Exploring respiratory chain glycoproteomics in mitochondrial disease

。 原发性线粒体呼吸链疾病与先天性糖基化异常 (CDG)是共同的常见代谢性疾病，可引起重叠的 扰乱了大脑发育和多系统疾病。关于这一点，还有很多有待发现 线粒体糖蛋白质组的性质和调节及其与 遗传性RC功能或糖基化障碍。我们的初步数据表明：[1] 不同物种的线粒体具有独特的N-连接糖基修饰 MALDI-TOF质谱(MS)；[2]几种RC蛋白是N-糖基化的；以及[3] 缺乏N-糖聚糖酶的患者成纤维细胞系(FCL)、线虫和小鼠品系 NGLY1存在线粒体耗竭、RC功能障碍和氧化应激增加。这个 这项建议的总体目标是表征线粒体RC糖蛋白组及其 调节，并阐明其与遗传性RC功能和NGLY1活性障碍的相关性。 我们假设线粒体糖蛋白的糖基化和去糖化， 包括RC复合体的亚单位，对于维持正常的线粒体是必不可少的 功能。这项建议的具体目的是表征N-连接的糖蛋白质组 RC以及RC疾病改变它的方式。在电子计算机预测中，特定的RC蛋白是 N-糖基化将通过凝集素反应性和PNGaseF、内切糖苷酶H和F进行检测 蓝色-天然凝胶分离的RC络合物的感度。糖蛋白及其相互作用 糖基化位点和糖链结构将通过质谱学鉴定。 遗传病或药物抑制对糖蛋白的抑制作用 RC病患者的FCL将表现为线粒体RC的修饰 和NGLY1缺陷患者，以及来自NGLY1缺陷的小鼠MEF模型。这些 研究将鉴定N-糖基化RC蛋白，并阐明RC如何影响它们 疾病。

. Primary mitochondrial respiratory chain (RC) diseases and congenital disorders of glycosylation (CDG) are collectively common metabolic diseases that cause an overlapping spectrum of disrupted brain development and multi-systemic disease. Much remains to be discovered about the nature and regulation of the mitochondrial glycoproteome and its potential relevance to inherited disorders of RC function or glycosylation. Our preliminary data suggest that: [1] Mitochondria from diverse species have unique N-linked glycome modifications assessed by MALDI-TOF mass spectrometry (MS); [2] Several RC proteins are N-glycosylated; and [3] Patient fibroblast cell lines (FCL), C. elegans, and mouse strains lacking the N-glycanase NGLY1 have mitochondrial depletion, RC dysfunction, and increased oxidative stress. The overall goal of this proposal is to characterize the mitochondrial RC glycoproteome and its regulation, and clarify its relevance to inherited disorders of RC function and NGLY1 activity. We hypothesize that glycosylation and deglycosylation of mitochondrial glycoproteins, including subunits of RC complexes, are essential for maintenance of normal mitochondrial function. The Specific Aim of this proposal is to characterize the N-linked glycoproteome of the RC and the ways it is altered by RC disease. In silico predictions that specific RC proteins are N-glycosylated will be tested by lectin reactivity and PNGaseF, endoglycosidase H and F sensitivity of blue-native gel separated RC complexes. The glycoproteins and their glycosylation sites as well as the glycan structure will be identified by mass spectrometry. Effects of RC inhibition due to either genetic disease or pharmacologic inhibition on glycoprotein modifications of the mitochondrial RC will be characterized in patient FCLs from RC disease and NGLY1 deficient patients, as well as from mouse NGLY1 deficient MEF models. These studies will identify N-glycosylated RC proteins and elucidate how they are impacted by RC disease.