The results described in the accompanying article support the model in which glucosylphosphoryldolichol (Glc-P-Dol) is synthesized on the cytoplasmic face of the ER, and functions as a glucosyl donor for three Glc-P-Dol:Glc0-2Man9-GlcNAc2P-P-Dol glucosyltransferases (GlcTases) in the lumenal compartment. In this study, the enzymatic synthesis and structural characterization by NMR and electrospray-ionization tandem mass spectrometry of a series of water-soluble β-Glc-P-Dol analogs containing 2-4 isoprene units with either the cis- or trans-stereoconfiguration in the β-position are described. The water-soluble analogs were (1) used to examine the stereospecificity of the Glc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) and (2) tested as potential substrates for a membrane protein(s) mediating the transbilayer movement of Glc-P-Dol in sealed ER vesicles from rat liver and pig brain. The Glc-P-Dol-mediated GlcTases in pig brain microsomes utilized [3H]Glc-labeled Glc-P-Dol10, Glc-P-(ω,c)Dol15, Glc-P(ω,t,t)Dol20, and Glc-P-(ω,t,c)Dol20 as glucosyl donors with [3H]Glc3Man9GlcNAc2-P-P-Dol the major product labeled in vitro. A preference was exhibited for C15-20 substrates containing an internal cis-isoprene unit in the β-position. In addition, the water-soluble analog, Glc-P-Dol10, was shown to enter the lumenal compartment of sealed microsomal vesicles from rat liver and pig brain via a protein-mediated transport system enriched in the ER. The properties of the ER transport system have been characterized. Glc-P-Dol10 was not transported into or adsorbed by synthetic PC-liposomes or bovine erythrocytes. The results of these studies indicate that (1) the internal cis-isoprene units are important for the utilization of Glc-P-Dol as a glucosyl donor and (2) the transport of the water-soluble analog may provide an experimental approach to assay the hypothetical 'flippase' proposed to mediate the transbilayer movement of Glc-P-Dol from the cytoplasmic face of the ER to the lumenal monolayer.
|Number of pages||11|
|State||Published - 1998|
Bibliographical noteFunding Information:
We thank Dr. Austin H.Cantor and Dr. Mike Ford, University of Kentucky Department of Animal Sciences, for their help in obtaining fresh hen oviducts and Mr. James R.May, University of Kentucky Department of Animal Sciences, for providing us with fresh pig brains. This work was supported by NIH Grant GM36065 awarded to C.J.W. We also thank Professor E.de Hoffmann, University of Louvain, for his assistance with the mass spectrometry measurements.
- Glc-P-Dol synthesis
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