Abstract
Purpose :
Age-related cataracts are the most prevalent type of cataract and have protein aggregation as a hallmark. Yet, very little is known about the aggregates themselves that cause light scattering. The purpose of this research was to determine if the age-related modification, deamidation, induces aggregation directly without requiring further perturbation of the structure of gammaS-crystallin and if deamidation increases susceptibility to other modifications.
Methods :
In vivo deamidations located on the surface of gammaS-crystallin and associated with cataracts were mimicked using site-directed mutagenesis replacing Asn with Asp residues. To mimic physiological oxidation, proteins were incubated in oxidized glutathione to induce disulfide bonding. In order to determine if deamidation initiates crystallin aggregation, increases in the size and shape of deamidated crystallins compared to controls were determined by dynamic and static light scattering.
Results :
Light scattering aggregates of recombinantly expressed crystallins and their deamidation mimics were detected under physiological conditions by multiangle light scattering in-line with size-exclusion chromatography. Two light scattering peaks eluted, the main monomer peak and a high molecular weight (HMW) peak. This HMW peak was confirmed to be gammaS by mass spectrometry. Using dynamic light scattering without prior fractionation, the major population of gammaS was 2-3 nm reflecting a monomer with a minor component of a larger population between 40-50 nm. In the N14D/N76D/N143D mutant, both populations had a board size distribution and the larger population contributed more than 50% of the scatter intensity, but was less than 1% of the total mass. We also found that the thermal-induced aggregation of oxidized, deamidated human gammaS was greater than in the nondeamidated controls. When CuSO4 was added instead of glutathione, aggregation of the deamidated mutants was acheived without heating.
Conclusions :
Our findings support that deamidation directly leads to light scattering aggregates and expose buried Cys residues, increasing their propensity to form disulfide crosslinks leading to aggregation associated with cataracts.
This is a 2020 ARVO Annual Meeting abstract.