pH-Driven Distinct Aggregation Pathways of Human γD-Crystallin

Human γD-Crystallin is an eye lens protein that helps maintain transparency in combination with α- and other βγ-crystallins. Despite its extreme stability, the protein aggregates in the lens, causing cataracts. Herein, we present different aggregation pathways of human γD-Crystallin at pH 2.0, 4.5, 7.4, and 65 °C under static conditions. At pH 2.0 and 65 °C, γD-Crystallin forms amyloid fibrils, as monitored by Thioflavin T (ThT) and Nile red fluorescence, and its morphology is confirmed by transmission electron microscopy (TEM). Secondary structure analysis by Fourier transform infrared (FT-IR) spectroscopy confirms the presence of a cross-β-sheet structure, a hallmark of amyloid fibrils. Interestingly, the protein shows fragmentation when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which is further confirmed by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF). At pH 4.5 and 65 °C, it also forms amyloid fibrils, however, without fragmentation due to conformational change in the monomer and subsequent exposure of hydrophobic surfaces, as observed by circular dichroism (CD) spectroscopy and 8-anilinonaphthalene-1-sulfonic acid (ANS) fluorescence, respectively. In contrast to the acidic pH, at physiological pH 7.4 and 65 °C, the protein forms amorphous aggregates, as observed by TEM, negligible ThT fluorescence intensity, and the absence of the cross-β-sheet structure as monitored by FT-IR spectroscopy. Our findings establish pH-driven distinct aggregation pathways of human γD-Crystallin, which may provide mechanistic insights into γD-Crystallin aggregation.