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P. Sundaresan, J. Kanagavalli, P. Pandaranayaka, S. Krishnadas, S. Krishnaswamy; In vitro and in vivo Study on the Secretion of Gly367Arg Mutant Myocilin Protein. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5898.
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Mutations in the Myocilin gene (MYOC) leading to a perturbed outflow of aqueous in the trabecular meshwork (TM) has been associated with the pathophysiology of glaucoma. This study examines the expression of normal and mutant Myocilin (Gly367Arg) in cultured TM cells.
Normal and mutant MYOC cDNA constructs were used to transfect the TM cells. In order to confirm the method of transfection, RTPCR was carried out. Further, confocal microscopic analysis was used to determine the cellular localization of Myocilin protein. The extracellular nature of Myocilin in the culture supernatant and cell lysates of the transfected cells was analyzed by western blot. Myocilin protein in the aqueous humor of POAG patients, including patients with Gly367Arg was quantified by dot blot. Molecular Modeling and Dynamics for the mutant was demonstrated with the native Myocilin model using GROMACS.
Gly367Arg mutation causes accumulation of Myocilin protein within TM cells with extensively reduced secretion, while wild type Myocilin was characterized by intracellular localization and extracellular secretion. The secreted Myocilin in the aqueous humor of patients with Gly367Arg mutation correlated with the in vitro findings, confirming the disease-causing glaucomatous phenotype. Further, Gly367Arg mutation occurs in a hydrophobic region causing aggregation, leading to burial of a charged residue resulting in the conformational change to accommodate the mutation.
We suggest that Gly367Arg is a potential mutation causing malfunction of TM cells either by dominant negative effect or gain of function of mutant Myocilin. The structural model indicatesthe aggregation of Myocilin protein confirming the pathogenic significance of Gly367Arg.
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