TAILIEUCHUNG - Báo cáo khoa học: Recombinant bovine zona pellucida glycoproteins ZP3 and ZP4 coexpressed in Sf9 cells form a sperm-binding active hetero-complex

The zona pellucida (ZP) is a transparent envelope that surrounds the mam-malian oocyte and mediates species-selective sperm–egg interactions. Por-cine and bovine ZPs are composed of the glycoproteins ZP2, ZP3, and ZP4. We previously established an expression system for porcine ZP glyco-proteins (ZPGs) using baculovirus in insect Sf9 cells. | ễFEBS Journal Recombinant bovine zona pellucida glycoproteins ZP3 and ZP4 coexpressed in Sf9 cells form a sperm-binding active hetero-complex Saeko Kanai1 Naoto Yonezawa1 Yuichiro Ishii1 Masaru Tanokura2 and Minoru Nakano1 1 Graduate Schoolof Science and Technology Chiba University Japan 2 Graduate Schoolof Agriculture and Life Science The University of Tokyo Japan Keywords baculovirus-Sf9 fertilization glycoprotein zona pellucida ZP domain Correspondence M. Nakano Graduate Schoolof Science Chiba University 1-33 Yayoi-cho Inage-ku Chiba 263-8522 Japan Fax 81 43 290 2874 Tel 81 43 290 2794 E-mail mnakano@ Received 18 April2007 revised 27 July 2007 accepted 24 August 2007 doi The zona pellucida ZP is a transparent envelope that surrounds the mammalian oocyte and mediates species-selective sperm-egg interactions. Porcine and bovine ZPs are composed of the glycoproteins ZP2 ZP3 and ZP4. We previously established an expression system for porcine ZP glycoproteins ZPGs using baculovirus in insect Sf9 cells. Here we established a similar method for expression of bovine ZPGs. The recombinant ZPGs were secreted into the medium and purified by metal-chelating column chromatography. A mixture of bovine recombinant ZP3 rZP3 and rZP4 coexpressed in Sf9 cells exhibited inhibitory activity for bovine sperm-ZP binding similar to that of a native bovine ZPG mixture whereas neither bovine rZP3 nor rZP4 inhibited binding. An immunoprecipitation assay revealed that the coexpressed rZP3 rZP4 formed a hetero-complex. We examined the functional domain structure of bovine rZP4 by constructing ZP4 mutants lacking the N-terminal domain or lacking both the N-termi-nal and trefoil domains. When either of these mutant proteins was coexpressed with bovine rZP3 the resulting mixtures exhibited inhibitory activity comparable to that of the bovine rZP3 rZP4 complex. Hetero-com-plexes of bovine rZP3 and porcine rZP4 or porcine rZP3 and bovine rZP4 .

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