Rush J S, van Leyen K, Ouerfelli O, Wolucka B, Waechter C J
Department of Biochemistry, University of Kentucky College of Medicine, Lexington, KY 40536, USA.
Glycobiology. 1998 Dec;8(12):1195-205. doi: 10.1093/glycob/8.12.1195.
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-GlcNAc2-P-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 beta-Glc-P-Dol analogs containing 2-4 isoprene units with either the cis - or trans -stereoconfiguration in the beta-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-(omega, c )Dol15, Glc-P(omega, t,t )Dol20, and Glc-P-(omega, t,c )Dol20as 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 beta-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-Dol10was 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.
随附文章中描述的结果支持这样一种模型，即糖基磷酸多萜醇（Glc-P-Dol）在内质网的胞质面合成，并作为内质网腔室中三种Glc-P-Dol:Glc0-2Man9-GlcNAc2-P-P-Dol糖基转移酶（GlcTases）的糖基供体发挥作用。在本研究中，描述了一系列在β位含有2-4个异戊二烯单元且具有顺式或反式立体构型的水溶性β-Glc-P-Dol类似物的酶促合成以及通过核磁共振和电喷雾电离串联质谱进行的结构表征。这些水溶性类似物被用于：（1）检测Glc-P-Dol:Glc0-2ManMancMan9GlcNAc2-P-P-Dol糖基转移酶（GlcTases）的立体特异性；（2）作为潜在底物，用于检测介导大鼠肝脏和猪脑封闭内质网囊泡中Glc-P-Dol跨膜运动的膜蛋白。猪脑微粒体中Glc-P-Dol介导的GlcTases利用[3H]Glc标记的Glc-P-Dol10、Glc-P-(ω,c)Dol15、Glc-P(ω,t,t)Dol20和Glc-P-(ω,t,c)Dol20作为糖基供体，[3H]Glc3Man9GlcNAc2-P-P-Dol是体外标记的主要产物。对β位含有内部顺式异戊二烯单元的C15-20底物表现出偏好。此外，水溶性类似物Glc-P-Dol10被证明通过内质网中富集的蛋白质介导转运系统进入大鼠肝脏和猪脑封闭微粒体囊泡的腔室。内质网转运系统的特性已得到表征。Glc-P-Dol10未被转运到合成的PC脂质体或牛红细胞中，也未被它们吸附。这些研究结果表明：（1）内部顺式异戊二烯单元对于将Glc-P-Dol用作糖基供体很重要；（2）水溶性类似物的转运可能提供一种实验方法，用于检测推测的介导Glc-P-Dol从内质网胞质面到腔面膜单层跨膜运动的“翻转酶”。
