Gandhi R, Elble R C, Gruber A D, Schreur K D, Ji H L, Fuller C M, Pauli B U
Department of Molecular Medicine, Cancer Biology Laboratories, Cornell University College of Veterinary Medicine, Ithaca, New York 14853, USA.
J Biol Chem. 1998 Nov 27;273(48):32096-101. doi: 10.1074/jbc.273.48.32096.
A protein (mCLCA1) has been cloned from a mouse lung cDNA library that bears strong sequence homology with the recently described bovine tracheal, Ca2+-sensitive chloride channel protein (bCLCA1), bovine lung endothelial cell adhesion molecule-1 (Lu-ECAM-1), and the human intestinal Ca2+-sensitive chloride channel protein (hCLCA1). In vitro, its 3.1-kilobase message translates into a 100-kDa protein that can be glycosylated to an approximately 125-kDa product. SDS-polyacrylamide gel electrophoresis from lysates of mCLCA1 cDNA-transfected transformed human embryonic kidney cells (HEK293) reveals proteins of 130, 125, and 90 kDa as well as a protein triplet in the 32-38 kDa size range. Western analyses with antisera raised against Lu-ECAM-1 peptides show that the N-terminal region of the predicted open reading frame is present only in the larger size proteins (i.e. 130, 125, and 90 kDa), whereas the C-terminal region of the open reading frame is observed in the 32-38 kDa size proteins, suggesting a posttranslational, proteolytic processing of a precursor protein (125/130 kDa) into 90 kDa and 32-38 kDa components similar to that reported for Lu-ECAM-1. Hydrophobicity analyses predict four transmembrane domains for the 90-kDa protein. The mCLCA1 mRNA is readily detected by Northern analysis and by in situ hybridization in the respiratory epithelia of trachea and bronchi. Transient expression of mCLCA1 in HEK293 cells was associated with an increase in whole cell Cl- current that could be activated by Ca2+ and ionomycin and inhibited by 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid, dithiothreitol, and niflumic acid. The discovery of mCLCA1 opens the door for further investigating the possible contribution of a Ca2+-sensitive chloride conductance to the pathogenesis of cystic fibrosis.
一种蛋白质（mCLCA1）已从小鼠肺cDNA文库中克隆出来，它与最近描述的牛气管钙敏感氯通道蛋白（bCLCA1）、牛肺内皮细胞黏附分子-1（Lu-ECAM-1）以及人肠道钙敏感氯通道蛋白（hCLCA1）具有很强的序列同源性。在体外，其3.1千碱基的信使核糖核酸（mRNA）翻译成一种100千道尔顿（kDa）的蛋白质，该蛋白质可被糖基化形成约125 kDa的产物。对转染了mCLCA1 cDNA的转化人胚肾细胞（HEK293）裂解物进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE），可显示出130 kDa、125 kDa和90 kDa的蛋白质，以及大小在32 - 38 kDa范围内的一组蛋白质条带。用针对Lu-ECAM-1肽段产生的抗血清进行蛋白质免疫印迹分析表明，预测的开放阅读框的N端区域仅存在于较大分子量的蛋白质（即130 kDa、125 kDa和90 kDa）中，而开放阅读框的C端区域则在32 - 38 kDa大小的蛋白质中观察到，这表明一种前体蛋白质（125/130 kDa）经过翻译后蛋白水解加工形成90 kDa和32 - 38 kDa的成分，这与报道的Lu-ECAM-1情况类似。疏水性分析预测90 kDa的蛋白质有四个跨膜结构域。通过Northern印迹分析和原位杂交很容易在气管和支气管的呼吸道上皮中检测到mCLCA1 mRNA。mCLCA1在HEK293细胞中的瞬时表达与全细胞氯离子电流增加有关，该电流可被钙离子（Ca2+）和离子霉素激活，并被4,4'-二异硫氰酸芪-2,2'-二磺酸、二硫苏糖醇和尼氟酸抑制。mCLCA1的发现为进一步研究钙敏感氯电导对囊性纤维化发病机制的可能作用打开了大门。
