Monitoring mutant myocilin secretion and localization in trabecular meshwork cell cultures using a protein...

Area of Science:

• Ophthalmology
• Genetics
• Cell Biology

Background:

• Primary open angle glaucoma (POAG) is linked to mutations in the MYOC gene.
• Pathogenic MYOC mutants are typically not secreted from trabecular meshwork cells and accumulate in the endoplasmic reticulum.
• Current methods to assess MYOC protein secretion rely on immunoblotting, which can lack sensitivity.

Purpose of the Study:

• To develop and validate a sensitive luminescence-based assay for detecting MYOC protein secretion defects.
• To confirm secretion defects in previously identified MYOC variants associated with POAG.

Main Methods:

• A HiBiT tag was appended to MYOC to enable complement-based luminescence detection.
• Assays were performed in an immortalized trabecular meshwork cell line using 96-well and 6-well plates.
• MYOC variants (T377R, D384G, D395ins, C433Y, T455K, L486F) were tested for secretion and intracellular accumulation.

Main Results:

• The luminescence assay demonstrated poor secretion of tested MYOC mutants compared to wild-type MYOC.
• Intracellular accumulation of MYOC mutants was observed in cellular fractions.
• Luminescence signals correlated well with immunofluorescence and immunoblotting, showing higher sensitivity than immunoblotting.

Conclusions:

• The complement-based luminescence assay provides a facile and sensitive method for assessing MYOC protein localization.
• The study confirmed secretion defects in seven MYOC variants, reinforcing their role in POAG pathogenesis.