Study On The Interaction Between PiT2 And FBLN5 In Mouse Placenta

PiT2 is type III sodium-phosphorus co-transporter 2 encoded by the SLC20A2,which has 12 transmembrane domains,including two PD domains and one intracellular domain(known as loop7 domain).PD domains play an important role in the transport of inorganic phosphorus,and loop7 domain can participate in the regulation of neural development.However,it is not clear that the functions of loop7 domain in other tissues.Previous studies have shown that placental calcification was the deposition of calcium phosphate minerals in placental,and PiT2 deficiency would lead to calcification in mouse placenta,but the exact mechanism of placental calcification remains unclear.In order to explore the mechanism of placenta calcification in PiT2 deficient mice,this study used yeast two hybrid system to screen proteins interacting with PiT2,which aimed to reveal the pathogenic mechanism of placenta calcification at the molecular level.In this study,the loop7 domain of PiT2 was selected as the bait protein,aiming to screen the cDNA libraries of mouse placenta from E8.5 to E18.5 by yeast two-hybrid.128 clones were initially screened,72 positive clones were obtained by rigorous screening of X-?-gal.Sequencing analysis and retransformating validation of positive clones showed that the loop7 domain of PiT2 may interact with fibulin protein 5(FBLN5),UMP-CMP kinase 1(CMPK1),galactose lectin 1(GAL1),and kinase A interaction protein 1(AKIP1).FBLN5 is an extracellular matrix glycoprotein 5 in the FBLN family,the researchers have found that FBLN5 could affect the production of intracellular reactive oxygen species by binding to integrin on the cell surface,and homologous protein FBLN3 could inhibit vascular calcification,so we speculated that PiT2 might affect the occurrence of placental calcification through its interaction with FBLN5.Therefore,we further studied interaction between PiT2 and FBLN5,and explore the role of this interaction in placental calcification.By the GST-pulldown,co-immunoprecipitation,immunofluorescence,Western blot and immunohistochemistry,we fo und direct interaction between PiT2 and FBLN5 in mouse placenta in vitro and in vivo.In summary,four PiT2 interacting proteins,including FBLN5,CMPK1,GAL1 and AKIP1 in mouse placenta,were discovered for the first time through the yeast two-hybrid system,then the interaction between PiT2 and FBLN5 was further confirmed in vitro and in vivo.The mechanism of interaction between PiT2 and FBLN5 in placental calcification was further discussed in this study,which may provide an important theoretical basis for studying the molecular mechanism of placental calcification.