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Terete Borras, K.David Kennedy, M.Grazia Spiga, LaKisha K. Buie; Cystatin A (stefin A), A Cysteine Protease Inhibitor, Is Involved In The Processing Of Myocilin. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3092.
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© ARVO (1962-2015); The Authors (2016-present)
Myocilin (MYOC) is cleaved intracellularly by calpain II, a calcium-dependent cysteine protease (1). This cleavage is inhibited in the MYOC mutants (1). Microarray studies from our laboratory comparing the effects of overexpressing MYOC mutants (MUTs) and WT in primary human trabecular meshwork cells (HTM) identified cystatin A. Our goal was to investigate whether cystatin A influenced MYOC processing in the HTM.
HTM-72 cells were infected with adenoviral vectors carrying no gene (Null), WT, Q368X, R342K, D380N and K423E MYOC mutants. At 48h post-infection, RNA was hybridized to Affymetrix U133plus 2.0 chips (n= 17) and analyzed with GSv7 and v10. Expression confirmation was performed by TaqManPCR. To evaluate processing, the full coding WT MYOC was cloned into expression vector pcDNA 3.1.V5-His-TOPO fused to its C-tag (pMYOC3’fused). An expression vector carrying full coding cystatin A under the control of a CMV promoter was obtained from Origene (pCSTA). Co-transfection of MYOC3’fused and cystatin plasmids was performed in 293 and primary HTM cells by calcium precipitation and nucleofector electroporation respectively. An empty plasmid was used as a control. Intracellular and extracellular proteins were analyzed by WB at 48h using an anti-V5 monoclonal antibody (1:400) that targeted the C-terminus of the WT MYOC protein.
Compared to AdNull, VennMaps revealed that cystatin expression was elevated in all MYOC MUTs but unchanged in the WT. TaqManPCR showed significant elevation of cystatin expression in cells infected with MYOC mutants. In pMYOC3'fused/pCSTA co-transfection experiments, WBs of cell lysate proteins probed with anti-V5 showed a marked reduction of the 35kDa MYOC processed proteolytic fragment in both cell lines. In the extracellular fraction, the effect was more pronounced, and the 35kDa secreted fragment was barely detected. Proteins from cells transfected with only MYOC WT or co-transfected with WT and empty plasmid, showed an intense 35kDa band in both intracellular and secreted fractions, which indicated normal processing.
The cysteine protease cleavage of MYOC appears to be inhibited by the cysteine protease inhibitor cystatin A. Induction of the expression of this inhibitor could represent the common pathway used by MYOC mutants to cause the glaucoma disease in the eye. Silencing cystatin A might be investigated as a potential treatment for MYOC-linked glaucoma.(1) Escribano’s lab JBC (2005) 280:21043-51; JBC (2007) 282:27810-24; Mol Vis (2008) 14, 2097
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