In vitro and in vivo study on the secretion of the Gly367Arg mutant myocilin protein

Janakaraj Kanagavalli, P J Eswari Pandaranayaka, Subbaiah Ramasamy Krishnadas, Sankaran Krishnaswamy, Periasamy Sundaresan
Molecular Vision 2007, 13: 1161-8

PURPOSE: 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.

METHODS: Normal and mutant MYOC cDNA constructs were used to transfect the TM cells. In order to confirm the method of transfection, reverse transcriptase polymerase chain reaction (RT-PCR) 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. Molecular modeling was done earlier using a knowledge based consensus method which involved threading the protein into the identified pentein fold for the COOH-terminal part. Molecular dynamics was carried out using GROMACS for the mutant model which was built using the native myocilin model.

RESULTS: The Gly367Arg mutation causes accumulation of myocilin protein within TM cells with extensively reduced secretion contrary to wild type myocilin being characterized by intracellular localization and extracellular secretion. Further, Gly367Arg mutation occurs in a hydrophobic region which leads to burial of a charged residue. The dynamics suggests large conformational change is required to accommodate the mutation favoring aggregation of the protein.

CONCLUSIONS: Our results suggest that Gly367Arg is a potential mutation that causes malfunction of TM cells either by dominant negative effect or gain of function of mutant myocilin. The structural model suggests that the mutated myocilin could aggregate, implying the possible role of Gly367Arg in causing Primary open angle glaucoma (POAG).

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