Nehrke K, Hagen F K, Tabak L A
Department of Dental Research, School of Medicine and Dentistry, University of Rochester, Rochester, New York 14642, USA.
J Biol Chem. 1996 Mar 22;271(12):7061-5. doi: 10.1074/jbc.271.12.7061.
The elements that regulate O-glycosylation are poorly understood. We have developed a novel in vivo system to analyze the role of flanking sequence on the modification of a single well characterized O-glycosylation site derived from human von Willebrand factor (PHMAQVTVGPGL). A secreted chimeric reporter protein, containing the human von Willebrand factor sequence, an antibody recognition epitope, and a heart muscle kinase site, was engineered and expressed in COS7 and MCF-7 cells. Glycosylated and non-glycosylated forms of the immunoprecipitated reporter were resolved electrophoretically and their relative amounts quantitated. Using mutational analysis we find that the glycosylation apparatus of COS7 cells can accommodate a broad range of changes in the flanking sequence without compromising glycosylation, but that the distribution of charged amino acids flanking the O-glycosylation site can have a profound influence on glycosylation with position -1 relative to the glycosylation site being particularly sensitive. A combination of acidic residues at positions -1 and +3 almost completely eliminates glycosylation of the reporter in both COS7 and MCF-7 cells. The overall density of charged amino acids is less important since substitution of acidic residues at position -2, +1, and +2 had no effect in the level of glycosylation observed.
调节O-糖基化的因素目前还知之甚少。我们开发了一种新型的体内系统，用于分析侧翼序列对源自人血管性血友病因子（PHMAQVTVGPGL）的单个特征明确的O-糖基化位点修饰的作用。构建了一种分泌型嵌合报告蛋白，其包含人血管性血友病因子序列、一个抗体识别表位和一个心肌激酶位点，并在COS7和MCF-7细胞中表达。对免疫沉淀后的报告蛋白的糖基化和非糖基化形式进行电泳分离，并对其相对含量进行定量。通过突变分析，我们发现COS7细胞的糖基化装置能够适应侧翼序列的广泛变化而不影响糖基化，但O-糖基化位点侧翼的带电氨基酸分布对糖基化有深远影响，相对于糖基化位点的-1位尤为敏感。在-1和+3位的酸性残基组合几乎完全消除了COS7和MCF-7细胞中报告蛋白的糖基化。带电氨基酸的总体密度不太重要，因为在-2、+1和+2位替换酸性残基对观察到的糖基化水平没有影响。
