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.

Peptidylarginine deiminase and protein citrullination in prion diseases

“…26 Finally, PAD6 is expressed only in male and female germ cells. 27 It participates in oocyte cytoskeletal sheet formation and female fertility, 28 is a critical factor for cytoplasmic lattice formation in growing oocytes, 29 and regulates microtubulemediated organelle positioning and movement. 30 believe that PAD isotypes have distinct substrate specificities, [45][46][47] which results in the prevalent citrullination of certain protein groups.…”

Peptidylarginine deiminase (PAD) 6 is essential for oocyte cytoskeletal sheet formation and female fertility

“…These modifications have a profound effect on protein stability and degradation and play a crucial role during development and cell differentiation (rev. in ) and is expressed uniquely in mammalian oocytes, sperm cells and early embryo and appears to be associated to the cytoskeletal sheets (Wright et al, 2003).…”

The subcortical maternal complex: multiple functions for one biological structure?

“…Despite a considerable effort by different research groups, the identification of additional members of the SCMC has not been straightforward. So far, the only valid candidate is protein PADI6, an oocyte-specific, 77-kDa peptidylarginine deiminase that is preferentially located in the cortex of eggs and preimplantation embryos [26]. It was identified as a potential SCMC component by tandem mass spectrometry after immunoprecipitation with anti-FLOPED antibodies [10].…”

“…Fig. 1 Schematic representation of the hypothesized structure of the SCMC and localization within the MII oocyte Interestingly, PADI6 was seen to localize to [26] and be essential for the formation of the oocyte cytoplasmic lattices (CPLs), a fibrillar matrix composed of a proteinaceous component and RNA [27,28]. The CLPs contain five to seven parallel fibers with repeating units of about 20 nm [29].…”