Chung Hye Jin, Steplewski Andrzej, Uitto Jouni, Fertala Andrzej
Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Biochem Biophys Res Commun. 2009 Dec 18;390(3):662-6. doi: 10.1016/j.bbrc.2009.10.024. Epub 2009 Oct 12.
Fluorescent proteins are powerful markers allowing tracking expression, intracellular localization, and translocation of tagged proteins but their effects on the structure and assembly of complex extracellular matrix proteins has not been investigated. Here, we analyzed the utility of fluorescent proteins as markers for procollagen VII, a triple-helical protein critical for the integrity of dermal-epidermal junction. DNA constructs encoding a red fluorescent protein-tagged wild type mini-procollagen VII alpha chain and green fluorescent protein-tagged alpha chains harboring selected mutations were genetically engineered. These DNA constructs were co-expressed in HEK-293 cells and the assembly of heterogeneous triple-helical mini-procollagen VII molecules was analyzed. Immunoprecipitation and fluorescence resonance energy transfer assays demonstrated that the presence of different fluorescent protein markers at the C-termini of individual alpha chains neither altered formation of triple-helical molecules nor affected their secretion to the extracellular space. Our study provides a basis for employing fluorescent proteins as tags for complex structural proteins of extracellular matrix.
荧光蛋白是强大的标记物，可用于追踪标记蛋白的表达、细胞内定位和转运，但它们对复杂细胞外基质蛋白的结构和组装的影响尚未得到研究。在此，我们分析了荧光蛋白作为前胶原VII标记物的效用，前胶原VII是一种对真皮-表皮连接完整性至关重要的三螺旋蛋白。对编码红色荧光蛋白标记的野生型微型前胶原VIIα链和绿色荧光蛋白标记的携带选定突变的α链的DNA构建体进行了基因工程改造。这些DNA构建体在HEK-293细胞中共同表达，并分析了异质三螺旋微型前胶原VII分子的组装。免疫沉淀和荧光共振能量转移分析表明，在单个α链的C末端存在不同的荧光蛋白标记既不会改变三螺旋分子的形成，也不会影响它们分泌到细胞外空间。我们的研究为将荧光蛋白用作细胞外基质复杂结构蛋白的标签提供了基础。
