Wright Paul W, Bolling Laura C, Calvert Meredith E, Sarmento Olga F, Berkeley Elizabeth V, Shea Margaret C, Hao Zhonglin, Jayes Friederike C, Bush Leigh Ann, Shetty Jagathpala, Shore Amy N, Reddi Prabhakara P, Tung Kenneth S, Samy Eileen, Allietta Margaretta M, Sherman Nicholas E, Herr John C, Coonrod Scott A
Department of Cell Biology and Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VA 22908, USA.
Dev Biol. 2003 Apr 1;256(1):73-88. doi: 10.1016/s0012-1606(02)00126-4.
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.
从2850个无透明带中期II期小鼠卵母细胞的考马斯亮蓝染色二维蛋白质凝胶中选取了一个相对丰度较高、分子量约为75 kDa、等电点为5.5的蛋白质斑点进行切取，并用串联质谱法（TMS）进行分析，鉴定出了新的微序列，这些微序列表明这是一种先前未被表征的卵母细胞蛋白质。然后通过RACE-PCR从鼠卵巢衔接子连接的cDNA文库中扩增出一个2.4 kb的cDNA，并确定了一个独特的2043 bp开放阅读框，其编码一个681个氨基酸的蛋白质。将推导的氨基酸序列与非冗余数据库进行比较，结果表明该蛋白质与钙依赖性肽基精氨酸脱亚氨酶（PAD）酶家族约40%相同。Northern印迹、RT-PCR和原位杂交分析表明，该蛋白质在卵巢中大量表达，在睾丸中弱表达，在其他组织中未表达。基于与PADs的同源性及其在卵母细胞中丰富的表达模式，该蛋白质被命名为ePAD，即卵母细胞和胚胎丰富的肽基精氨酸脱亚氨酶样蛋白。抗重组ePAD单特异性抗体将该分子定位到卵巢切片中原始卵泡、初级卵泡、次级卵泡和格拉夫卵泡中卵母细胞的细胞质中，而其他卵巢细胞类型均未被染色。ePAD在未成熟卵母细胞、成熟卵母细胞以及胚胎发育的囊胚阶段也有表达，在囊胚阶段其表达水平开始下降。免疫电子显微镜将ePAD定位到卵母细胞胞质片层，这是一种仅在哺乳动物卵母细胞和早期胚胎中发现的独特的含角蛋白中间丝结构，已知在发育的关键阶段会发生重组。先前有报道称PAD介导的上皮细胞角蛋白脱亚氨基作用会导致细胞骨架重塑，这表明ePAD在卵母细胞和早期胚胎的细胞骨架重组中可能发挥作用。
