Abstract
Purpose: :
Myocilin is found in the trabecular meshwork, the anatomical region of the eye involved in regulating intraocular pressure. Wild-type (WT) myocilin has been associated with steroid-induced glaucoma, and variants of myocilin have been linked to early-onset, inherited glaucoma. Elevated levels and aggregation of myocilin are linked to a hastening of increased intraocular pressure and glaucoma-characteristic vision loss due to irreversible damage to the optic nerve. In spite of the reports of intracellular accumulation of mutant and WT myocilin in vitro, cell culture and model organisms, these aggregates have not been structurally characterized. We now provide biophysical evidence for the hallmarks of amyloid fibrils in aggregated forms of WT and mutant myocilin, localized to the C-terminal olfactomedin (myoc-OLF) domain.
Methods: :
Recombinant expression of the myoc-OLF domain in E. coli followed by affinity purification; tinctorial affinity for the thioflavin T (ThT) dye in vitro and in mammalian cell culture; resistance to proteolysis by the promiscuous protease proteinase K; visualization by transmission electron microscopy; recognition by a protofibril-specific antibody; electrophoretic mobility in SDS-PAGE; spontaneous and seeded kinetics.
Results: :
WT myoc-OLF can spontaneously form fibrils that appear as intertwined ropes within days, in the nucleation-specific manner expected of an amyloid when incubated under physiological conditions. Destabilizing conditions form ThT-positive fibrils that exhibit a familiar signature by circular dichroism, and these seeds accelerate myoc-OLF fibril. The less stable myoc-OLF(Y437H) variant forms fibrils more readily than WT. Cross-seeding reactivity with WT is suggestive of a common amyloidogenic peptidic core that can recruit new monomer. ThT-positive amyloid fibrils accumulate intracellularly when full-length myocilin, particularly the P370L variant, is expressed in mammalian cells.
Conclusions: :
WT myoc-OLF is a stable, monomeric globular domain with high β-sheet content that can be converted to a fibrillar morphology under mild conditions. Mutations or reagents that destabilize myoc-OLF accelerate fibril formation. Our observations of ThT-positive aggregates in cell culture link our conclusions in vitro to the body of studies that point to the disease-relevance of myocilin aggregation.
Keywords: protein purification and characterization • microscopy: electron microscopy