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ePAD, an oocyte and early embryo-abundant peptidylarginine deiminase-like protein that localizes to egg cytoplasmic
Paul W Wright1, Laura C Bolling, Meredith E Calvert
1Department of Cell Biology and Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VA 22908, USA.
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.
Insights
Researchers identified a novel egg protein, ePAD, abundant in mouse ovaries and eggs. This peptidylarginine deiminase-like protein may play a role in cytoskeletal reorganization during early development.
Area of Science:
- Reproductive Biology
- Molecular Biology
- Protein Biochemistry
Background:
- Mammalian eggs and early embryos possess unique cellular structures crucial for development.
- Intermediate filament networks, like keratin, are vital for cytoskeletal integrity and undergo dynamic remodeling.
- Peptidylarginine deiminases (PADs) are enzymes known to modify proteins, including keratin, potentially affecting cytoskeletal structure.
Purpose of the Study:
- To identify and characterize a novel protein highly expressed in mouse oocytes and early embryos.
- To determine the homology and expression pattern of this newly identified protein.
- To investigate the subcellular localization and potential function of the protein in the context of cytoskeletal elements.
Main Methods:
- Proteomic analysis of mouse eggs using two-dimensional gel electrophoresis and tandem mass spectrometry (TMS).
- Cloning of the corresponding cDNA using RACE-PCR and sequence analysis.
- Gene expression analysis via Northern blotting, RT-PCR, and in situ hybridization.
- Immunolocalization studies using anti-recombinant antibodies and immunoelectron microscopy.
Main Results:
- A novel 75 kDa protein, designated ePAD (egg and embryo-abundant peptidylarginine deiminase-like protein), was identified in mouse eggs.
- ePAD shares homology with calcium-dependent peptidylarginine deiminases (PADs) and is abundantly expressed in the ovary and weakly in the testis.
- ePAD localizes to the cytoplasm of oocytes and is present in egg cytoplasmic sheets, a keratin-containing structure, suggesting a role in cytoskeletal remodeling.
Conclusions:
- A novel PAD-like protein, ePAD, is specifically expressed in mouse oocytes and early embryos.
- ePAD's localization to egg cytoplasmic sheets suggests a role in the dynamic reorganization of the egg's cytoskeleton.
- Further research into ePAD's enzymatic activity and substrate interactions is warranted to elucidate its precise function in mammalian reproduction and development.
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