Effect of glycation on alpha-crystallin structure and chaperone-like function

Abstract

The chaperone-like activity of alpha-crystallin is considered to play an important role in the maintenance of the transparency of the eye lens. However, in the case of aging and in diabetes, the chaperone function of alpha-crystallin is compromized, resulting in cataract formation. Several post-translational modifications, including non-enzymatic glycation, have been shown to affect the chaperone function of alpha-crystallin in aging and in diabetes. A variety of agents have been identified as the predominant sources for the formation of AGEs (advanced glycation end-products) in various tissues, including the lens. Nevertheless, glycation of alpha-crystallin with various sugars has resulted in divergent results. In the present in vitro study, we have investigated the effect of glucose, fructose, G6P (glucose 6-phosphate) and MGO (methylglyoxal), which represent the major classes of glycating agents, on the structure and chaperone function of alpha-crystallin. Modification of alpha-crystallin with all four agents resulted in the formation of glycated protein, increased AGE fluorescence, protein cross-linking and HMM (high-molecular-mass) aggregation. Interestingly, these glycation-related profiles were found to vary with different glycating agents. For instance, CML [N(epsilon)-(carboxymethyl)lysine] was the predominant AGE formed upon glycation of alpha-crystallin with these agents. Although fructose and MGO caused significant conformational changes, there were no significant structural perturbations with glucose and G6P. With the exception of MGO modification, glycation with other sugars resulted in decreased chaperone activity in aggregation assays. However, modification with all four sugars led to the loss of chaperone activity as assessed using an enzyme inactivation assay. Glycation-induced loss of alpha-crystallin chaperone activity was associated with decreased hydrophobicity. Furthermore, alpha-crystallin isolated from glycated TSP (total lens soluble protein) had also increased AGE fluorescence, CML formation and diminished chaperone activity. These results indicate the susceptibility of alpha-crystallin to non-enzymatic glycation by various sugars and their derivatives, whose levels are elevated in diabetes. We also describe the effects of glycation on the structure and chaperone-like activity of alpha-crystallin.

Figure 1. Non-tryptophan AGE fluorescence of α-crystallin

(A) Non-tryptophan AGE fluorescence of native and glycated α-crystallin. Native α-crystallin (trace 1); α-crystallin modified with 0.5 M glucose (trace 2), 0.1 M fructose (trace 3), 0.05 M G6P (trace 4) and 0.005 M MGO (trace 5). (B) Non-tryptophan AGE fluorescence of α-crystallin isolated from native TSP (trace 1) and α-crystallin isolated from TSP glycated with fructose (trace 2).

Figure 2. SDS/PAGE analysis of in-vitro-glycated α-crystallin

Native α-crystallin (lane 1), and α-crystallin modified with 0.1 M fructose (lane 2), 0.005 M MGO (lane 3), 0.05 M G6P (lane 4) and 0.5 M glucose (lane 5).

Figure 3. Characterization of AGEs by immunoblotting

Immunoblotting of α-crystallin with anti-CML (A, B and E), anti-AGE-BSA (C) and anti-MGO-BSA (D) antibodies. (A) Glycation of α-crystallin with glucose resulted in formation of CML. M, molecular-mass markers (in kDa); C, control; G1 and G2, α-crystallin glycated with 0.5 M and 1.0 M glucose respectively. (B) Glycation of α-crystallin with fructose resulted in formation of CML. M, molecular-mass markers; C, control; F, α-crystallin glycated with 0.1 M fructose. (C) Glycation of α-crystallin with G6P resulted in formation of AGE-BSA. C, control; G3, α-crystallin glycated with 0.05 M G6P. (D) Glycation of α-crystallin with MGO results in formation of MGO-AGE. M, molecular-mass markers; C, control; M1, modified with 0.005 M MGO. (E) α-Crystallin isolated from control TSP (C) and 0.1 M fructose-glycated TSP (F).

Figure 4. Phenylboronate affinity chromatogram

α-Crystallin (5 mg), native or glycated with the various agents, was applied to column. After washing the unbound fraction with 0.25 mM ammonium acetate, the bound fraction was eluted with 0.1 M Tris/HCl (pH 7.5) containing 0.2 M sorbitol.

Figure 5. Chaperone-like activity of α-crystallin

(A) Chaperone-like activity of α-crystallin as assessed by the suppression of heat-induced aggregation of β_L-crystallin. β_L-crystallin (0.2 mg/ml in 50 mM phosphate buffer pH 7.4) was incubated at 65 °C in the absence (trace 1) or in the presence of 0.025 mg/ml native α-crystallin (trace 2) or α-crystallin glycated with glucose (trace 3), fructose (trace 4), G6P (trace 5) or MGO (trace 6). The results shown were an average of three assays. (B) Chaperone-like activity of α-crystallin as assessed by the suppression of heat-induced aggregation of CS. CS (0.05 mg/ml) was incubated at 45 °C in the absence (trace 1) or presence of 0.025 mg/ml native α-crystallin (trace 2) or α-crystallin glycated with glucose (trace 3), fructose (trace 4), G6P (trace 5) or MGO (trace 6). The results shown were an average of three assays. (C) Chaperone-like activity of α-crystallin isolated from TSP as assessed by the suppression of heat-induced aggregation of β_L-crystallin. β_L-crystallin aggregation in the absence of α-crystallin (trace 1), in the presence of α-crystallin isolated from control TSP (trace 2) or in the presence of α-crystallin isolated from TSP glycated with fructose (trace 3).

Figure 6. UV CD spectra of α-crystallin

Far-UV CD spectra (A) and near-UV CD (B) spectra of α-crystallin. Trace 1, native α-crystallin; traces 2–5 correspond to α-crystallin modified with glucose, fructose, G6P and MGO respectively.

Figure 7. α-Crystallin fluorescence

Intrinsic tryptophan fluorescence (A) and ANS fluorescence (B) of α-crystallin. Trace 1, native α-crystallin; traces 2–5 correspond to α-crystallin modified with glucose, fructose, G6P and MGO respectively.