Ware F E, Lehrman M A
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041, USA.
J Biol Chem. 1996 Jun 14;271(24):13935-8. doi: 10.1074/jbc.271.24.13935.
Lec15 and Lec35 are recessive Chinese hamster ovary (CHO) cell glycosylation mutations characterized by inefficient synthesis and utilization, respectively, of mannose-P-dolichol (MPD). Consequently, Lec15 and Lec35 cells accumulate Man5GlcNAc2-P-P-dolichol and glucosaminyl-acylphosphatidylinositol. This report describes the cloning of a suppressor (termed SL15) of the Lec15 and Lec35 mutations from a CHO cDNA library by functional expression in Lec15 cells, employing phytohemagglutinin/swainsonine selection. The SL15 protein has a predicted molecular weight of 26,693 with two potential membrane spanning regions and a likely C-terminal endoplasmic reticulum retention signal (Lys-Lys-Glu-Gln). Lec15 cells transfected with SL15 have normal levels of MPD synthase activity in vitro and convert Man5GlcNAc2-P-P-dolichol to Glc0-3Man9GlcNAc2-P-P-dolichol in vivo. Surprisingly, SL15 also corrects the defective mannosylation in Lec35 cells. The SL15 protein bears no apparent similarity to Saccharomyces cerevisiae MPD synthase (the DPM1 protein), but is highly similar to the hypothetical F38E1.9 protein encoded on Caenorhabditis elegans chromosome 5. These results indicate a novel function for the SL15 protein and suggest that MPD synthesis is more complex than previously suspected.
Lec15和Lec35是隐性中国仓鼠卵巢（CHO）细胞糖基化突变，其特征分别是甘露糖-P-多萜醇（MPD）合成效率低下和利用效率低下。因此，Lec15和Lec35细胞积累了Man5GlcNAc2-P-P-多萜醇和葡糖胺酰基-酰基磷脂酰肌醇。本报告描述了通过在Lec15细胞中进行功能表达，利用植物血凝素/苦马豆素选择从CHO cDNA文库中克隆Lec15和Lec_{35}突变的一个抑制子（称为SL15）。SL15蛋白的预测分子量为26,693，有两个潜在的跨膜区域和一个可能的C端内质网滞留信号（Lys-Lys-Glu-Gln）。用SL15转染的Lec15细胞在体外具有正常水平的MPD合酶活性，并在体内将Man5GlcNAc2-P-P-多萜醇转化为Glc0-3Man9GlcNAc2-P-P-多萜醇。令人惊讶的是，SL15还纠正了Lec35细胞中缺陷的甘露糖基化。SL15蛋白与酿酒酵母MPD合酶（DPM1蛋白）没有明显的相似性，但与秀丽隐杆线虫5号染色体上编码的假想F38E_{1.9}蛋白高度相似。这些结果表明了SL15蛋白的一种新功能，并表明MPD合成比以前怀疑的更为复杂。
