Abstract
Purpose :
αA66-80 peptide accumulates in the nucleus of old human and guinea pig lenses and binds to αA-crystallin, but its effects on intact αA-crystallin oligomers are not well understood. We used transmission electron microscopy (TEM) to study the binding of the peptide to three recombinant αA-crystallins: human (173 amino acids, 2 –SH groups), guinea pig (173 amino acids, 1 –SH group) and guinea pig αAins (196 amino acids, 1 –SH group, comprising 10-20% of total αA-crystallin present in a guinea pig lens)
Methods :
Recombinant αA-crystallins (0.25 mg/ml) were incubated with synthetic αA66-80 peptide (0.05 mg/ml) in PBS, 37oC, for 0 and 24 hrs, with and without 50 mM dithiothreitol (DTT), and analyzed by TEM on carbon-coated copper grids following negative staining.
Results :
Each of the three αA-crystallins exhibited a well-ordered oligomer structure at 0 time; however, guinea pig αAins crystallin lost its ordered structure after 24 hrs, forming amorphous aggregates. Addition of αA66-80 peptide to the incubation medium for 24 hrs induced the formation of distinct amyloid fibrils in guinea pig αA-crystallin and guinea pig αAins crystallin, with the fibrils being much more highly organized with αAins crystallin. The mechanisms of formation of the two types of fibrils were very different. With guinea pig αA-crystallin, αA oligomers bound randomly to peptide fibrils to form peptide/oligomer fibrils of various lengths and thicknesses. With guinea pig αAins crystallin, peptide/oligomer tetramers first formed fibril chains which later lengthened, and then became compressed and significantly wider. In contrast, when αA66-80 peptide was incubated with human αA-crystallin for 24 hrs, formation of linear aggregate chains of peptide/αA oligomers, without fibril formation, was observed. Inclusion of DTT in the incubation mixture completely prevented αA66-80 peptide-induced formation of either amyloid fibrils or linear aggregate chains with the three αA-crystallins.
Conclusions :
αA66-80 peptide can induce either aggregate or amyloid fibril formation in human and guinea pig αA-crystallins, but free protein sulfhydryl groups appear to be required for each. The results highlight the importance of having a highly reduced environment present in the lens nucleus in order to maintain normal αA-crystallin function.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.