A Sensitive Assay for Monitoring Wild-Type and Mutant Myocilin Secretion

Date

2016-01-19

Authors

Zadoo, Serena

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Abstract

Primary open angle glaucoma (POAG)-associated mutations in myocilin (MYOC) cause protein 'non-secretion', rendering secreted MYOC difficult to quantitatively detect using time-consuming conventional techniques. This study focused on developing an assay which could be used to quickly and easily detect mutant and wild-type (WT) MYOC secretion. We fused Gaussia luciferase (eGLuc2) to MYOC variants and expressed the constructs in HEK-293T cells. The secretion, intracellular soluble and insoluble portions of WT and Y437H MYOC eGLuc2 constructs were evaluated by western blotting and compared to FLAG-tagged constructs. Secreted and soluble MYOC eGLuc2 was measured by a GLuc assay. The secretion of nine additional MYOC mutants was assayed in conditioned media from HEK-293T and NTM-5 cells to test the general applicability of the assay. MYOC eGLuc2 behaved similarly to FLAG MYOC with respect to secretion, soluble intracellular levels, and in response to drug treatment. The GLuc assay could sensitively detect Y437H MYOC secretion 30 min after a media change. eGLuc2 fused variants followed predicted trends; non-pathogenic variants (D208E, G244V) were secreted at WT-like levels, whereas predicted disease-causing variants (C245Y, G246R, E300K, Y437H, I477N) demonstrated substantial secretion defects ranging from 0.20 - 4.0% of WT MYOC levels in HEK-293T cells. These variants were slightly more tolerated in NTM-5 cells, resulting in secretion levels ranging from 1.0 - 12% of WT MYOC levels. Secretion defects caused by the C245Y, G246R, and Y437H mutations were partially rescued by permissive growth temperatures. Interestingly, a combination of growth temperature reduction and cycloheximide treatment of transfected cells indicate that the pool of protein that is rescued at lower temperatures arises from intracellular stores, and is not from newly synthesized MYOC under permissive temperature. Fusion of eGLuc2 to MYOC does not significantly change the behavior of MYOC. The use of the eGLuc2-tagged version of MYOC can be utilized to develop a deeper understanding of MYOC folding and secretion. This newly developed MYOC reporter system has the potential to be used in small molecule and/or genetic high-throughput screens to identify modulators of MYOC secretion.

General Notes

The 54th Annual Medical Student Research Forum at UT Southwestern Medical Center (Monday, January 19, 2016, 2-5 p.m., D1.700)
Each year the Medical Student Research Program awards students for the best oral presentation and the best poster presentation as judged by faculty across campus. This author received an award as one of the best poster presentations at this forum.

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Citation

Zadoo, S., Nguyen, A., Zode, G., & Hullerman, J. D. (2016, January 19). A sensitive assay for monitoring wild-type and mutant myocilin secretion. Poster presented at the 54th Annual Medical Student Research Forum, Dallas, TX. Retrieved from https://hdl.handle.net/2152.5/3262

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