Amyloid β-Sheet Secondary Structure Identified in UV-Induced Cataracts of Porcine Lenses using 2D IR Spectroscopy.

Cataracts are formed by the aggregation of crystallin proteins in the eye lens. Many in vitro studies have established that crystallin proteins precipitate into aggregates that contain amyloid fibers when denatured, but there is little evidence that ex vivo cataracts contain amyloid. In this study, we collect two-dimensional infrared (2D IR) spectra on tissue slices of porcine eye lenses. As shown in control experiments on in vitro αB- and γD-crystallin, 2D IR spectroscopy can identify the highly ordered β-sheets typical of amyloid secondary structure even if the fibers themselves are too short to be resolved with TEM. In ex vivo experiments of acid-treated tissues, characteristic 2D IR features are observed and fibers >50nm in length are resolved by transmission electron microscopy (TEM), consistent with amyloid fibers. In UV-irradiated lens tissues, fibers are not observed with TEM, but highly ordered β-sheets of amyloid secondary structure is identified from the 2D IR spectra. The characteristic 2D IR features of amyloid β-sheet secondary structure are created by as few as four or five strands and so identify amyloid secondary structure even if the aggregates themselves are too small to be resolved with TEM. We discuss these findings in the context of the chaperone system of the lens, which we hypothesize sequesters small aggregates, thereby preventing long fibers from forming. This study expands the scope of heterodyned 2D IR spectroscopy to tissues. The results provide a link between in vitro and ex vivo studies and support the hypothesis that cataracts are an amyloid disease.