Amyloid fibril formation by the glaucoma-associated olfactomedin domain of myocilin

Abstract

Myocilin is a protein found in the extracellular matrix of trabecular meshwork tissue, 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 hasten increased intraocular pressure and glaucoma-characteristic vision loss due to irreversible damage to the optic nerve. In spite of reports on the intracellular accumulation of mutant and WT myocilin in vitro, cell culture, and model organisms, these aggregates have not been structurally characterized. In this work, we provide biophysical evidence for the hallmarks of amyloid fibrils in aggregated forms of WT and mutant myocilin localized to the C-terminal olfactomedin (OLF) domain. These fibrils are grown under a variety of conditions in a nucleation-dependent and self-propagating manner. Protofibrillar oligomers and mature amyloid fibrils are observed in vitro. Full-length mutant myocilin expressed in mammalian cells forms intracellular amyloid-containing aggregates as well. Taken together, this work provides new insights into and raises new questions about the molecular properties of the highly conserved OLF domain, and suggests a novel protein-based hypothesis for glaucoma pathogenesis for further testing in a clinical setting.

Figure 1

MBP-OLF fibrils and pre-fibrillar oligomers isolated from E. coli. (a) Overlay of Superdex 75 chromatographs from WT MBP-OLF, MBP-OLF(P370L), MBP-OLF(Y437H), and PK-treated MBP-OLF. (b) Corresponding ThT fluorescence of monomeric and aggregate MBP-OLF variants, as well as de novo formed myoc-OLF fibrils (asterisk, see text). (c) Micrographs of aggregates isolated in (a). Scale bar = 100 nm with two measured fibril widths indicated. (d) Detection of protofibillar oligomers in aggregates isolated in (a) using the A11 antibody. Anti-MBP used as a positive control where possible.

Figure 2

De novo and seeded fibril formation of myoc-OLF. (a) Representative nucleation-dependent fibrillization of myoc-OLF incubated at 37 °C monitored by ThT fluorescence. (b) Micrograph of de novo formed myoc-OLF fibrils generated in the same procedure as in (a). Scale bar = 100 nm with measured fibril width indicated (c) CD spectrum of myoc-OLF in vitro fibrils prepared by overnight incubation at 37 °C with 0.5 mM SDS (see text). (d) Representative multiple-round seeding kinetics with myoc-OLF. (e) Seeding kinetics with different quantities of seeds. (f) Representative de novo fibril formation of myoc-OLF(Y437H) and fibril formation initiated with WT myoc-OLF seeds. (g) Micrograph after treatment of myoc-OLF with 1.5% H₂O₂ incubated at 37 °C.

Figure 3

Myocilin fibrils isolated from CHO cells. (a) Gel mobility of Triton X-100 soluble and insoluble isolates from vector control (VC), WT myocilin, and myocilin(P370L). Left: gel without boiling. Accumulation of material appears at the stacking interface. Right: gel after steaming. Accumulated protein from stacking interface has entered the resolving gel. S = soluble, I = insoluble fraction, as designated by TX-100 fractionation. (b) In-cell ThT fluorescence of WT and P370L variants of myocilin. Overlay of differential interference contrast (DIC) and ThT fluorescence confirm intracellular aggregation.

Figure 4

Assembly of MBP-OLF fragments to identify regions of high amyloid propensity. (a) Myocilin sequence. Shaded area comprises the N-terminal signal sequence and coiled-coil not present in our OLF construct. Blue and green denote myoc-OLF. (b) Pictorial representation of MBO-OLF fragments discussed in text. (c) Overlay of Superdex 75 chromatograph of five MBP-OLF fragment constructs. (d) ThT fluorescence of void volume species isolated in (c). (e) ThT fluorescence of MBP and MBP-OLF_228-234 after incubation in a 37 °C water bath for 95 h with 0.5 mM SDS.