Conserved furin cleavage site not essential for secretion and integration of ZP3 into the extracellular egg coat of transgenic mice.
The extracellular zona pellucida surrounding mammalian eggs is formed by interactions of the ZP1, ZP2, and ZP3 glycoproteins. Female mice lacking ZP2 or ZP3 do not form a stable zona matrix and are sterile. The three zona proteins are synthesized in growing oocytes and secreted prior to incorporation into the zona pellucida. A well-conserved furin site upstream of a transmembrane domain near the carboxyl terminus of each has been implicated in the release of the zona ectodomains from oocytes. However, mutation of the furin site (RNRR --> ANAA) does not affect the intracellular trafficking or secretion of an enhanced green fluorescent protein (EGFP)-ZP3 fusion protein in heterologous somatic cells. After transient expression in growing oocytes, normal EGFP-ZP3 and mutant EGFP-ZP3 associate with the inner aspect of the zona pellucida, which is distinct from the plasma membrane. These in vitro results are confirmed in transgenic mice expressing EGFP-ZP3 with or without the mutant furin site. In each case, EGFP-ZP3 is incorporated throughout the width of the zona pellucida and the transgenic mice are fertile. These results indicate that the zona matrix accrues from the inside out and, unexpectedly, suggest that cleavage at the furin site is not required for formation of the extracellular zona pellucida surrounding mouse eggs.
Zhao
Ming
M
Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-8028, USA.
Gold
Lyn
L
Ginsberg
Ann M
AM
Liang
Li-Fang
LF
Dean
Jurrien
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D017124:Conserved Sequence; D004527:Egg Proteins / Q000276:immunology / Q000378:metabolism*; D005260:Female; D045683:Furin; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000166:cytology / Q000378:metabolism*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011956:Receptors, Cell Surface; D013381:Subtilisins / Q000378:metabolism*; D015044:Zona Pellucida / Q000737:chemistry / Q000378:metabolism*; D000072216:Zona Pellucida Glycoproteins
D004527:Egg Proteins; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D000072216:Zona Pellucida Glycoproteins; C000606519:Zp1 protein, mouse; C000606520:Zp2 protein, mouse; C000606521:Zp3 protein, mouse; D049452:Green Fluorescent Proteins; D013381:Subtilisins; D045683:Furin