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pH-Driven Distinct Aggregation Pathways of Human γD-Crystallin
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DOI：10.1021/acs.jpcb.5c06855.png)
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Abstract
En 中文
Human gamma D-Crystallin is an eye lens protein that helps maintain transparency in combination with alpha- and other beta gamma-crystallins. Despite its extreme stability, the protein aggregates in the lens, causing cataracts. Herein, we present different aggregation pathways of human gamma D-Crystallin at pH 2.0, 4.5, 7.4, and 65 degrees C under static conditions. At pH 2.0 and 65 degrees C, gamma D-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-beta-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 degrees 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 degrees C, the protein forms amorphous aggregates, as observed by TEM, negligible ThT fluorescence intensity, and the absence of the cross-beta-sheet structure as monitored by FT-IR spectroscopy. Our findings establish pH-driven distinct aggregation pathways of human gamma D-Crystallin, which may provide mechanistic insights into gamma D-Crystallin aggregation.
Keywords:
AGE-RELATED-CHANGES
LENS CRYSTALLINS
AMYLOID FIBRILS
IN-VITRO
PROTEINS
SPECTROSCOPY
OXIDATION
CYSTEINE
REVEALS
FTIR
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