Arroyo-Flores B L, Calvo-Méndez C, Flores-Carreón A, López-Romero E
Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad Autonoma de Guanajuato, Mexico.
Microbiology (Reading). 1995 Sep;141 ( Pt 9):2289-94. doi: 10.1099/13500872-141-9-2289.
A mixed membrane fraction (MMF) was isolated from yeast cells of Candida albicans with the ability to synthesize dolichol phosphate mannose (Dol-P-Man) from GDP-Man and dolichol phosphate (Dol-P) and transfer the sugar to proteins. Temperature of incubation (20-37 degrees C) did not affect the synthesis of Dol-P-Man but protein mannosylation occurred better at physiological temperatures (28 degrees C and 37 degrees C). Most of the sugar (87-93%) in the mannoproteins was O-linked as judged by its release by beta-elimination. Mannose was identified as the sole product after this treatment. Following incubation of MMF with the sugar donor, parallel levels of Dol-P-Man and mannosylated proteins were detected up to 30 min. Thereafter, Dol-P-Man levels reached a steady value whereas mannoproteins rapidly accumulated. Lipid-linked oligosaccharides were also detected in incubation mixtures, though in much lower amounts than those of Dol-P-Man or mannoproteins. Dol-P-Man synthase activity increased proportionally in response to increasing concentrations of either of the two enzyme substrates. A Km value of 0.36 microM for GDP-Man was calculated. MMF failed to use exogenous Dol-P-Man for protein glycosylation. Specific inhibition of Dol-P-Man synthesis with amphomycin was concomitant with a parallel decrease in protein mannosylation, indicating that most of the sugar is transferred to protein via the carrier lipid. Results are discussed in terms of the role of Dol-P-Man in protein glycosylation in C. albicans.
从白色念珠菌酵母细胞中分离出一种混合膜组分（MMF），它能够从GDP-甘露糖和磷酸多萜醇（Dol-P）合成磷酸多萜醇甘露糖（Dol-P-Man），并将糖基转移到蛋白质上。孵育温度（20-37℃）不影响Dol-P-Man的合成，但在生理温度（28℃和37℃）下蛋白质甘露糖基化效果更好。通过β-消除法释放的甘露糖蛋白中的大部分糖（87-93%）是O-连接的。经此处理后，甘露糖被鉴定为唯一产物。将MMF与糖供体孵育后，在30分钟内检测到Dol-P-Man和甘露糖基化蛋白的平行水平。此后，Dol-P-Man水平达到稳定值，而甘露糖蛋白迅速积累。在孵育混合物中也检测到了脂质连接的寡糖，但其含量远低于Dol-P-Man或甘露糖蛋白。Dol-P-Man合酶活性随两种酶底物中任一种浓度的增加而成比例增加。计算出GDP-Man的Km值为0.36 microM。MMF不能利用外源性Dol-P-Man进行蛋白质糖基化。两性霉素对Dol-P-Man合成的特异性抑制伴随着蛋白质甘露糖基化的平行下降，这表明大部分糖是通过载体脂质转移到蛋白质上的。本文从Dol-P-Man在白色念珠菌蛋白质糖基化中的作用方面对结果进行了讨论。
