Molecular Cloning And Characterization Of IZUMO4and TSSK6and Their Interaction With Other Sperm-Egg Fusion Related Proteins In Cashmere Goat And Sheep

Binding and fusion of mammalian sperm with egg is a multistep process mediated by interactions among multiple proteins on egg and sperm, including Izumo family members, testis specific serine/threonine kinases (TSSKs) family member TSSK6, protein disulfide isomerase (PDI) family member PDIA3(ERp57). The mechanism underlying the protein interaction in the gamete fusion remains unknown. In the present study we cloned cDNA of Izumo4and Tssk6of cashmere goat and sheep, expressed them in E. coli BL21(DE3) and collected the recombinant HIS-Izumo4and HIS-TSSK6fusion proteins to generate anti-Izumo4and anti-TSSK6ascites polyclonal antibodies. With the antibodies we examined the expression pattern and localization of Izumo4and TSSK6in cashmere goat testes and sperm cells by immunohistochemistry and immunocytochemistry. We also identified the interactions among cashmere goat Izumo4, TSSK6, Izumo1and PDIA3by coimmuneprecipitation (Co-IP) and yeast two hybridization (Y2H).We designed the primers according to the conservative regions revealed by aligning multiple mammalian Izumo4and TSSK6cDNA sequences released in GenBank. The cloned cashmere goat and sheep Izumo4and Tssk6cDNA sequences, which are highly homologous with the orthologues in other mammals, contain ORF of465bp, encoding a putative peptide of154aa, and822bp, encoding a putative peptide of273aa, respectively, both proteins without transmembrane domain.We then expressed recombinant cashmere goat HIS-Izumo4and HIS-TSSK6fusion proteins in E. coli BL21(DE3) by inserting their ORFs into a prokaryotic expression vector pET-32a, respectively. The mouse anti-Izumo4and anti-TSSK6ascites polyclonal antibody, which could recognize Izumo4and TSSK6protein specifically in the total protein extract from goat testes and sperms, were generated and purified successfully by immunizing KM mouse with purified HIS-izumo4and HIS-TSSK6fusion proteins followed by celiacly injecting the mouse with SI80cells.Izumo4was detected to be expressed widely in testis sections, and localized under the outer membrane of intact sperm acrosome, while TSSK6was mainly detected in the meiotic spermatocytes and the round sperm cells, and localized in acrosome of the intact sperms, both disappeared after the acrosome reaction.To investigate the interaction of TSSK6with the key sperm-egg fusion protein Izumol and other important sperm-egg fusion related proteins, we constructed eukaryotic expression vectors pcDNA3carrying Izumol, Izumo4and PDIA3with the HA tag, and Izumo4, TSSK6with the HIS tag. These vectors were transiently co-transfected into the human293T cell line in various combinations to perform co-immunoprecipitation experiments. Meanwhile, we also constructed the yeast two-hybrid vectors:pGAD-Izumol/Izumo4/Tssk6and pGBK-Tssk6/Pdia3, and transfected them into yeast cells in four combinations for yeast hybrid screening. The results of thes experiments strongly suggested that stable binding interactions exist between Izumol and TSSK6; Izumo4and TSSK6; Izumo4and PDIA3; as well as between TSSK6and PDIA3in vivo.