Impact of mannosylation on structure and functionality of ER and Golgi

Many proteins undergo glycosylation in the endoplasmic reticulum (ER) and the Golgi apparatus. Altered glycosylation can manifest in serious, sometimes fatal malfunctions. We recently showed that mutations in the cytoplasmic protein GDP-mannose pyrophosphorylase A (GMPPA) cause a syndrome characterized by alacrima, achalasia, mental retardation and myopathic alterations. GMPPA acts as feedback inhibitor of GDP-mannose pyrophosphorylase B (GMPPB), which provides GDP-mannose as a substrate for protein glycosylation. Loss of GMPPA enhances incorporation of mannose into glycochains of various proteins, including α-dystroglycan (α-DG), a protein that links the extracellular matrix with the cytoskeleton. Here, we show that loss of GMPPA affects the functionality of the Golgi apparatus using different approaches. First, we show a fragmentation of the Golgi apparatus in skeletal muscle fibers and in neurons of GMPPA KO mice. A major reorganization is also evident by mass spectrometry of KO tissues with a regulation of several ER- and Golgi-resident proteins. We further show that loss of GMPPA increases the retention of α-DG in the ER. Notably, mannose supplementation can mimic changes in ER and Golgi structure and function in WT cells. In summary, our data underline the importance of a balanced mannose homeostasis for structure and function of the secretory pathway.

众多蛋白质可在内质网（endoplasmic reticulum, ER）与高尔基体中发生糖基化修饰。异常糖基化可引发严重甚至致死性的功能障碍。我们此前的研究发现，胞质蛋白GDP-甘露糖焦磷酸化酶A（GDP-mannose pyrophosphorylase A, GMPPA）的突变可导致一种以泪液缺乏、贲门失弛缓症、智力障碍及肌病改变为特征的综合征。GMPPA可作为GDP-甘露糖焦磷酸化酶B（GDP-mannose pyrophosphorylase B, GMPPB）的反馈抑制剂，而后者可合成GDP-甘露糖作为蛋白质糖基化的底物。GMPPA功能缺失会增强甘露糖向多种蛋白质糖链中的掺入，其中包括α-肌营养不良聚糖（α-dystroglycan, α-DG）——一种连接细胞外基质与细胞骨架的蛋白质。本研究通过多种实验手段证实，GMPPA功能缺失会影响高尔基体的功能：其一，我们在基因敲除（knockout, KO）小鼠的骨骼肌纤维与神经元中观察到高尔基体发生碎裂；对基因敲除小鼠组织进行质谱分析同样显示出显著的蛋白质组重编程，多种内质网与高尔基体驻留蛋白的表达水平出现调控变化。我们还发现，GMPPA功能缺失会增加α-DG在内质网中的滞留。值得注意的是，向野生型（wild type, WT）细胞中补充甘露糖可模拟内质网与高尔基体的结构及功能改变。综上，本研究数据凸显了平衡的甘露糖稳态对于分泌通路结构与功能的重要意义。