Role And Mechanism Of Bone Morphogenetic Protein 5(BMP5) N-Glycosylation Modification In Trophoblast Cell Migration And Invasion

Background and Objective:Preeclampsia(PE)is a common complication of pregnancy with a prevalence of 2%-8%,posing a serious threat to maternal and fetal life and health.It is characterized by the new onset of hypertension and proteinuria in pregnant women after 20 weeks of gestation,which may be followed by systemic multi-organ system dysfunction,such as headaches,visual disturbances,pulmonary edema,liver and kidney abnormalities,thrombocytopenia or coagulation disorders,fetal growth restriction,and stillbirth.Studies have shown that impaired migration and invasion of trophoblast cells are important factors contributing to preeclampsia.During embryonic implantation,impaired trophoblast migration and invasion cause placental ischemia and hypoxia,and the placenta releases pro-inflammatory cytokines,exosomes,and anti-angiogenic agents into the maternal circulation,which in turn leads to preeclampsia.Currently,the mechanisms regulating the migration and invasion capacity of trophoblast cells are not fully elucidated.Protein glycosylation is a common post-translational modification process of proteins in living organisms,and there are two main forms: N-glycosylation and O-glycosylation.N-glycosylation modifications are present in many proteins in eukaryotes,and protein Nglycosylation modifications have important roles in protein structure and biological functions.Studies have shown that protein N-glycosylation modifications are associated with a variety of reproductive processes,such as fertilization,embryonic development,and placental formation.Bone morphogenetic protein 5(BMP5)is a secreted glycoprotein containing four potential N-glycosylation modification sites,has a molecular weight of 52 k Da,and is a member of the transforming growth factor β(TGF-β)superfamily.BMP5 plays an important role in many biological processes,such as regulating osteogenesis,cell proliferation,senescence,migration,and invasion.However,the effect of the N-glycosylation modification of the BMP5 protein on the migration and invasion of trophoblast cells and its mechanism of action have not been reported.In this study,we propose to investigate the effects of BMP5 N-glycosylation modification on trophoblast migration and invasion and its molecular mechanism by constructing BMP5 overexpression(BMP5 cDNA)and BMP5 mutant plasmids(BMP5 Mutant),aiming to provide a new theoretical basis for the pathogenesis and treatment of preeclampsia.Methods:1.The expression of N-glycans in normal and preeclamptic placental tissues was detected by Lectin fluorescent staining and Lectin blot.2.The GEO database(GSE114691)was used to screen for differentially expressed genes in normal and preeclamptic placental tissues,and the mRNA and protein expression levels of BMP5 in normal and preeclamptic placental tissues were detected by Immunofluorescent staining,Real-time PCR,and Western blot.3.BMP5 N-glycosylation site was predicted by the Net NG1 yc 1.0 website;BMP5 protein sequence was found and BMP5 overexpression(BMP5 cDNA)and BMP5 mutant plasmids(BMP5 Mutant)were constructed according to the Uni Prot website;Real-time PCR and Western blot were used to detect the levels of BMP5 in trophoblast HTR-8/SVneo cells and supernatants after transfection with BMP5 cDNA and BMP5 mutant.4.The effects of transfected BMP5 overexpression(BMP5 cDNA)and BMP5 mutant plasmids(BMP5 Mutant)on trophoblast HTR-8/SVneo cell migration,invasion,and EMT were detected by Western blot,Transwell,Wound healing,and Cell morphology assays.5.The effect of transfected BMP5 overexpression(BMP5 cDNA)and BMP5 mutant plasmids(BMP5 Mutant)on the angiogenesis of trophoblast HTR-8/SVneo cells was detected by Western blot and Tube formation assays.6.The effects of transfected BMP5 overexpression(BMP5 cDNA)and BMP5 mutant plasmids(BMP5 Mutant)on the proliferation of trophoblast HTR-8/SVneo cells were detected by Western blot,Ed U,and CCK-8 assays.7.The activation of signaling pathways mediated by trophoblast HTR-8/SVneo cells transfected with BMP5 overexpression(BMP5 cDNA)and BMP5 mutant plasmids(BMP5 Mutant)was detected by the KOBAS 3.0 website,the STRING website,Western blot,and Cell immunofluorescence.Results:1.The results of Lectin fluorescence staining and Lectin blot experiments showed that the expression levels of N-glycans were significantly reduced in preeclamptic placental tissues compared with normal placental tissues.2.The results of the GEO database(GSE114691),Immunofluorescence staining,Realtime PCR,and Western blot showed that the expression level of BMP5 was significantly reduced in preeclamptic placental tissues compared with normal placental tissues.3.Predictions from Net NG1 yc 1.0 showed that BMP5 has four potential N-glycosylation sites;transfection of BMP5 overexpression(BMP5 cDNA)and BMP5 mutant plasmids(BMP5 Mutant)into trophoblast HTR-8/SVneo cells,Real-time PCR,and western blot results showed that both plasmids increased the expression of BMP5 in HTR-8/SVneo cells and supernatant,and the apparent molecular weight of BMP5 was significantly reduced in the BMP5 Mutant transfected group.4.BMP5 cDNA and BMP5 Mutant were transfected into trophoblast HTR-8/SVneo cells,and the results of Western blot,Transwell,and Wound healing experiments showed that BMP5 N-glycosylation modification promoted the migration,invasion,and EMT of trophoblast HTR-8/SVneo cells;cells in the BMP5 cDNA transfected group became elongated mesenchymal-like cells,while cell morphology in the BMP5 Mutant group did not change significantly compared with the Vector group.5.BMP5 overexpression(BMP5 cDNA)and BMP5 mutant plasmids(BMP5 Mutant)were transfected into trophoblast HTR-8/SVneo cells,and Western blot and Tube formation assays showed that BMP5 N-glycosylation modification promoted angiogenesis in trophoblast HTR-8/SVneo cells.6.BMP5 overexpression(BMP5 cDNA)and BMP5 mutant plasmids(BMP5 Mutant)were transfected into trophoblast HTR-8/SVneo cells,and Western blot,Ed U,and CCK-8 assays showed that BMP5 N-glycosylation modification promoted the proliferation of trophoblast HTR-8/SVneo cells.7.KEGG pathway enrichment analysis was performed by the KOBAS 3.0 website using differential genes based on GSE114691;the STRING website was used to predict proteins that interact with BMP5;Western blot and cellular immunofluorescence were used to detect the expression of molecules downstream of BMP5;and the results showed that BMP5 N-glycosylation modification activates the BMP5/SMAD5 signaling pathway.Conclusion:1.Reduced expression of N-glycans and BMP5 in preeclamptic placental tissues.2.BMP5 N-glycosylation modification promotes implantation functions such as migration and invasion of trophoblast HTR-8/SVneo cells through activation of the BMP5/SMAD5 signaling pathway.