Oolemmal proteomics--identification of highly abundant heat shock proteins and molecular chaperones in the mature mouse egg and their localization on the plasma membrane.
Oolemmal proteomics--identification of highly abundant heat shock proteins and molecular chaperones in the mature mouse egg and their localization on the plasma membrane.
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DOI：
10.1186/1477-7827-1-27
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发表时间：
2003-02-14
期刊：
影响因子：
--
通讯作者：
Coonrod, Scott A
中科院分区：
文献类型：
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作者：
Calvert, Meredith E;Digilio, Laura C;Coonrod, Scott A
背景：成熟的小鼠卵子含有受精、向合子转录过渡以及胚胎发生起始阶段所需的全部母体蛋白质。其中许多蛋白质仍有待表征，因此在这项研究中，我们使用蛋白质组学方法鉴定了高度丰富的鸡蛋蛋白质，并发现其中一些蛋白质似乎也定位于鸡蛋表面。这些分子的表征将为了解受精和早期发育的细胞事件提供重要的见解。方法：为了鉴定一些更丰富的鸡蛋蛋白，在考马斯染色的二维 (2D) PAGE 凝胶上对全鸡蛋提取物进行解析。通过串联质谱 (TMS) 对几个高度丰富的蛋白质点进行核化和微测序，并确定为分子伴侣蛋白质。同时进行实验以使用 2D 亲和素印迹来鉴定卵膜蛋白。似乎通过生物素化进行表面标记的蛋白质斑点与最初的考马斯染色参考凝胶相关。令人惊讶的是，一些表面标记的蛋白质与之前鉴定的丰富的伴侣蛋白相对应。为了确认这些分子是否在鸡蛋的卵膜表面积累，我们使用 HSP70、HSP90、GRP94、GRP78、钙网蛋白和钙联蛋白的抗体对活的、无透明带的鸡蛋进行免疫荧光。结果：通过生物素化鉴定的推定表面标记蛋白包括分子伴侣 HSP70 (MW 70 KDa，pI 5.5)、HSP90a (MW 85 KDa，pI 4.9)、GRP94（MW 92 KDa，pI 4.7）、GRP78（MW 72 KDa，pI 5.0）、氧调节蛋白 150（ORP150；MW 111 KDa，pI 5.1）、钙网蛋白（MW 48 KDa，pI 4.3）、钙连接蛋白（MW 65） KDa，pI 4.5）和蛋白质二硫键异构酶（PDI；MW 57 KDa，pI 4.8）。免疫荧光结果显示，针对 HSP90、GRP94、GRP78 和钙网蛋白的抗体与油膜蛋白发生反应。我们无法通过这种方法确认 HSP70 或钙联蛋白的表面定位。结论：我们在此报告了小鼠卵蛋白质组中九个高度丰富的分子伴侣的鉴定。此外，我们提供的初步数据表明这些分子定位于成熟小鼠卵子的卵膜。
BACKGROUND: The mature mouse egg contains the full complement of maternal proteins required for fertilization, the transition to zygotic transcription, and the beginning stages of embryogenesis. Many of these proteins remain to be characterized, therefore in this study we have identified highly abundant egg proteins using a proteomic approach and found that several of these proteins also appear to localize to the egg surface. Characterization of such molecules will provide important insight into the cellular events of fertilization and early development.METHODS: In order to identify some of the more abundant egg proteins, whole egg extracts were resolved on coomassie-stained two-dimensional (2D) PAGE gels. Several highly abundant protein spots were cored and microsequenced by tandem mass spectrometry (TMS), and determined to be molecular chaperone proteins. Concurrent experiments were performed to identify oolemmal proteins using 2D avidin blotting. Proteins spots that appeared to be surface labeled by biotinylation were correlated with the initial coomassie-stained reference gel. Surprisingly, some of the surface labelled proteins corresponded to those abundant chaperone proteins previously identified. To confirm whether these molecules are accumulating at the oolemmal surface in eggs, we performed immunofluoresence on live, zona-free eggs using antibodies to HSP70, HSP90, GRP94, GRP78, calreticulin and calnexin.RESULTS: The putative surface-labeled proteins identified by biotinylation included the molecular chaperones HSP70 (MW 70 KDa, pI 5.5), HSP90a (MW 85 KDa, pI 4.9), GRP94 (MW 92 KDa, pI 4.7), GRP78 (MW 72 KDa, pI 5.0), Oxygen regulated protein 150 (ORP150; MW 111 KDa, pI 5.1), Calreticulin (MW 48 KDa, pI 4.3), Calnexin (MW 65 KDa, pI 4.5), and Protein disulfide isomerase (PDI; MW 57 KDa, pI 4.8). Immunofluoresence results showed that antibodies to HSP90, GRP94, GRP78 and calreticulin were reactive with oolemmal proteins. We were unable to confirm surface localization of HSP70 or calnexin by this method.CONCLUSIONS: We report here the identification of nine highly abundant molecular chaperones in the mouse egg proteome. In addition, we present preliminary data suggesting that these molecules localize to the oolemma of the mature mouse egg.