Structural Characterization of Native Mouse Zona Pellucida Proteins Using Mass Spectrometry

Abstract

The zona pellucida is an extracellular matrix consisting of three glycoproteins that surrounds mammalian eggs and mediates fertilization. The primary structures of mouse ZP1, ZP2, and ZP3 have been deduced from cDNA. Each has a predicted signal peptide and a transmembrane domain from which an ectodomain must be released. All three zona proteins undergo extensive coand post-translational modifications important for secretion and assembly of the zona matrix. In this report, native zonae pellucidae were isolated and structural features of individual zona proteins within the mixture were determined by high resolution electrospray mass spectrometry. Complete coverage of the primary structure of native ZP3, 96% of ZP2, and 56% of ZP1, the least abundant zona protein, was obtained. Partial disulfide bond assignments were made for each zona protein, and the size of the processed, native protein was determined. The N termini of ZP1 and ZP3, but not ZP2, were blocked by cyclization of glutamine to pyroglutamate. The C termini of ZP1, ZP2, and ZP3 lie upstream of a dibasic motif, which is part of, but distinct from, a proprotein convertase cleavage site. The zona proteins are highly glycosylated and 4/4 potential N-linkage sites on ZP1, 6/6 on ZP2, and 5/6 on ZP3 are occupied. Potential O-linked carbohydrate sites are more ubiquitous, but less utilized.The zona pellucida is an extracellular matrix surrounding mammalian eggs that functions in taxon-specific gamete binding, provides a post-fertilization block to polyspermy, and protects the developing pre-implantation embryo (1-3). The mouse zona pellucida (ZP) 1 is composed of three major glycoproteins (ZP1, ZP2, and ZP3) that are synthesized and secreted by oocytes during a 2-3 week growth period (4). The primary structures of ZP1 (623 amino acids), ZP2 (713 amino acids), and ZP3 (424 amino acids) have been deduced from cDNA (5-7). Each glycoprotein has a signal peptide directing it into a secretory pathway, a ϳ260 amino acid zona domain containing 8 conserved cysteine residues, and a transmembrane domain near the C terminus followed by a short cytoplasmic tail (8). The zona domain has been observed in multiple proteins (9) and has been implicated in the polymerization of extracellular matrices (10).During oocyte growth, ZP1, ZP2, and ZP3 traffick through the growing oocyte, and their ectodomains are released from a transmembrane domain at the surface of the cell (11, 12). A conserved hydrophobic patch upstream of the transmembrane domain is required for progression to the cell surface 2 and a consensus cleavage site (RX(K/R)R2) for the proprotein convertase furin is present upstream of the transmembrane domain. Although this site has been implicated in the release of the zona ectodomain (13-15), mutations (RNRR3 ANAA, or RNRR3 ANGE), do not prevent incorporation of reporter-ZP3 proteins into the zona pellucida in growing oocytes (12, 16) or transgenic mice (12) and secretion of recombinant human ZP3 with a similar mutation (RNRR3 ANAA) is not prevented (17).The three zo...

A duplicated motif controls assembly of zona pellucida domain proteins

“…The only invariant residues within the ZP domain are eight conserved C residues (2, 4); apparently, the domain consists of two halves, one containing the first four C residues and the other containing the remaining C residues (7,8). This finding is supported by the fact that the sequence between the two groups of C residues can be glycosylated (7,8) and is susceptible to protease digestion (34), as expected for a solvent-exposed polypeptide fragment linking independently folded regions. Moreover, PLAC1 (35) and OOSP1 (36) are proteins whose N-terminal sequences share homology with the first half of the ZP3 ZP domain, including C residues 1-4 (Fig.…”

“…1 A). Because the other 12 C residues of ZP3 are present as intramolecular disulfides in the extracellular portion of the protein (7,8), and because cytoplasmatic C residues can be important for assembly of transmembrane protein complexes (25, 26), we asked whether mutation of the C residue to an S residue would affect secretion and assembly of mouse ZP3. The mutant C413S protein was secreted and assembled to the same extent as wild-type ZP3; also, its secretion was unaffected in transfected cell lines (data not shown).…”

“…Cleavage of the C-terminal portion of ZP domain protein precursors is required for secretion and assembly into extracellular structures (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). To determine whether truncation of ZP3 before its TMD affected proteolytic processing, a truncated construct with a Cterminal FLAG-tag was produced (ZP3-373-FLAG).…”

“…1A) that are replaced by a glycosyl phosphatidylinositol anchor in some ZP domain proteins. During secretion, but before incorporation into the ZP, a furin-like enzyme excises the propeptide from ZP precursors by cleavage at the CFCS (5)(6)(7)(8). C-terminal processing of precursors also occurs for ZP homologues from fish to birds (9)(10)(11), as well as for other ZP domain proteins (12)(13)(14).…”

Established Preblastocyst- and Blastocyst-Derived ES Cell Lines Have Highly Similar Gene Expression Profiles, Despite Their Differing Requirements for Derivation Culture Conditions

“…The zona pellucidae is an extracellular matrix surrounding mammalian eggs that blocks polyspermy and protects the developing preimplantation embryo (Boja et al, 2003). It has been reported that early implantation stage embryos from which the zona pellucida was removed developed into blastocysts by in vitro culture without feeders (Mintz, 1962).…”