PURPOSE: The present study compared properties of wild-type and mutated mouse and human myocilin (Myoc) proteins as a prerequisite for development of a mouse model of glaucoma. METHODS: cDNA encoding full-length mouse Myoc was cloned into the p3XFLAG-CMV-14 vector. Tyr423His and Ile463Ser mutations were introduced into the mouse Myoc protein by in vitro mutagenesis. Intracellular localization and secretion of wild-type and mutated mouse Myoc proteins were studied in immunostaining and Western blotting experiments, respectively, after transfection into COS-7 cells. RESULTS: Similar to human MYOC, wild-type and mutated mouse Myoc demonstrated vesicular staining in transfected cells. However, while wild-type human and mouse Myoc were preferentially located in both the endoplasmic reticulum and Golgi, mutated human and mouse Myoc were located mainly in the endoplasmic reticulum and were excluded from Golgi. Similar to mutations in human MYOC, mutations in mouse Myoc dramatically reduced its secretion from transfected cells. Secretion of mutated Myoc was partially restored by culturing cells at 30 degrees C instead of 37 degrees C. The presence of mutated human MYOC prevented secretion of wild-type mouse Myoc but did not dramatically affect secretion of alkaline phosphatase, thrombospondin, Timp3 or olfactomedin-1. CONCLUSIONS: Properties of the mouse Myoc protein are similar to those of the human MYOC. The presence of mutated mouse or human Myoc does not block a general secretory pathway. Expression of mutated Myoc in the eye in mice may mimic human glaucoma and lead to development of a genetic mouse model of glaucoma.
PURPOSE: The present study compared properties of wild-type and mutated mouse and humanmyocilin (Myoc) proteins as a prerequisite for development of a mouse model of glaucoma. METHODS: cDNA encoding full-length mouseMyoc was cloned into the p3XFLAG-CMV-14 vector. Tyr423His and Ile463Ser mutations were introduced into the mouseMyoc protein by in vitro mutagenesis. Intracellular localization and secretion of wild-type and mutated mouseMyoc proteins were studied in immunostaining and Western blotting experiments, respectively, after transfection into COS-7 cells. RESULTS: Similar to humanMYOC, wild-type and mutated mouseMyoc demonstrated vesicular staining in transfected cells. However, while wild-type human and mouseMyoc were preferentially located in both the endoplasmic reticulum and Golgi, mutated human and mouseMyoc were located mainly in the endoplasmic reticulum and were excluded from Golgi. Similar to mutations in humanMYOC, mutations in mouseMyoc dramatically reduced its secretion from transfected cells. Secretion of mutated Myoc was partially restored by culturing cells at 30 degrees C instead of 37 degrees C. The presence of mutated humanMYOC prevented secretion of wild-type mouseMyoc but did not dramatically affect secretion of alkaline phosphatase, thrombospondin, Timp3 or olfactomedin-1. CONCLUSIONS: Properties of the mouseMyoc protein are similar to those of the humanMYOC. The presence of mutated mouse or humanMyoc does not block a general secretory pathway. Expression of mutated Myoc in the eye in mice may mimic humanglaucoma and lead to development of a genetic mouse model of glaucoma.
Authors: Athéna C Patterson-Orazem; Shannon E Hill; Yiming Wang; Iramofu M Dominic; Carol K Hall; Raquel L Lieberman Journal: Biochemistry Date: 2019-03-12 Impact factor: 3.162
Authors: Afia Sultana; Naoki Nakaya; Vladimir V Senatorov; Stanislav I Tomarev Journal: Invest Ophthalmol Vis Sci Date: 2011-04-20 Impact factor: 4.799