POST TRANSLATIONAL MOD STRUCT OF CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REG

主要翻译后修饰的结构解释 提出了重组CFTR(RCFTR)的构建方法。多肽的定位研究 将确定rCFTR的磷酸化和糖基化位点 使用灵敏的基于质谱学的策略。Cftr蛋白和 核心1的蛋白质片段将被利用。我们的预赛 对另一种膜糖蛋白CHIP-28的研究表明， 证明了我们的方法可以用于pmo1的分析。 CFTR的数量。原状上磷酸盐基团的数量 在大肠杆菌中表达的RR结构域将用以下方法确定 基质辅助激光解吸飞行时间质谱仪。 来自RR结构域的蛋白水解酶的磷酸化肽， 将被识别和表征(例如，磷酸盐的数量 组)使用微孔高效液相色谱/电喷雾电离质量 光谱分析。磷酸基丝氨酸残基的定位 将通过使用串联对磷酸基化肽进行测序来确定 高能碰撞诱导位错的质谱学。一个 将使用类似的方法来定位磷酸化残基。 从哺乳动物细胞和昆虫细胞中提取rCFTR。多肽的定位 的N-和O-糖基化位点将从准确的质量中推断出来 测定(凌晨1点内)蛋白水解物中的糖肽 在昆虫(Sf9)和哺乳动物细胞中表达的rCFTR的酶切片段。这个 在昆虫细胞中表达的来自rCFTR的寡糖将被 在它们释放后，使用自动联氨分解进行测序。这些 研究将确定昆虫细胞糖基化的程度 可作为哺乳动物细胞糖基化CFTR的模型。 哺乳动物CFTR寡糖的结构特征 来源将使用“低聚糖”的组合来确定 作图和外切糖苷酶分析。CFTR的调查 磷酸化，迄今为止产生了模棱两可的发现，是 对理解它作为氯离子通道的功能和它的 受蛋白激酶和磷酸酶的调节。更详细的研究 CFTR碳水化合物的含量是更好地理解 不同突变体在细胞内转运的异常 Cf患者。

Structural elucidation of major post-translational modifications of recombinant CFTR(rCFTR) is proposed. Polypeptide localization of phosphorylation and glycosylation sites of rCFTR will be determined using a sensitive mass spectrometric-based strategy. CFTR proteinand protein fragments from Core 1 will be utilized. Our preliminary studies with another membrane glycopro-tein, CHIP-28, have demonstrated that our methodology can be used for the analysis of pmol quantities of CFTR. The number of phos-phate groups on intact rR-domain, expressed in E. coli, will be determined using matrix-assisted laser desorption, time of flight massspectrometry. Phosphorylated peptides, from proteolytic digests of the rR-domain, will be identified and characterized (e.g., number of phosphate groups) using micro-bore HPLC/electro-spray ionization mass spectrometry. The location of the phos-phorylated serine residues will be determined by sequencing phos-phorylated peptides using tandem mass spectrometry with high energy collision induced dissocation. A similar approach will be used to localize the phosphorylated residues of rCFTR from mammal-ian and insect cells. The polypeptide location of the N- and O-glycosylation sites will be deduced from accurate mass determina-tion (within 1 a.m.u.) of glycopeptides from proteolytic digests of rCFTR, expressed in insect (Sf9) and mammalian cells. The oligosaccharides from rCFTR, expressed in insect cells, willbe sequenced after their release using automated hydrazinolysis. These studies will ascertain the extent to which insect cell glycosylation can be used as a model for mammalian cell glycosyla-tion of CFTR. Structural features of the oligosaccharides of CFTR from mammalian sources will be determined using a combination of "oligosaccharide mapping" and exoglycosidase analysis. Investiga-tions of CFTR phosphorylation, which heretofore has yielded ambiguous findings, is essential for understanding its function as a chloride channel and its regulation by protein kinases and phosphatases. More detailed studies of CFTR carbohydrate are necessary to understand better the abnormalities of intracellular trafficking of the various mutants in CF patients.