Abstract
Abstract: :
Purpose: Some mutant myocilins are not secreted. The relationship of this phenomenon to the observed toxic gain of function phenotype associated with glaucoma in heterozygotes is uncertain. We engineered panels of cell lines that stably express WT and Gln368stop myocilin (alone or in combination) by 2 methods: linearized plasmid transfection and lentiviral transduction (feline immunodeficiency virus vectors). Methods: Myocilin-negative human 293T cells were transfected or transduced to express myocilin, Gln368stop, or myocilin and Gln368stop, and stably selected by use of internal ribosome entry site-linked puromycin and phleomycin markers. Northern and Western analyses for myocilin were performed to determine mRNA and protein expression levels and to identify complex formation in cellular lysates and media (denatured, non-reducing SDS-PAGE immunoblots). Similarly transfected and transduced 293 GFPu-1 indicator cells, which assay proteasome dysfunction through GFP linked to a yeast degron, were analyzed for GFP expression. Results: In cells producing WT myocilin only, the protein was found in minimal amounts in cell lysates but at high levels in culture supernatants. In contrast, Gln368stop was found only in the lysates, confirming lack of secretion. Notably, in the double-expressing lines (analogous to the heterozygous clinical setting), much less myocilin was secreted and a correspondingly large increase in intracellular WT myocilin was observed. Northern blots showed equivalent mRNAs levels, indicating that these protein variations resulted post-translationally. Complex formation was also altered in the double-expressing cells, suggesting heterodimer formation. However, 293 GFPu-1 indicator cells showed no increase in fluorescence, whether transiently or stably over-expressing these genes singly or in combination. Conclusion: Co-expression of myocilin and Gln368stop causes altered myocilin complex formation and a profound reduction in WT myocilin secretion coupled with an increase in intracellular WT myocilin. This phenomenon does not affect the ubiquitin-proteasome system as assayed in GFPu-1 cells.
Keywords: genetics • mutations • trabecular meshwork