enzymes to transfer the L-Fucose(GDP-Fuc)to the sugar acceptor substrates,yielding fucosylated glycans.Thirteen FUTs(N-FUTs and O-FUTs)have been identified based on the linkage mode of the sugar chain with the peptide.N-FUTs are classified into α1,2-,α1,3/4-and α1,6FUTs which are responsible for the corresponding α1,2-,α1,3-/4-and α1,6-fucosylation epitopes.Fucosyltransferase Ⅳ(FUT4)catalyzes the α1,3-fucosylation linkage of difucusylated Lewis Y(LeY,Fuc α1-2 Gal-α1-4[Fuc α1-3]GlcNAcβ1).Mesoderm-specific transcript(MEST),also named as PEG 1,belongs to the alpha/beta hydrolase superfamily.MEST is a single chain glycoprotein composed of 335 amino acids.As a maternal imprint gene,MEST is widely expressed throughout the embryonic developmental period.MEST plays a crucial role in the development of embryo and placenta,as well as fetal growth.Inappropriate MEST expression is linked to the increased probability of early spontaneous miscarriage and severe fetal defects,such as growth abnormality,low birth weight(LBW),or metabolic disorders in human.In addition,Mest-deficient mice exhibited embryonic and placental development disorders.MEST deficiency also causes postnatal growth inhibition,weight gain and multiple organ hypertrophy.Furthermore,decreased expression of MEST has been linked to cancerpathogenesis,such as choriocarcinoma,breast cancer,lung adenocarcinomas and lymphoblastoid,etc.MEST is an essential molecule in embryo development and maturation.The expression and regulation of MEST influence the trophoblast function.Although MEST has three molecular subforms produced by the alternative splicing,the full-length protein has only a N-glycosylation site at Asn163.Using Lectin array analysis,we have compared the trophoblast glycosylation state in miscarriage patients and early normal pregnancy control.The results showed that the 1,3-linkage fucose glycan,especially LeY,was significantly decreased on the trophoblast of miscarriage patients.Applying proteomics and translatomics,we identified MEST as one of the differentially expressed scaffold proteins of LeY.We further found that that the α1,3-fucosylation of MEST mediates its binding with eukaryotic initiation factor eIF4E2,and initiated the embryo implantation-related gene translation.This study provides a new insight of function and mechanism of protein fucosylation of trophoblast during normal embryo implantation.Ⅱ.Methods A.FUT4 catalyzes the synthesis of LeY oligosaccharides to promote the proliferation,migration and invasion of trophoblast cells.1.Lectin microarray analysis of the global glycosylation expression levels and differences in the villous tissues from early normal pregnancy women and miscarriage patients.2.The expression and localization of LTL(Lotus Tetragonolobus Lectin)in the villous tissue of 10 pairs from early pregnancy and miscarriage patients were analyzed by Lectin blot and immunohistofluorescence.To analyze the expression of fucosyltransferase(FUT3-7,9-11)gene that catalyzes the synthesis of α1,3-fucosylation in the villous tissue of 10 pairs from early pregnancy and miscarriage patients by qPCR.3.Western blot analysis of the protein expression of fucosyltransferase Ⅳ in the villous tissue of 10 pairs from early pregnancy and miscarriage patients.The expression and localization of LeY in the villus tissue of 10 pairs from early pregnancy and miscarriage patients were analyzed by Lectin blot and immunohistofluorescence.4.The protein expressions of LTL,LeY and FUT4 in serum of 5 pairs from early pregnancy and miscarriage patients were detected by Lectin blot and Western blot.5.Silencing FUT4 by specific siRNA significantly inhibited FUT4 expression at both mRNA and protein levels;whereas transfecting FUT4 cDNA partially reversed the inhibitory effects of FUT4 siRNA.The expression of LTL and LeY was detected by down-regulating and restoring FUT4 by Lectin blot analysis.6.The expression and localization of LeY and FUT4 after down-regulation and restoration of FUT4 by confocal immunofluorescent analysis.7.Detect the outgrowth ability of villous trophoblast cells after down-regulation and restoration of FUT4.8.Detect the proliferation ability of trophoblast cells after down-regulating and restoring FUT4 by flow cytometry,EdU and Western blot.The migration and invasion ability of trophoblast cells after down-regulation and restoration of FUT4 were detected by cell scratch assay,transewll,gelatin zymography and Western blot.B.LeY on MEST mediates the interaction of MEST and eIF4E2 to stimulate translation initiation and expression of embryo implantation related genes.1.Protein profiling and GEO database(GSE76862)analysis to identification of LeY scaffold glycoproteins.2.The immunocoprecipitation(IP)with anti-LeY antibody and immunoblotting of tissue samples further confirmed the MEST expression variations in the villous trophoblastic tissues from both NP and MP.Examined the alteration of LeY/α1,3-fucosylation LTL on MEST after MEST cDNA or MEST mut transfection.Examined the alteration of LeY/α1,3-fucosylation LTL on MEST after FUT4 cDNA or FUT4 siRNA transfection.3.The gene and protein expressions of MEST after transfection of MEST cDNA and MEST mut were analyzed by qPCR,Western blot and cellular immunofluorescence.4.The proliferation ability of embryonic trophoblast cells after transfection of MEST cDNA and MEST mut was analyzed by flow cytometry,EdU,and Western blot.The migration and invasion ability of embryonic trophoblast cells after transient transfection with MEST cDNA and MEST mut were analyzed by transwell,cell scratch,gelatin zymography and Western blot.5.Analysis of interacting proteins with MEST by protein profiling,CBB and bioinformatics.The binding of MEST to eIF4E2 was regulated by LeY of MEST through co-IP and confocal immunofluorescent analysis.6.Analysis of mRNA bound by eIF4E2 after transfection of FUT4 cDNA and FUT4 siRNA by RIP-seq.R language analysis of eIF4E2 binding position and functional classification of mRNA.7.The ability of eIF4E2 to recruit 4E-T and GIGYF2 after transfection of MEST cDNA or MEST mut and FUT4 cDNA or FUT4 siRNA was analyzed by co-IP.8.The efficiency of gene translation after transfection of FUT4 cDNA and FUT4 siRNA was analyzed by ribosome isolation experiments.The ribosome isolated samples were analyzed by qPCR to detect the content of seven genes.9.In embryos cultured in vitro,the gene and protein expression of FUT4 after down-regulation of FUT4 was detected by qPCR,Western blot and immunofluorescence.In cultured embryos,the expressions of LeY and LTL after down-regulation of FUT4 were detected by Lectin blot and immunofluorescence.Embryos were transfected in vitro with FUT4 siRNA and blocked with LeY antibody and injected through the uterine horn again to analyze the effects of FUT4 and LeY on embryo implantation.C.Epiregulin affects embryonic trophoblast syncytialization and autophagy activation by promoting α1,3-fucosylation.1.Three cases of villus tissue from early normal pregnancy women and miscarriage patients to detected the level of syncytization and autophagy activation by immunohistofluorescence staining,qPCR and Western blot.2.Western blot and MDC staining were used to detect the levels of trophoblast maturation and autophagy activation after treatment with Rapamycin and 3-MA.3.The gene and protein expressions of FUT4 were detected by qPCR,Western blot and cellular immunofluorescence in HTR-8/SVneo treated with different concentrations of epiregulin.Lectin blot was used to detect the expression of α1,3-fucosylation after epiregulin treatment.4.Western blot,immunofluorescence,MDC and Lysotracker were used to detect the effect of FUT4 on trophoblast syncytization and autophagy activation.5.Western blot,immunofluorescence,MDC and Lysotracker were used to detect the effects of epiregulin-recovered FUT4 siRNA on trophoblast syncytization and autophagy activation.Ⅲ.ResultsA.FUT4 catalyzed the synthesis of LeY oligosaccharides to promote the proliferation,migration and invasion of trophoblast cells.1.Decreased α1,3-fucosylation in the villous trophoblast and serum of miscarriage patients.2.FUT4 mainly regulated the synthesis of α1,3-fucosylation and LeY.3.Silencing FUT4 inhibited the expansion of embryonic trophoblast cells.4.Silencing FUT4 inhibited LeY biosynthesis,impaired the potential of trophoblast implantation.B.LeY on MEST mediates the interaction of MEST and eIF4E2 to stimulate translation initiation and expression of embryo implantation related genes.1.MEST mutation inhibited the proliferation,migration and invasion of embryonic trophoblast cells.2.LeY on MEST mediated the interaction of MEST and eIF4E2.3.LeY on MEST preferentially stimulated the translation initiation and expression of embryo implantation related genes.4.α1,3-fucosylation of MEST at Asn163 preferentially stimulated the translation and expression of embryo implantation related genes.5.Silencing FUT4 and anti-LeY antibody blockage inhibited embryo implantation in vivo.C.Epiregulin affects embryonic trophoblast syncytialization and autophagy activation by promoting α1,3-fucosylation.1.Epiregulin promoted the expression of FUT4 and α1,3-fucosylation.2.The FUT4 siRNA impaired trophoblast syncytialization and inhibited autophagy activation under FSK treatment.3.FUT4 siRNA could inhibit autophagy activation during trophoblast syncytialization,and FUT4 cDNA could activate autophagy activation.4.Epiregulin restored the inhibitory effect of FUT4 siRNA on trophoblast syncytialization and autophagy.IV.ConclusionsA.FUT4 catalyzes the synthesis of LeY oligosaccharides to promote the proliferation,migration and invasion of trophoblast cells.1.Decreased α1,3-fucosylation/FUT4/LeY in the villous trophoblast of miscarriage patients.2.FUT4 mainly regulated the synthesis of α1,3-fucosylation and LeY biosynthesis,promoting the potential of trophoblast implantation.B.LeY on MEST mediates the interaction of MEST and eIF4E2 to stimulate translation initiation and expression of embryo implantation related genes.1.MEST has α1,3--fucosylation and LeY oligosaccharide modification.MEST mutation inhibits the proliferation,migration and invasion of embryonic trophoblast cells.2.LeY on MEST preferentially stimulates the translation initiation and expression of embryo implantation related genes.3.Silencing FUT4 and anti-LeY antibody blockage inhibits embryo implantation in vivo.C.Epiregulin affects embryonic trophoblast syncytialization and autophagy activation by promoting α1,3-fucosylation.1.The trophoblast syncytialization and autophagy activation were reduced in miscarriage patients.2.Epiregulin promotes FUT4 and α1,3-fucosylation.3.Epiregulin promotes FUT4 expression to stimulate trophoblast syncytialization and autophagy activation. |
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