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A Novel Luciferase Assay For Sensitively Monitoring Myocilin Variants in Cell Culture
Serena Zadoo1, Annie Nguyen1, Gulab Zode2
1Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas, United States.
Investigative Ophthalmology & Visual Science
|April 20, 2016
Summary
A novel reporter assay effectively detects mutant myocilin (MYOC) secretion in glaucoma research. This tool aids in studying MYOC variants and identifying potential therapeutic modulators.
Area of Science:
- Biochemistry
- Genetics
- Ophthalmology
Background:
- Mutations in myocilin (MYOC) are linked to primary open-angle glaucoma.
- These MYOC mutations often result in protein "nonsecretion," complicating detection with standard methods.
Purpose of the Study:
- To develop and validate a rapid and sensitive assay for detecting mutant MYOC secretion.
- To facilitate the study of MYOC variants and their role in glaucoma pathogenesis.
Main Methods:
- Fused Gaussia luciferase (eGLuc2) to MYOC variants for expression in cell lines.
- Evaluated secreted and intracellular MYOC eGLuc2 levels using Western blotting and a GLuc assay.
- Assayed secretion of nine additional MYOC mutants to confirm assay applicability.
Main Results:
- The MYOC eGLuc2 reporter system accurately reflected MYOC secretion and intracellular levels.
- The GLuc assay detected Y437H MYOC secretion within 30 minutes.
- Disease-associated MYOC mutants showed significant secretion defects, while nonpathogenic variants were secreted normally.
- Partial rescue of secretion defects was observed for some mutants at a permissive temperature, independent of new protein synthesis.
Conclusions:
- Fusion of eGLuc2 to MYOC does not alter its biological behavior.
- This novel reporter system is a valuable tool for studying MYOC variants and discovering modulators of MYOC secretion and function in glaucoma.
