Abstract
Purpose :
Previously, we have shown that the deletion of 54FLRAPSW61 region containing the primary phosphorylation site (S59) in αB-Crystallin results in a 2-fold decrease in oligomeric mass and a 10-fold increase in chaperone activity. This study aimed to see if the deletion or a phosphomimetic substitution of Ser59 residue will also have a similar effect on αB-Crystallin's oligomeric state and chaperone function.
Methods :
Recombinant αB-WT, αBΔS59, αBS59D, and αBS59A purified to over > 95% was used. The oligomeric mass of the proteins was estimated using a multi-angle light scattering detector. The chaperone activities of the recombinant proteins were evaluated using luciferase (0.5 μM) and alcohol dehydrogenase (ADH) (100 mg) substrates. The chaperone assay was carried out in 0.25 mL of PBS containing 50 mM EDTA using multiple concentrations of the chaperone proteins (0 – 50 μM) on a plate reader set at 37oC, and the absorbance was monitored at 360 nm for up to 1h.
Results :
The average oligomeric mass of αB-WT decreased marginally from 565 kDa to 549 kDa in αBΔS59, and in αBS59D, the oligomeric mass dropped significantly to 451 kDa. The αBS59A showed an average mass of 487 kDa. At 10 μM concentration, the WT protein showed 28% and 57% protection against luciferase and ADH aggregation, respectively. When used at the same concentration, the αBΔS59, αBS59D, and αBS59A showed 21%, 45%, and 25% protection, respectively, against luciferase, and with ADH, 30%, 78%, and 46% protection, respectively, were seen. At 40 μM, the WT and the mutants showed over 90% protection of both substrates.
Conclusions :
Our study shows that S59 residue has no direct role in the oligomer formation or chaperone function of αB-Crystallin but can regulate these features. When phosphorylated, it decreases the oligomeric size and increases the chaperone function. The extent of reduction in the oligomeric size and increase in chaperone activity seen with αBS59D is much less when compared to the αBD54-61 mutant.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.