Formation of amyloid fibrils in vitro by human γD-crystallin and its isolated domains

PURPOSE: Amyloid fibrils are associated with a variety of human protein misfolding and protein deposition diseases. Previous studies have shown that bovine crystallins form amyloid fibers under denaturing conditions and amyloid fibers accumulate in the lens of mice carrying mutations in crystallin genes. Within differentiating lens fiber cells, crystallins may be exposed to low pH lysosome compartments. We have investigated whether human γD-crystallin forms amyloid fibrils in vitro, when exposed to low pH partially denaturing conditions. METHODS: Human γD-crystallin expressed and purified from E. coli, is stable and soluble at 37 °C, pH7, and refolds from the fully denatured state back to the native state under these conditions. Purified Human γD-crystallin as well as its isolated NH2- and COOH-terminal domains were incubated at acid pH and subsequently examined by transmission electron microscopy, absorption spectroscopy in the presence of Congo red, FTIR, and low-angle X-ray scattering. RESULTS: Incubation of the intact protein at 37 °C in 50 mM acetate buffer pH 3 at 50 mg/ml for 2 days, led to formation of a viscous, gel-like solution. Examination of negatively stained samples by transmission electron microscopy revealed linear, non-branching fibrils of variable lengths, with widths ranging from 15 to 35 nm. Incubation with the dye Congo red generated the spectral red shift associated with dye binding to amyloid. Low-angle X-ray scattering from samples showed clear meridional reflection at 4.7 Å and a more diffuse reflection on the equator between 10 and 11 Å which is the typical “cross-β” X-ray fiber diffraction pattern for amyloid fibers. FTIR was used to follow the evolution of the secondary structure of γD-crystallin with time during incubation of the protein at pH 3. The native protein displayed a major band at 1640 cm-1 that converted during incubation at 37 °C to a band at 1616 cm-1. An additional band at 1689 cm-1 also appeared with time. The presence of bands in the regions about 1620 cm-1 and about 1680 cm-1 has been attributed to the formation of intermolecular β-sheet structure that characterizes the fibrillar amyloid motif. The isolated NH2-terminal 1–82 and COOH-terminal 86–174 domains of HγD-crystallin also formed amyloid fibrils after incubation under the same conditions, but to a lesser extent than the full length. CONCLUSIONS: HγD-crystallin, as well as its isolated NH2-terminal 1–82 and COOH-terminal 86–174 domains of HγD-crystallin formed amyloid fibrils upon incubation at acid pH. Investigations of early stages in cataract formation within the lens will be required to assess whether amyloid fibrils play a role in the initiation of cataract in vivo.