Abstract
Purpose :
We previously reported that deletion of 54FLRAPSWF61 sequence from αB-crystallin results in gain of function. Since αA- and αB- have significant (~54%) sequence homology, we hypothesized that deletion of 52LFRTVL57 from αA- will result in a gain of function. Thus, the present study was undertaken to see the effect of deleting 52-57 region on the structure-function of αA-crystallin.
Methods :
Chaperone activities of the purified recombinant αA-wt and the deletion mutant (αAΔ52-57) were evaluated using one µM luciferase and 100 µg alcohol dehydrogenase (ADH) substrates. The ability of the proteins to inhibit Aβ1-42 oligomerization and fibril formation in-vitro was examined under TEM. Cell integrity and ROS detection assays were performed to access the cytoprotective activity of the crystallins in sodium iodate-challenged ARPE-19 cells. The molecular size, hydrophobicity, and stability of the WT and mutant proteins were compared by standard methods.
Results :
The average molar mass of αA oligomers increased from 710 kDa to 1036 kDa in αAΔ52-57. The hydrodynamic radii (Rh) of the mutant also showed a similar increase in size. The αAΔ52-57 mutant exhibited a 7-fold and 5-fold increase in chaperone activity compared to αA-wt with the unfolding luciferase and ADH, respectively. αA- mutant also suppressed Aβ1-42 fibril formation in-vitro and showed 11 ± 0.39 % higher protection against Aβ1-42-induced cytotoxicity in ARPE-19 cells compared to WT. Sodium iodate-induced cytotoxicity and ROS detection studies showed that the mutant αA protein has 20 ± 1.06 % greater anti-apoptotic activity than WT with equal concentration of chaperone tested and also showed 15 ± 0.68 % higher anti-oxidative activity than the wild-type in oxidatively stressed cells.
Conclusions :
Our findings suggest that the residues 52-57 in αA-crystallin modulate oligomer size and chaperone activity. The deletion of the conserved 52-57 residues activates αA-crystallin. Further studies are required to understand the molecular basis for the activation of αA-crystallin after deletion of 52-57 sequence.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.