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Abstract #21548 Published in IGR 10-3
Upregulation of copine1 in trabecular meshwork cells of POAG patients: A membrane proteomics approach
Zhang Y; Gao Q; Duan S; He Y; Sun X; Jiang R; Duan Y; Zhong X; Ge JMolecular Vision 2008; 14: 1028-1036
PURPOSE: Primary open-angle glaucoma (POAG) is a leading cause of irreversible blindness worldwide, and its pathogenesis is still unknown. The purpose of this study was to determine molecular changes in membrane proteins in trabecular meshwork (TM) cells from POAG patients compared to those of age-matched normal controls. METHODS: Two-dimensional (2-D) gel electrophoresis profiles of membrane extracts from normal and glaucomatous TM cells were compared. The desired spots were identified after trypsin digestion and mass spectrometric analysis. Based on the results, a calcium-dependant membrane-binding protein, copine1, was further approached for a possible role in glaucomatous TM cells. The intracellular calcium concentration ([Ca2+]i) of TM cells was increased by incubating with calcium ionophore, A23187. Relative quantification real-time polymerase chain reaction (PCR) and western blot analysis measured copine1 expression and localization both in untreated and A23187-treated TM cells. RESULTS: Real-time PCR and western blot analysis confirmed that copine1 mRNA and protein expression were upregulated in glaucomatous TM cells when compared to normal ones. The cell distribution studies further showed that copine1 existed both in the membrane and cytoplasm fractions of glaucomatous TM cells but existed exclusively in cytoplasm fractions of their normal counterparts. More importantly, an influx of Ca2+ markedly promoted the translocation of copine1 from the cytoplasm to membranes in glaucomatous TM cells. CONCLUSIONS: Copine1 is upregulated in plasma membranes of TM cells in individuals with POAG, which may be partly explained by its Ca2+-dependent translocation from the cytoplasm to the membranes. Investigation of the role and functions of copine1 in TM cells should offer new insight into the abnormal intracellular Ca2+-signaling pathway in glaucomatous TM and help to clarify the molecular mechanism of POAG.
Dr. J. Ge, Zhongshan Ophthalmic Center, Sun Yat-Sen University, 510060 Guangzhou, China. gejian@mail.sysu.edu.cn
Classification:
3.12 Proteomics (Part of: 3 Laboratory methods)
2.5.1 Trabecular meshwork (Part of: 2 Anatomical structures in glaucoma > 2.5 Meshwork)
3.6 Cellular biology (Part of: 3 Laboratory methods)
3.5 Molecular biology incl. SiRNA (Part of: 3 Laboratory methods)
