Mutations in Gamma-Crystallins Lead to Amyloid Fibre Formation

Abstract

Abstract: : Purpose:To determine if gamma-crystallin mutants can form amyloid fibrils.Methods:Lenses were collected from the Crygb_nop mouse. This mouse contains a truncated Crygb. The lenses were processed for electron microscopy. Crygb_nop was cloned by RT-PCR, sequenced and subcloned into mammalian (pcDNA3.1, pEGFP) and bacterial expression vectors. A GFP-fusion construct was also generated. Contructs were transfected into PtK2 cells. Crygb_nop was expressed in E. coli and the protein purified by ion exchange chromatography for in vitro studies.Results:Transfection of Crygb_nop into cells induced the formation of protein inclusions. These were both cytoplasmic and intranuclear. Electron microscopy revealed a filamentous substructure to the inclusions. These were similar in morphology to the intranuclear inclusions in E17.5 Crygb_nop mouse lenses. In vitro studies showed that Crygb_nop formed 6-15nm filaments with amyloid fibril-like characteristics. The filaments were further characterized in terms of Congo Red staining, thioflavin T binding and x-ray diffraction.Conclusion:The data show the value of careful analysis of established maouse mutations. These data suggest a dramatic change in our perception of cataract etiologies and future treatment strategies.

Keywords: 378 crystallins • 527 protein structure/function • 528 proteins encoded by disease genes