Abstract
Purpose :
The aging human lens accumulates protein modifications that are dramatically elevated in cataracts. Among chemical processes involved ascorbylation protein adducts carboxymethyl-lysine (CML), carboxyethyl-lysine (CEL) and methylglyoxal hydroimidazole (MG-H1) (Fan et al Aging Cell 2020) are thought to destabilize the proteins based on Ortwerth(1968) who showed that crystallins incubated with ascorbate precipitate under both aerobic and anaerobic conditions. Here we determined ascrobylation sites present solely in insoluble protein exposed to ascorbate.
Methods :
Calf lens crystallin homogenate was fractionated by Sephadex G200 gel filtration into Pool 1-4 (P1-P4) corresponding to alpha-, beta-H, beta-L and gamma crystallin, respectively. Pool 3 and 4 were incubated sterile in 50 mM K+/PO4 buffer for 7days with 10 mM ascorbate or dehydroascorbate and centrifuged. Precipitates and supernatants were processed for proteomics analysis. Electrospray MS/MS spectra were obtained and tryptic peptides were analyzed for modified lysine (CML (m/z: +58), CEL (m/z: +72)) and arginine residues by MG-H1 (m/z: +54).
Results :
30-40% of P3 proteins precipitated upon incubation with ASA or DHA. Presence of chelator DTPA suppressed precipitation from DHA, but not ASA. Precipitation of P4 proteins was suppressed by DTPA suggesting ASA/DHA fragmentation are necessary for precipitation. Robust CML modifications sites were K88 (CRYAA), K23 (CRYBB1), K168 (CRYBB2) and K163 (CRYGD). For CEL K168 (CRYBB2). For MG-H1: R188 (CRYBB2), R147 (CRYGC) and R10 (CRYGD). Multiple other sites were also modified, but deemed less important for insolubilization. The significance of the ascorbylation sites for cataract remains to be established. However, K88 is in the minichaperone domain of CRYAA, K23 in the highly conserved sequence GPDGKGK(23)G of CRYBB1, K168 in the conserved QYLLEKGD domain of CRYBB2. R188 in CRYBB2 is in the last strand of βB2-crystallin Greek-key motif 4 and its mutation to R188H promotes βB2-crystallin aggregation via perturbation of the dynamic equilibrium by dissociating the dimer and stabilizing the tetramer (Zhang et al. BBA 1842, 2014). R10 in CRYGD is highly conserved and so is the positive charge at K/R163.
Conclusions :
The uncovered robust ascorbylation sites in various bovine crystallins are hypothesized to play a destabilizing role in age-related human cataract.
This is a 2021 ARVO Annual Meeting abstract.