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RA Quinlan, A Sandilands, H Long, V Pigaga, A Prescott, G Vrensen, J Löster, J Graw, C MacPhee, C Dobson; Mutations in Gamma-Crystallins Lead to Amyloid Fibre Formation . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1922.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:To determine if gamma-crystallin mutants can form amyloid fibrils.Methods:Lenses were collected from the Crygbnop mouse. This mouse contains a truncated Crygb. The lenses were processed for electron microscopy. Crygbnop 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. Crygbnop was expressed in E. coli and the protein purified by ion exchange chromatography for in vitro studies.Results:Transfection of Crygbnop 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 Crygbnop mouse lenses. In vitro studies showed that Crygbnop 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.
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