Mutation in myocilin affect it's processing and secretion in the trabedular meshwork cell




Jacobson, Nasreen


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Jacobson, Nasreen, Mutations in myocilin affect it secretion and processing in the cell. Doctor of Philosophy (Cell Biology and Ginetics), May 2003, 157 pp., 6 tables, 46 illustrations, 17 movies. Introduction. Myocilin is the protein product of the glaucoma gene MYOC whose function is unknown. Structural predictions of the protein indicate myocilin is secreted. This study uses several techniques to determine whether myocilin is synthesized and processed through the secretory pathway. Methods. Agents known to disrupt the secretory pathway at specific organelles will be used to examine the effect on myocilin secretion. Also, constructs for chimeric myocilin and fluorescent proteins (myoc.504DsRED and myoc.504EGFP) will be used in conjunction with EGFP directed to specific organelles to determine colocalization of myocilin in the cell. The disruption of wild type and disease-causing mutants (myocQ368X.DsRED, myocG364V.504DsRED and myocY437H.504DsRED) of myocilin will be compared. Then in vivo studies will be used to try to determine if myocilin is associated with increased intraocular pressure (IOP). Results. Myocilin appears as a doublet on SDS-PAGE western blots when probed with anti-myocilin antibody (AB129). Treatment of cells with tunicamycin prevents secretion of the upper band of the myocilin doublet, but not secretion of the lower band. Brefelden A prevents secretion of both bands of the myocilin doublet indicating that both bands are processed in the Golgi. Monensin treatment indicates there is no post-Golgi processing of myocilin prior to secretion. Colocalization of fluorescent myocilin with cellular organelles tagged with EGFP indicated that myocilin travels through the ER, Golgi and is secreted from the cell. Disease-causing mutations in myocilin are not secreted. The Q368X associates with wild type myocilin and appears to be degraded. The G364V and Y437H mutants can apparently be retained in the ER and also are closely associated with peroxisomes. Experiments designed to determine if myocilin can be correlated with increased IOP suggest an association of myocilin with increased IOP in an ex vivo human anterior segment perfusion system, but in vivo experiments gave inconclusive results. Conclusions. Myocilin is a secreted glycoprotein in the TM. Glaucomatous mutations in myocilin cause non-secretion. TM cells handle different myocilin mutations differently.