The goal of this research is to characterize in detail a number of enzymes and/or enzymatic processes involved in the synthesis of dolichyl phosphate (Dol-P) and Dol-P linked saccharides involved in N-linked glycoprotein synthesis. The first overall objective is to characterize the pathway of biosynthesis and the subcellular sites of deposition of Dol and Dol-P. Studies will be carried out in an embryonic Drosophila cell line, KC. The rationale for using insect cells is that they lack the """"""""cholesterol branch"""""""" in the polyisoprenoid pathway. Hence, the only lipid end products arising from mevalonic acid are Dol and coenzyme Q. Pulse-chase experiments will be carried out using labeled mevalonic acid and cells in culture to determine if Dol of Dol-P is the end product of the de novo pathway. Subcellular fractionation at various times during the chase period will be used to determine the subcellular localization of Dol and Dol-P. If the above studies reveal that, as is the case in liver, the majority of the Dol is deposited in lysosomes, extended chase experiments will be carried out under conditions in which the supply or the demand for Dol or Dol-P is perturbed in order to determine if lysosomal Dol can be """"""""mobilized"""""""" by conversion to Dol-P. The second overall objective is to isolate and characterize several of the enzymes in the dolichol-mediated pathway in hen oviduct. The four enzymes to be investigated are those that catalyze synthesis of GlcNAc-PP-Dol, GlcNAc2-PP-Dol, Man-P-Dol, and Glc-P-Dol. These synthases will be solubilized and purified by either conventional methods or by antibody affinity chromatography. In the latter case antibody will be prepared against the enzymes isolated in radioactive form by photoaffinity tagging. The antibodies will be used to study the regulation of synthesis of these enzymes in developing chick oviduct, and to screen for a cDNA probe in order to study regulation of the expression of mRNAs encoding for these enzymes. The purified enzymes, along with Dol-P (or GlcNAc-PP-Dol), will be reconstituted in unilamellar liposomes to study localization of the end products upon external addition of the appropriate sugar nucleotide. The possible formation of a glycosyl-enzyme intermediate from NDP-glycose in the absence of the other substrate, Dol-P, will be investigated. To study the topology of the synthases in the lipid bilayer of the RER, proteases and antitransferase antibodies will be used as probes. Finally, new probes will be developed to determine the transmembrane orientation of Dol-P and its glycosylated derivatives in sealed RER.
Showing the most recent 10 out of 45 publications
