Nazarian Javad, Hathout Yetrib, Vertes Akos, Hoffman Eric P
Research Center for Genetic Medicine, Children's National Medical Center, Washington, DC, USA.
Department of Chemistry, George Washington University, Washington, DC, USA.
Proteomics. 2007 Feb;7(4):617-627. doi: 10.1002/pmic.200600686.
Torpedo californica is a species in class Chondrichthyes. Electric rays have evolved the electric organ, which is similar to the mammalian neuromuscular junction (NMJ). Here, we took a combined cDNA sequencing and proteomic approach to define the molecular constituents of the T. californica electric organ. For soluble proteins, 2-DE was used and 224 protein spots were mapped. Plasma membrane fractions were analyzed using the shotgun approach (LC-MS/MS). A Torpedo cDNA library was constructed and 607 cDNA clones were sequenced. Identification of electric organ proteins was done using cross-species comparisons, and a custom database was constructed from cDNA translations. We unambiguously identified 121 proteins and transcripts, 103 of which were novel additions to the existing databases of Torpedo fish. Fifteen proteins of known function, but not previously associated with either the electroplaque or NMJ, were present at high abundance. These included the heat shock and oxidative stress proteins, annexin V (calelectrin), and plectin 1. Most interesting were the unambiguous matches to 11 human ORFs of unknown function, including four potential RNA splicing proteins, a vacuolar sorting protein, and a tetraspanin containing protein. This analysis identified proteins that may play a role in the higher vertebrate neuromuscular junction or other electrical synapses.
加州电鳐是软骨鱼纲中的一个物种。电鳐进化出了电器官,该器官类似于哺乳动物的神经肌肉接头(NMJ)。在此,我们采用cDNA测序和蛋白质组学相结合的方法来确定加州电鳐电器官的分子组成成分。对于可溶性蛋白质,使用了双向电泳(2-DE)并绘制了224个蛋白质点。采用鸟枪法(液相色谱-串联质谱法,LC-MS/MS)分析质膜组分。构建了一个电鳐cDNA文库并对607个cDNA克隆进行了测序。通过跨物种比较对电器官蛋白质进行鉴定,并从cDNA翻译产物构建了一个定制数据库。我们明确鉴定出了121种蛋白质和转录本,其中103种是电鳐鱼类现有数据库中的新增内容。有15种已知功能但先前未与电板或神经肌肉接头相关联的蛋白质大量存在。这些包括热休克蛋白和氧化应激蛋白、膜联蛋白V(钙电蛋白)和网蛋白1。最有趣的是与11个人类未知功能开放阅读框的明确匹配,包括四种潜在的RNA剪接蛋白、一种液泡分选蛋白和一种含四跨膜蛋白的蛋白质。该分析鉴定出了可能在高等脊椎动物神经肌肉接头或其他电突触中发挥作用的蛋白质。