In vitro and in vivo study on the secretion of the Gly367Arg mutant myocilin protein.
OBJECTIVE
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).
Department of Genetics, Aravind Medical Research Foundation, Tamilnadu, India.
Janakaraj
J
Kanagavalli
P J Eswari
PJ
Pandaranayaka
Subbaiah Ramasamy
SR
Krishnadas
Sankaran
S
Krishnaswamy
Periasamy
P
Sundaresan
D001120:Arginine / Q000235:genetics*; D015153:Blotting, Western; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005136:Eye Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D005998:Glycine / Q000235:genetics*; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D050505:Mutant Proteins / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D017433:Protein Structure, Secondary; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D014129:Trabecular Meshwork / Q000166:cytology; D014162:Transfection; D000099179:Myocilin
D001120:Arginine; D003598:Cytoskeletal Proteins; D005136:Eye Proteins; D005998:Glycine; D006023:Glycoproteins; D050505:Mutant Proteins; D012333:RNA, Messenger; D000099179:Myocilin