A Novel Luciferase Assay For Sensitively Monitoring Myocilin Variants in Cell Culture

Abstract

PurposePrimary 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.MethodsWe 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.ResultsMyocilin 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.ConclusionsFusion 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.

Clinically-identified C-terminal mutations in fibulin-3 are prone to misfolding and destabilization

“…Mapping and testing F3 mutations as we have performed, combined with comprehensive ocular phenotypic data, will provide more information regarding whether there are certain 'hot-spots' in F3 that are prone to secretion-compromising or pathogenic mutations. For example, the combination of analogous studies [40][41][42][43] have led to the determination that the olfactomedin (OLF) domain is a 'hot-spot' for many pathogenic mutations in myocilin (MYOC). Likewise, we noticed a trend that C-terminal mutations (i.e., mutations occuring after N249, as an approximate midpoint) were more prone to either secretion defects (e.g., R345W and L451F), or further destabilization (i.e., N249Q/Y397H).…”

Endoplasmic Reticulum Stress Response of Trabecular Meshwork Stem Cells and Trabecular Meshwork Cells and Protective Effects of Activated PERK Pathway

“…Although our results show that TUN, BreA, and Thap can effectively induce ER stress in TMSCs and TM cells in vitro, the mechanism is different from the actual pathology of glaucoma. Zadoo et al46 reported successful transfection of mutant myocilin into TM cells using plasmids. Jain et al47 used CRISPR-Cas9 technology to transfect human TM cells with mutant myocilin and successfully induced ER stress in those cells.…”

Stem cell transplantation rescued a primary open-angle glaucoma mouse model

“…Recombinant Lentiviral Vector encoding Myoc Y437H was constructed from plasmids pLenti CMV-GFP (Addgene 17448) ( Campeau et al, 2009 ), p CAGIG2 (Addgene 111159) ( Matsuda and Cepko, 2004 ) and pcDNA3 Myoc Y437H ( Zadoo et al, 2016 ) (a kind gift from Dr. Hulleman at UT Southwestern). Briefly, pLenti CMV-GFP-Puro was digested with BamHI and SalI (NEB) to remove GFP cassette and served as the vector backbone, which was utilized to generate lentivirus encoding plasmid (pLenti CMV-IRES-GFP ) by insertion of IRES-EGFP cassette (obtained from p CAGIG2 ) into it.…”