Department of Neurology, School of Health Sciences, Faculty of Medicine, University of Crete, Heraklion, Crete 71003, Greece.
J Biol Chem. 2010 May 28;285(22):16748-56. doi: 10.1074/jbc.M109.092999. Epub 2010 Mar 1.
Mammalian glutamate dehydrogenase (GDH) is an allosterically regulated enzyme that is expressed widely. Its activity is potently inhibited by GTP and thought to be controlled by the need of the cell for ATP. In addition to this housekeeping human (h) GDH1, humans have acquired (via a duplication event) a highly homologous isoenzyme (hGDH2) that is resistant to GTP. Although transcripts of GLUD2, the gene encoding hGDH2, have been detected in human neural and testicular tissues, data on the endogenous protein are lacking. Here, we developed an antibody specific for hGDH2 and used it to study human tissues. Western blot analyses revealed, to our surprise, that endogenous hGDH2 is more densely expressed in testis than in brain. At the subcellular level, hGDH2 localized to mitochondria. Study of testicular tissue using immunocytochemical and immunofluorescence methods revealed that the Sertoli cells were strongly labeled by our anti-hGDH2 antibody. In human cerebral cortex, a robust labeling of astrocytes was detected, with neurons showing faint hGDH2 immunoreactivity. Astrocytes and Sertoli cells are known to support neurons and germ cells, respectively, providing them with lactate that largely derives from the tricarboxylic acid cycle via conversion of glutamate to alpha-ketoglutarate (GDH reaction). As hGDH2 is not subject to GTP control, the enzyme is able to metabolize glutamate even when the tricarboxylic acid cycle generates GTP amounts sufficient to inactivate the housekeeping hGDH1 protein. Hence, the selective expression of hGDH2 by astrocytes and Sertoli cells may provide a significant biological advantage by facilitating metabolic recycling processes essential to the supportive role of these cells.
哺乳动物谷氨酸脱氢酶（GDH）是一种变构调节酶，广泛表达。其活性被 GTP 强烈抑制，被认为是由细胞对 ATP 的需求控制的。除了这种管家人类（h）GDH1 外，人类还通过（通过重复事件）获得了一种高度同源的同工酶（hGDH2），它对 GTP 有抗性。虽然编码 hGDH2 的 GLUD2 基因的转录本已在人类神经和睾丸组织中检测到，但缺乏内源性蛋白质的数据。在这里，我们开发了一种针对 hGDH2 的特异性抗体，并使用它研究人类组织。Western blot 分析令人惊讶地表明，内源性 hGDH2 在睾丸中的表达密度高于大脑。在亚细胞水平上，hGDH2 定位于线粒体。使用免疫细胞化学和免疫荧光方法研究睾丸组织时，我们发现 Sertoli 细胞被我们的抗 hGDH2 抗体强烈标记。在人类大脑皮层中，检测到星形胶质细胞的强烈标记，神经元显示出微弱的 hGDH2 免疫反应性。众所周知，星形胶质细胞和 Sertoli 细胞分别支持神经元和生殖细胞，为它们提供主要来自三羧酸循环的乳酸，通过将谷氨酸转化为α-酮戊二酸（GDH 反应）。由于 hGDH2 不受 GTP 控制，因此即使三羧酸循环产生足以使管家 hGDH1 蛋白失活的 GTP 量，该酶也能够代谢谷氨酸。因此，星形胶质细胞和 Sertoli 细胞选择性表达 hGDH2 可能通过促进这些细胞支持作用所必需的代谢回收过程提供重要的生物学优势。
