Peptide-induced formation of protein aggregates and amyloid fibrils in human and guinea pig αA-crystallins under physiological conditions of temperature and pH.

αA peptide forms with age in the lens nucleus of humans and guinea pigs， and binds to αA-crystallin by hydrophobic bonds to produce protein aggregates. The purpose of the present study was to use negative-stain transmission electron microscopy (TEM) to investigate peptide/crystallin structures that form when αA peptide is incubated with recombinant human (HU) and guinea pig (GP) αA-crystallin， and GP αA-crystallin， under physiological conditions of temperature and pH. HU and GP αA-crystallin each consists of 173 amino acid residues with only eight differences. GP αA-crystallin contains an insertion of 23 amino acids into the sequence for normal GP αA-crystallin. Synthetic αA peptide was incubated with each crystallin for 24 h in phosphate buffered saline at 37 C， pH 7.4. Samples were loaded onto electron microscopy grids and analyzed by TEM. With GP αA-crystallin， the αA peptide appeared to first form up to 700 nm long， independent peptide fibrils， which subsequently bound numerous αA-crystallin oligomers along the entire length of the fibril to form up to 30 nm thick peptide/oligomer fibril structures. In contrast， HU αA-crystallin somehow prevented independent αA peptide fibrillation， and instead joined with the peptides to form amorphous aggregates and linear chains of αA oligomers. When incubated alone， GP αA-crystallin eventually lost all oligomeric structure and formed amorphous aggregates. However， addition of αA peptide to the incubation medium resulted in the formation of highly-organized peptide/αA oligomer amyloid fibrils， nearly 400 nm in length， with oligomer dimers appearing to stack one on top of another. Mature peptide/αA oligomer fibrils were highly compressed and 40% thicker than observed protofibrils (17 nm vs. 12 nm thickness). Use of a control V72P αA-crystallin peptide， as well as addition of the disulfide bond reducing agent DTT to the incubation medium， prevented fibril formation. Turbidity and Thioflavin T fluorescence analyses confirmed the observed peptide-induced fibril formation. This study represents the first time that fibrillation of αA-crystallin has been accomplished under physiological conditions of temperature and pH without use of denaturants. Fibrillation of GP αA-crystallin was linked with possible intermolecular disulfide-crosslinking of oligomers. The finding that αA peptide induces aggregate formation with HU αA-crystallin supports a role for this peptide in the development of HU nuclear cataract.