Mutation R120G in alphaB-crystallin, which is linked to a desmin-related myopathy, results in an irregular structure and defective chaperone-like function

Abstract

alphaB-crystallin, a member of the small heat shock protein family, possesses chaperone-like function. Recently, it has been shown that a missense mutation in alphaB-crystallin, R120G, is genetically linked to a desmin-related myopathy as well as to cataracts [Vicart, P., Caron, A., Guicheney, P., Li, A., Prevost, M.-C., Faure, A., Chateau, D., Chapon, F., Tome, F., Dupret, J.-M., et al. (1998) Nat. Genet. 20, 92-95]. By using alpha-lactalbumin, alcohol dehydrogenase, and insulin as target proteins, in vitro assays indicated that R120G alphaB-crystallin had reduced or completely lost chaperone-like function. The addition of R120G alphaB-crystallin to unfolding alpha-lactalbumin enhanced the kinetics and extent of its aggregation. R120G alphaB-crystallin became entangled with unfolding alpha-lactalbumin and was a major portion of the resulting insoluble pellet. Similarly, incubation of R120G alphaB-crystallin with alcohol dehydrogenase and insulin also resulted in the presence of R120G alphaB-crystallin in the insoluble pellets. Far and near UV CD indicate that R120G alphaB-crystallin has decreased beta-sheet secondary structure and an altered aromatic residue environment compared with wild-type alphaB-crystallin. The apparent molecular mass of R120G alphaB-crystallin, as determined by gel filtration chromatography, is 1.4 MDa, which is more than twice the molecular mass of wild-type alphaB-crystallin (650 kDa). Images obtained from cryoelectron microscopy indicate that R120G alphaB-crystallin possesses an irregular quaternary structure with an absence of a clear central cavity. The results of this study show, through biochemical analysis, that an altered structure and defective chaperone-like function of alphaB-crystallin are associated with a point mutation that leads to a desmin-related myopathy and cataracts.

Figure 1

Gel filtration chromatography performed on a Superose HR-6 column of human wild-type and R120G αB-crystallin.

Figure 2

Near and far UV CD of human wild-type and R120G αB-crystallin. (A) Near UV CD spectra of wild-type and R120G αB-crystallin. Each spectrum represents the average of 32 scans, and the concentration for each sample was 2.2 mg/ml. (B) Far UV CD spectra of wild-type and R120G αB-crystallin. Each spectrum represents the average of 16 scans with each sample containing a 1.0-mg/ml concentration of wild-type or R120G αB-crystallin. Estimates of secondary structure were performed as described in Materials and Methods. For wild-type αB-crystallin, the secondary structure was predicted to contain 10% α-helix, 44% β-sheet, 16% β-turns, and 30% other. For R120G αB-crystallin, the secondary structure was predicted to contain 15% α-helix, 33% β-sheet, 19% β-turns, and 33% other.

Figure 3

Cryo-EM of wild-type and R120G αB-crystallin. Shown are seven representative images of wild-type and R120G αB-crystallin assemblies after low-pass filtering to 4-nm resolution, translationally aligning, and masking. The lower rightmost image of each set is a rotationally averaged sum of 200 filtered and translationally aligned images. The protein density appears white. (Bars = 10 nm.)

Figure 4

Aggregation of α-lactalbumin after the addition of 20 mM DTT at 37°C and pH 6.7. Curve 1, 0.25 mg of α-lactalbumin; curve 2, 0.25 mg of α-lactalbumin plus 0.25 mg of wild-type αB-crystallin; curve 3, 0.25 mg of α-lactalbumin plus 0.25 mg of R120G αB-crystallin. The Inset is an SDS/polyacrylamide gel that illustrates the particulate and soluble fractions obtained after a 60-min incubation of R120G αB-crystallin with α-lactalbumin in the presence of DTT, as shown in curve 3. Lane A, αB-crystallin; lane B, α-lactalbumin; lane C, soluble fraction of α-lactalbumin plus R120G αB-crystallin; lane D, particulate fraction of α-lactalbumin plus R120G αB-crystallin.

Figure 5

Aggregation of alcohol dehydrogenase induced by heat (42°C) and 2 mM EDTA. Curve 1, 0.2 mg of alcohol dehydrogenase; curve 2, 0.2 mg of alcohol dehydrogenase plus 0.2 mg of wild-type αB-crystallin; curve 3, 0.2 mg of alcohol dehydrogenase plus 0.2 mg of R120G αB-crystallin; curve 4, 0.2 mg of alcohol dehydrogenase plus 0.4 mg of R120G αB-crystallin.

Figure 6

Aggregation of the insulin B chain after the addition of 20 mM DTT at 37°C. Curve 1, 0.1 mg of insulin; curve 2, 0.1 mg of insulin plus 0.1 mg of wild-type αB-crystallin; curve 3, 0.1 mg of insulin plus 0.1 mg of R120G αB-crystallin; curve 4, 0.1 mg of insulin plus 0.2 mg of R120G αB-crystallin.

Figure 7

Chaperone-like function of heterooligomers containing wild-type and R120G αB-crystallin with α-lactalbumin as a substrate. Wild-type αB-crystallin was premixed with R120G αB-crystallin for 2 h at 37°C to form complexes containing different ratios of wild-type αB-crystallin/R120G αB-crystallin subunits. Curve 1, 0.25 mg of α-lactalbumin; curve 2, 0.25 mg of α-lactalbumin plus 0.125 mg of wild-type αB-crystallin; curve 3, 0.25 mg of α-lactalbumin plus 0.25 mg of R120G αB-crystallin; curve 4, 0.25 mg of α-lactalbumin plus a complex containing 0.125 mg of wild-type αB-crystallin and 0.125 mg of R120G αB-crystallin; curve 5, 0.25 mg of α-lactalbumin plus a complex containing 0.167 mg of wild-type αB-crystallin and 0.083 mg of R120G αB-crystallin.