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

Abstract

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 amyloidogenic β-sheet secondary structure because this structure has exceptionally strong coupling and a high degree of ordering that creates characteristic diagonal and cross peaks. In ex vivo experiments of acid treated tissues, characteristic 2D IR features are observed and fibers >50 nm in length are resolved by TEM, consistent with amyloid fibers. In UV-irradiated lens tissues, fibers are not observed with TEM, but amyloidogenic β-sheet 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 4 or 5 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 is an amyloid disease.

Amyloid found in human cataracts with two-dimensional infrared spectroscopy

“…The frequency along ω probe also differs, signifying a change in the a+ vibrational mode that is created by couplings along the β-strands. The cross-peak frequencies are consistent with the presence of amyloid structure as established previously by in vitro crystallin and ex vivo porcine lens experiments (31).…”

“…At least 50 β-strands are necessary to create fibrils of sufficient elongation to be identified with in vitro TEM (41), whereas only 4 to 5 β-strands are needed to create the three spectroscopic observables of amyloid in 2DIR spectra (26,31). Previously, we resolved amyloid fibrils in TEM images of acid-treated porcine lenses (31), suggesting that TEM images would reveal amyloid in human tissue if fibrils were formed that are extended and well structured. A possible explanation for unidentifiable amyloid fibrils via TEM is that the amyloid β-sheets might reside in protofibrils or fibrils shorter than 50 nm.…”

“…These peaks correspond to the fundamental and overtone vibrational transitions. Native β-sheets exhibit an amide I absorption feature between 1,630 cm −1 and 1,640 cm −1 in a 2D slice (31,33). These two peaks are elongated along the diagonal, indicating that this mode is inhomogeneously broadened, presumably due to the distribution of structures from the many different crystallin proteins present in the lens sample.…”

“…Amide I 2DIR spectra have not previously been reported for human tissues. We followed our recently developed protocol for porcine lenses with some modifications (31). We sectioned unfixed lenses frozen in optimum cutting temperature medium, and placed each 25-μm-thick slice on a poly-L-lysine−treated CaF 2 window.…”

Not All β-Sheets Are the Same: Amyloid Infrared Spectra, Transition Dipole Strengths, and Couplings Investigated by 2D IR Spectroscopy

“…For example, a recent study used transition dipole strengths to estimate amyloid formation in the tissues of pigs, providing evidence that cataract formation may be an amyloid disease. 74 Transition dipole strengths can also be useful for determining secondary structure in congested regions of the spectrum where multiple secondary structures can absorb at the same frequency (e.g., random coil vs. α helix). With knowledge of both the transition dipole strength and frequency, secondary structures can be assigned with much higher confidence.…”