title: Identification of distinctive interdomain interactions among ZP-N, ZP-C and other domains of zona pellucida glycoproteins underlying association of chicken egg-coat matrix.
abstract: The vertebrate egg coat, including mammalian zona pellucida, is an oocyte-specific extracellular matrix comprising two to six zona pellucida (ZP) glycoproteins. The egg coat plays important roles in fertilization, especially in species-specific interactions with sperm to induce the sperm acrosome reaction and to form the block to polyspermy. It is suggested that the physiological functions of the egg coat are mediated and/or regulated coordinately by peptide and carbohydrate moieties of the ZP glycoproteins that are spatially arranged in the egg coat, whereas a comprehensive understanding of the architecture of vertebrate egg-coat matrix remains elusive. Here, we deduced the orientations and/or distributions of chicken ZP glycoproteins, ZP1, ZP3 and ZPD, in the egg-coat matrix by confocal immunofluorescent microscopy, and in the ZP1-ZP3 complexes generated in vitro by co-immunoprecipitation assays. We further confirmed interdomain interactions of the ZP glycoproteins by far-Western blot analyses of the egg-coat proteins and pull-down assays of ZP1 in the serum, using recombinant domains of ZP glycoproteins as probes. Our results suggest that the ZP1 and ZP3 bind through their ZP-C domains to form the ZP1-ZP3 complexes and fibrils, which are assembled into bundles through interactions between the repeat domains of ZP1 to form the ZP1-ZP3 matrix, and that the ZPD molecules self-associate and bind to the ZP1-ZP3 matrix through its ZP-N and ZP-C domains to form the egg-coat matrix. Based on these results, we propose a tentative model for the architecture of the chicken egg-coat matrix that might be applicable to other vertebrate ones.
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agriculture, Meijo University, Nagoya 468-8502, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Okumura
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agriculture, Meijo University, Nagoya 468-8502, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Sato
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agriculture, Meijo University, Nagoya 468-8502, Japan.
authors.forename: Rio
authors.initials: R
authors.lastname: Sakuma
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agriculture, Meijo University, Nagoya 468-8502, Japan.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Fukushima
authors.affiliation: Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
authors.forename: Tsukasa
authors.initials: T
authors.lastname: Matsuda
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agriculture, Meijo University, Nagoya 468-8502, Japan.
authors.forename: Minoru
authors.initials: M
authors.lastname: Ujita
keywords: CBB, Coomassie Brilliant Blue; DIC, differential interference contrast; DTT, dithiothreitol; EGF, epidermal growth factor; EHP, external hydrophobic patch; Egg coat; Extracellular matrix; Fertilization; His6, hexahistidine; IHP, internal hydrophobic patch; Interdomain interaction; MBP, maltose binding protein; RT, room temperature; TGFR, transforming growth factor-β receptor; THP, Tamm–Horsfall protein; Trx, thioredoxin; ZP, zona pellucida; Zona pellucida