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 I 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 mass spectrometry. 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 0 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, will be 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.
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