title: ePAD, an oocyte and early embryo-abundant peptidylarginine deiminase-like protein that localizes to egg cytoplasmic sheets.
abstract: Selected for its high relative abundance, a protein spot of MW approximately 75 kDa, pI 5.5 was cored from a Coomassie-stained two-dimensional gel of proteins from 2850 zona-free metaphase II mouse eggs and analyzed by tandem mass spectrometry (TMS), and novel microsequences were identified that indicated a previously uncharacterized egg protein. A 2.4-kb cDNA was then amplified from a mouse ovarian adapter-ligated cDNA library by RACE-PCR, and a unique 2043-bp open reading frame was defined encoding a 681-amino-acid protein. Comparison of the deduced amino acid sequence with the nonredundant database demonstrated that the protein was approximately 40% identical to the calcium-dependent peptidylarginine deiminase (PAD) enzyme family. Northern blotting, RT-PCR, and in situ hybridization analyses indicated that the protein was abundantly expressed in the ovary, weakly expressed in the testis, and absent from other tissues. Based on the homology with PADs and its oocyte-abundant expression pattern, the protein was designated ePAD, for egg and embryo-abundant peptidylarginine deiminase-like protein. Anti-recombinant ePAD monospecific antibodies localized the molecule to the cytoplasm of oocytes in primordial, primary, secondary, and Graafian follicles in ovarian sections, while no other ovarian cell type was stained. ePAD was also expressed in the immature oocyte, mature egg, and through the blastocyst stage of embryonic development, where expression levels began to decrease. Immunoelectron microscopy localized ePAD to egg cytoplasmic sheets, a unique keratin-containing intermediate filament structure found only in mammalian eggs and in early embryos, and known to undergo reorganization at critical stages of development. Previous reports that PAD-mediated deimination of epithelial cell keratin results in cytoskeletal remodeling suggest a possible role for ePAD in cytoskeletal reorganization in the egg and early embryo.
authors.affiliation: Department of Cell Biology and Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Paul W
authors.initials: PW
authors.lastname: Wright
authors.forename: Laura C
authors.initials: LC
authors.lastname: Bolling
authors.forename: Meredith E
authors.initials: ME
authors.lastname: Calvert
authors.forename: Olga F
authors.initials: OF
authors.lastname: Sarmento
authors.forename: Elizabeth V
authors.initials: EV
authors.lastname: Berkeley
authors.forename: Margaret C
authors.initials: MC
authors.lastname: Shea
authors.forename: Zhonglin
authors.initials: Z
authors.lastname: Hao
authors.forename: Friederike C
authors.initials: FC
authors.lastname: Jayes
authors.forename: Leigh Ann
authors.initials: LA
authors.lastname: Bush
authors.forename: Jagathpala
authors.initials: J
authors.lastname: Shetty
authors.forename: Amy N
authors.initials: AN
authors.lastname: Shore
authors.forename: Prabhakara P
authors.initials: PP
authors.lastname: Reddi
authors.forename: Kenneth S
authors.initials: KS
authors.lastname: Tung
authors.forename: Eileen
authors.initials: E
authors.lastname: Samy
authors.forename: Margaretta M
authors.initials: MM
authors.lastname: Allietta
authors.forename: Nicholas E
authors.initials: NE
authors.lastname: Sherman
authors.forename: John C
authors.initials: JC
authors.lastname: Herr
authors.forename: Scott A
authors.initials: SA
authors.lastname: Coonrod
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D002970:Cleavage Stage, Ovum / Q000201:enzymology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D018507:Gene Expression Regulation, Developmental; D006867:Hydrolases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D017403:In Situ Hybridization; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009865:Oocytes / Q000201:enzymology* / Q000648:ultrastructure; D010053:Ovary / Q000201:enzymology; D011247:Pregnancy; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012995:Solubility
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; D006867:Hydrolases; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; C552151:peptidylarginine deiminase 4, mouse