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.

青光眼（Glaucoma）是一类神经退行性疾病，亦是全球范围内不可逆失明的首要致病诱因。肌纤蛋白相关性青光眼（Myocilin-associated glaucoma）作为该疾病的遗传性亚型，由错误折叠的突变型肌纤蛋白在细胞内聚集所引发。体外实验中，作为青光眼发病机制相关核心结构域的肌纤蛋白C端嗅素结构域（OLF），可在温和条件下被诱导形成淀粉样蛋白样纤维。本研究对体外纤维形成过程中存在的中间物种进行了系统表征：将纯化后的OLF按照后续电子显微镜成像与核磁共振波谱学检测所需的浓度进行纤维形成诱导；全程对OLF寡聚化反应的各组分开展硫黄素T（ThT）荧光检测与分析。结果显示，终末纤维混合物中含有硫黄素T阳性的纤维聚集体，上清液组分则主要包含硫黄素T阴性的物种；过滤后的上清液样品荧光信号微弱，证实体系中主要存在的物种为天然状态的OLF单体。