A Novel Luciferase Assay For Sensitively Monitoring Myocilin Variants in Cell Culture.
OBJECTIVE
Primary open angle glaucoma-associated mutations in myocilin (MYOC) cause protein "nonsecretion," rendering secreted MYOC difficult to detect using conventional techniques. This study focused on developing and using an assay that can quickly and easily detect mutant MYOC secretion.


METHODS
We fused Gaussia luciferase (eGLuc2) to MYOC variants and expressed the constructs in HEK-293T and NTM-5 cells. Secreted and intracellular levels of MYOC eGLuc2 variants were evaluated by Western blotting and compared to untagged and FLAG-tagged MYOC constructs. Secreted and soluble intracellular MYOC eGLuc2 were measured by a GLuc assay. The secretion of nine additional MYOC mutants was assayed in conditioned media from transfected cells to test the applicability of the assay for monitoring other MYOC variants.


RESULTS
Myocilin eGLuc2 behaved similarly to untagged and FLAG-tagged MYOC with respect to secretion, soluble intracellular levels, and in response to drug treatment. The GLuc assay could sensitively detect Y437H MYOC secretion 30 minutes after media change. Gaussia luciferase fused variants followed anticipated trends; nonpathogenic variants (D208E, G244V) were secreted at wild-type (WT) levels, whereas predicted disease-causing variants (C245Y, G246R, E300K, Y437H, I477N) demonstrated substantial secretion defects. Secretion defects caused by the C245Y, G246R, and Y437H mutations were partially rescued by permissive growth temperature. Interestingly, however, this increase in secretion was independent of newly synthesized protein.


CONCLUSIONS
Fusion of eGLuc2 to MYOC does not significantly change the behavior of MYOC. This newly developed MYOC reporter system can be used to study engineered MYOC variants and potentially to identify modulators of MYOC secretion and function.
Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas, United States.
Serena
S
Zadoo
Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas, United States.
Annie
A
Nguyen
Department of Cell Biology & Immunology and the North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, Texas, United States.
Gulab
G
Zode
Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas, United States 3Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas, United States.
John D
JD
Hulleman
D015153:Blotting, Western; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism*; D009154:Mutation / Q000235:genetics / Q000502:physiology; D060888:Real-Time Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012680:Sensitivity and Specificity; D000099179:Myocilin
D003598:Cytoskeletal Proteins; D005136:Eye Proteins; D006023:Glycoproteins; D008156:Luciferases; D011993:Recombinant Fusion Proteins; D000099179:Myocilin