Abstract
Purpose :
We developed 15N labeled standards for all 13 major human crystallins (αA, αB, βA2, βA3/A1, βA4, βB1, βB2, βB3, γA, γB, γC, γD, γS) and demonstrate their utility by measuring crystallin composition in young human lenses, and human WT and N101D αA in a transgenic mouse cataract model.
Methods :
All 13 major human lenses crystallins were expressed as His tagged N-terminal Sumo fusion proteins in minimal media supplemented with 15N. Each crystallin was then purified and quantified by protein assay. Linearity of quantitation was tested and crystallin abundances measured in quadruple digests of soluble protein from 5-day, 23-day, 18-month, and 18-year old human lenses, in addition to the soluble and insoluble fractions of lenses from 3-month old transgenic mice. Proteins were S-trap digested with trypsin, LC/MS data acquired using data-independent acquisition, and results analyzed using Skyline software. Quantification of both mouse αA and human αA transgene products was performed by comparison of unique and shared peptides to each species.
Results :
Quantification was linear over 2 orders of magnitude for all 13 major crystallins with R2 values >0.99. When individual digests of each aged lens were repeated, the average coefficient of variation was 2.3%. At 5-days of age, the most abundant lens crystallin was αA, comprising 22% of the total soluble fraction, while αB was present at only 3% (αA/αB ratio = 7.7). However, by 18 years, this ratio fell to 3.4. At 5-days age, the lowest abundance crystallins were βA2 (1.3%), γB (1.0%), and βB3 (0.07%). γB then increased 2-fold by 18-years, while both βb3 and βA2 decreased 15- and 4-fold, respectively, and γS increased from 9 to 17%. In transgenic mouse lenses αA comprised on average 20% of the soluble fraction, while the human αA transgene products made up approximately 0.7%. The presence of human αA N101D was associated with a decrease in the overall insolubilization of αA in mouse lenses from 14% of the total insoluble protein to 9.8% (p=0.05).
Conclusions :
The mixture of 15N labeled human crystallin standards provide an accurate measure of crystallin abundance in the human and mouse lenses. The αA N101D deamidation mimic may have decreased chaperone function, which may explain the lower abundance of αA in the lens insoluble fraction of αA N101D mice
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.