Leverkoehne Ina, Gruber Achim D
Department of Pathology, School of Veterinary Medicine Hannover, Hannover, Germany.
J Histochem Cytochem. 2002 Jun;50(6):829-38. doi: 10.1177/002215540205000609.
The putative anion channel mCLCA3 (alias gob-5) is the third murine member of the recently discovered family of calcium-activated chloride channels (CLCA family). Preliminary data suggest that mCLCA3 may play a significant role in diseases with secretory dysfunctions, including asthma and cystic fibrosis. In this study, the mCLCA3 protein was characterized biochemically and its cellular and subcellular distribution pattern was established in normal murine tissues. Polyclonal rabbit antibodies were generated and affinity-immunopurified using synthetic oligopeptides corresponding to the extracellular amino terminus of the mCLCA3 polypeptide. After in vitro translation and glycosylation, proteinase K protection assay, and heterologous expression in COS-7 or HEK 293 cells, SDS-PAGE and immunoblotting revealed a protein structure similar to that of previously characterized CLCA proteins. A systematic light, confocal laser scanning, and transmission electron microscopic immunolocalization study, including virtually all murine tissues, identified the mCLCA3 protein exclusively associated with mucin granule membranes of gastrointestinal, respiratory, and uterine goblet cells and other mucin-producing cells. The results suggest that mCLCA3 may be involved in the synthesis, condensation, or secretion of mucins.
推定的阴离子通道mCLCA3（别名gob-5）是最近发现的钙激活氯离子通道家族（CLCA家族）的第三个小鼠成员。初步数据表明，mCLCA3可能在包括哮喘和囊性纤维化在内的分泌功能障碍性疾病中发挥重要作用。在本研究中，对mCLCA3蛋白进行了生化特性分析，并确定了其在正常小鼠组织中的细胞和亚细胞分布模式。使用与mCLCA3多肽细胞外氨基末端对应的合成寡肽制备并亲和免疫纯化了兔多克隆抗体。经过体外翻译和糖基化、蛋白酶K保护试验以及在COS-7或HEK 293细胞中的异源表达后，SDS-PAGE和免疫印迹显示出一种与先前鉴定的CLCA蛋白结构相似的蛋白质。一项系统的光镜、共聚焦激光扫描和透射电子显微镜免疫定位研究，涵盖了几乎所有的小鼠组织，结果表明mCLCA3蛋白仅与胃肠道、呼吸道和子宫杯状细胞以及其他产生粘蛋白的细胞的粘蛋白颗粒膜相关。结果提示mCLCA3可能参与了粘蛋白的合成、浓缩或分泌过程。
