Clarification Of The Interactions Among Sheep TMEM190,IZUMO1 And PDIA3

The fertilization of mammals would undergo four processes:sperm capacitation,binding to the zona pellucida,acrosome reaction,and fusion with egg,in which the final membrane fusion of sperm with egg is the key step including a large number of proteins on the membranes of both sperm and egg.So far,the most important protein interaction during the fusion of sperm and egg membrane is the weak binding between two sperm-egg fusion proteins,the IZUMO1 on the sperm and the JUNO on the egg.But the association between IZUMOl and JUNO is not stong enough to bring the sperm membrane closly to the egg membrane for fusion,rather it may be of a recognition.We speculated that there should be other interactions among sperm proteins and egg proteins,or between the surface proteins of the sperm and their ligant proteins on the egg.In this study,we used Co-immunoprecipitation and Yeast two-hybrid technique to address the interaction among three sperm proteins,IZUMO1,PDIA3 and TMEM190 of sheep.At first we cloned the sheep IZUMO1,PDIA3 and TMEM190 cDNA,of which the 779 bp cDNA fragment of sheep TMEM190 including 543 bp open reading frame was newly amplified by RT-PCR and was submitted to GenBank with the accession number:KY914491.The prokaryotic expression vectors were constructed by ligating the cDNAs of these three cDNAs with pGEX-4T-1,and were transformed into E.coli to express the TMEM190-GST(43.9 kDa),IZUMO1-GST(60.2 kDa)and PDIA3-GST(80.6 kDa)fusion proteins.Rabbit anti-sheep IZUMO1 antiserum was obtained by immunizing rabbits with purified IZUMOI-GST protein as antigen.Mouse anti-sheep PDIA3 antiserum was obtained by immunizing mice with purified PDIA3-GST protein as antigen.Western Blot analysis showed that both antisera could recognize GST and GST fusion proteins.For the Co-immunoprecipitation assay,we used plasmids pCMV-HA-C and pCMV-Flag-C to construct eukaryotic expression vectors:pCMV-IZUMOl-HA,pCMV-IZUMO1-Flag,pCMV-PDIA3-HA,pCMV-PDIA3-Flag,pCMV-TMEM190-HA and pCMV-TMEM 190-Flag.Then pCMV-IZUMO1-HA and pCMV-PDIA3-Flag,pCMV-TMEM190-HA and pCMV-PDIA3-Flag,pCMV-TMEM 190-HA and pCMV-IZUMO1-Flag were co-transfected into 293T cells,respectively.The rabbit anti-HA polyclonal antibody as IP antibody,the interaction between IZUMO1-HA and PDIA3-Flag,TMEM 190-HA and PDIA3-Flag was detected by mouse anti-sheep PDIA3 antiserum,and the expected coprecipitated protein bands appeared.The interaction between TMEM 190-HA and IZUMO1-Flag was detected by rabbit anti-sheep IZUMO1 antiserum,and no expected coprecipitated protein bands appeared.For Yeast two-hybrid experiments,we used pGAD-T7 and pGBK-T7 to construct Yeast two-hybrid vectors:pGAD-IZUMO1,pGBK-IZUMO1,pGAD-PDIA3,pGBK-PDIA3,pGAD-TMEM190 and pGBK-TMEM190.All recombinant pGAD vectors were transformed into Y2H Gold yeast strian while those recombinant pGBK vector transformation into Y187 strian.IZUMO1(Y187)and PDIA3(Y2H Gold),IZUMO1(Y2H Gold)and TMEM190(Y187),PDIA3(Y2H Gold)and TMEM190(Y187)hybridized,respectively.The IZUMO1(Y187)-PDIA3(Y2H Gold)and PDIA3(Y2H Gold)-TMEM190(Y187)hybrids could grow well on SD/-Trp-Leu two deficient plate and on SD/-Ade-His-Leu-Trp four deficient plates with lot of blue colonies,wheras IZUMO1(Y2H Gold)-TMEM190(Y187)hybrid plate did not grow colonies.The results indicated that IZUMO1 and PDIA3,PDIA3 and TMEM190 can interact with each other,but IZUMO1 and TMEM190 can not.Taken together,our results from both Co-immunoprecipitation and Yeast two-hybrid experiments suggested that there be interactions between IZUMO1 and PDIA3,PDIA3 and TMEM190,but not between IZUMO1 and TMEM190.