Hu Yong, Suarez Jorge, Fricovsky Eduardo, Wang Hong, Scott Brian T, Trauger Sunia A, Han Wenlong, Hu Ying, Oyeleye Mary O, Dillmann Wolfgang H
Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, California 92093 and the Scripps Research Institute, La Jolla, California 92037.
Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, California 92093 and the Scripps Research Institute, La Jolla, California 92037.
J Biol Chem. 2009 Jan 2;284(1):547-555. doi: 10.1074/jbc.M808518200. Epub 2008 Nov 12.
Increased nuclear protein O-linked beta-N-acetylglucosamine glycosylation (O-GlcNAcylation) mediated by high glucose treatment or the hyperglycemia of diabetes mellitus contributes to cardiac myocyte dysfunction. However, whether mitochondrial proteins in cardiac myocytes are also submitted to O-GlcNAcylation or excessive O-GlcNAcylation alters mitochondrial function is unknown. In this study, we determined if mitochondrial proteins are O-GlcNAcylated and explored if increased O-GlcNAcylation is linked to high glucose-induced mitochondrial dysfunction in neonatal rat cardiomyocytes. By immunoprecipitation, we found that several mitochondrial proteins, which are members of complexes of the respiratory chain, like subunit NDUFA9 of complex I, subunits core 1 and core 2 of complex III, and the mitochondrial DNA-encoded subunit I of complex IV (COX I) are O-GlcNAcylated. By mass spectrometry, we identified that serine 156 on NDUFA9 is O-GlcNAcylated. High glucose treatment (30 mm glucose) increases mitochondrial protein O-GlcNAcylation, including those of COX I and NDUFA9 which are reduced by expression of O-GlcNAcase (GCA). Increased mitochondrial O-GlcNAcylation is associated with impaired activity of complex I, III, and IV in addition to lower mitochondrial calcium and cellular ATP content. When the excessive O-GlcNAc modification is reduced by GCA expression, mitochondrial function improves; the activity of complex I, III, and IV increases to normal and mitochondrial calcium and cellular ATP content are returned to control levels. From these results we conclude that specific mitochondrial proteins of cardiac myocytes are O-GlcNAcylated and that exposure to high glucose increases mitochondrial protein O-GlcNAcylation, which in turn contributes to impaired mitochondrial function.
高糖处理或糖尿病高血糖介导的核蛋白O-连接β-N-乙酰葡糖胺糖基化（O-GlcNAcylation）增加会导致心肌细胞功能障碍。然而，心肌细胞中的线粒体蛋白是否也会发生O-GlcNAcylation，或者O-GlcNAcylation过度是否会改变线粒体功能尚不清楚。在本研究中，我们确定了线粒体蛋白是否发生O-GlcNAcylation，并探讨了O-GlcNAcylation增加是否与新生大鼠心肌细胞中高糖诱导的线粒体功能障碍有关。通过免疫沉淀，我们发现几种线粒体蛋白，即呼吸链复合物的成员，如复合物I的亚基NDUFA9、复合物III的核心1和核心2亚基以及复合物IV（COX I）的线粒体DNA编码亚基I发生了O-GlcNAcylation。通过质谱分析，我们确定NDUFA9上的丝氨酸156发生了O-GlcNAcylation。高糖处理（30 mM葡萄糖）会增加线粒体蛋白的O-GlcNAcylation，包括COX I和NDUFA9的O-GlcNAcylation，而O-GlcNAcase（GCA）的表达会降低它们的O-GlcNAcylation。线粒体O-GlcNAcylation增加除了会降低线粒体钙和细胞ATP含量外，还与复合物I、III和IV的活性受损有关。当通过GCA表达减少过度的O-GlcNAc修饰时，线粒体功能会改善；复合物I、III和IV的活性增加到正常水平，线粒体钙和细胞ATP含量恢复到对照水平。从这些结果我们得出结论，心肌细胞的特定线粒体蛋白发生了O-GlcNAcylation，暴露于高糖会增加线粒体蛋白的O-GlcNAcylation，进而导致线粒体功能受损。
