Myocilin-OLF oligomer reaction fractions ThT

Glaucoma is a group of neurodegenerative diseases that together are the leading cause of irreversible blindness worldwide. Myocilin-associated glaucoma is an inherited form of this disease caused by intracellular aggregation of misfolded mutant myocilin. In vitro, the myocilin C-terminal olfactomedin domain (OLF), the relevant domain for glaucoma pathogenesis, can be driven to form amyloid-like fibrils under mild conditions. Here we characterize a species present during in vitro fibrilization. Purified OLF was subjected to fibrillization at concentrations required for downstream electron microscopy imaging and NMR spectroscopy.ThT fluorescence measurements of OLF oligomer reactions fractions characterized throughout the work. The end-point fibril mixture contains the ThT-positive fibrils. The supernatant fractions contain largely non-ThT-positive species. The low fluorescence in the filtered supernatant sample confirms that the primary species is native OLF monomer.