Upregulation of Copine1 in trabecular meshwork cells of POAG patients: a membrane proteomics approach.
OBJECTIVE
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 ([Ca(2+)]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 Ca(2+) 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 Ca(2+)-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 Ca(2+)-signaling pathway in glaucomatous TM and help to clarify the molecular mechanism of POAG.
Zhang
Yuehong
Y
Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
Gao
Qianying
Q
Duan
Shan
S
He
Yuan
Y
Sun
Xuerong
X
Jiang
Ruzhang
R
Duan
Yongheng
Y
Zhong
Xiufeng
X
Ge
Jian
J
D000293:Adolescent; D000328:Adult; D015153:Blotting, Western; D020013:Calcium Signaling; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D005902:Glaucoma, Open-Angle / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D007150:Immunohistochemistry; D008875:Middle Aged; D040901:Proteomics / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013347:Subcellular Fractions / Q000378:metabolism; D014129:Trabecular Meshwork / Q000378:metabolism* / Q000473:pathology*; D015854:Up-Regulation
D005136:Eye Proteins; D012333:RNA, Messenger