Mechanism of insolubilization by a single-point mutation in alphaA-crystallin linked with hereditary human cataracts

Abstract

AlphaA-crystallin is a small heat shock protein that functions as a molecular chaperone and a lens structural protein. The R49C single-point mutation in alphaA-crystallin causes hereditary human cataracts. We have previously investigated the in vivo properties of this mutant in a gene knock-in mouse model. Remarkably, homozygous mice carrying the alphaA-R49C mutant exhibit nearly complete lens opacity concurrent with small lenses and small eyes. Here we have investigated the 90 degrees light scattering, viscosity, refractive index, and bis-ANS fluorescence of lens proteins isolated from the alphaA-R49C mouse lenses and found that the concentration of total water-soluble proteins showed a pronounced decrease in alphaA-R49C homozygous lenses. Light scattering measurements on proteins separated by gel permeation chromatography showed a small amount of high-molecular mass aggregated material in the void volume which still remains soluble in alphaA-R49C homozygous lens homogenates. An increased level of binding of beta- and gamma-crystallin to the alpha-crystallin fraction was observed in alphaA-R49C heterozygous and homozygous lenses but not in wild-type lenses. Quantitative analysis with the hydrophobic fluorescence probe bis-ANS showed a pronounced increase in fluorescence yield upon binding to alpha-crystallin from mutant as compared with the wild-type lenses. These results suggest that the decrease in the solubility of the alphaA-R49C mutant protein was due to an increase in its hydrophobicity and supra-aggregation of alphaA-crystallin that leads to cataract formation. Our study further shows that analysis of mutant proteins from the mouse model is an effective way to understand the mechanism of protein insolubilization in hereditary cataracts.

Figure 1. Gel permeation profile of lens proteins from wild type mouse lenses

(A) Light scattering (solid gray line) and refractive index (dotted black line) measurements on proteins eluting from the column. The column was calibrated using molecular weight markers 1, dextran blue (2 MDa); 2, thyroglobulin (669 kDa); 3, ferritin (440 kDa); 4, catalase (232 kDa); 5, aldolase (156 kDa); 6, bovine serum albumin (67 kDa); 7, chymotrypsin (25 kDa) and 8, ribonuclease A (12.5 kDa). (B) The dashed line indicates the viscosity of proteins eluted from the column. The first peak is α-crystallin (αA + αB), the second is β-heavy (βH) crystallin; the third peak is βL (β-light) crystallin; and the fourth is γ-crystallin.

Figure 2. Abundance of a high molecular weight soluble protein species in αA-R49C homozygous lenses detected by light scattering

Wild type (dashed line); αA-R49C heterozygous (dotted line); αA-R49C homozygous lenses (solid gray line). Note the protein peak in the void volume of the gel permeation chromatography column in αA-R49C homozygous lens proteins but not in wild type or αA-R49C heterozygous proteins.

Figure 3. Refractive index and differential molecular weight distribution of crystallins isolated from wild type and αA-R49C mutant lenses

(A) Refractive index profile of crystallins separated by gel permeation chromatography. Wild type (dashed line); αA-R49C heterozygous (dotted line); αA-R49C homozygous lenses (solid gray line). Note the dramatic decrease in βL-crystallin in the soluble fraction of αA-R49C homozygous lenses. (B) Differential molecular weight distribution. Wild type (dashed line); αA-R49C heterozygous (dotted line); αA-R49C homozygous lenses (solid gray line). Note the diminution of βL-crystallin (60 kDa) in the αA-R49C homozygous lenses (arrowhead). Also note the increase in a 15 kDa protein in the αA-R49C homozygous lenses (arrow).

Figure 4. UV absorbance and immunoblot analysis of lens protein fractions from wild type and αA-R49C mutant lenses

Wild type lenses (dashed line); αA-R49C heterozygous lenses (dotted line); αA-R49C homozygous lenses (solid gray line). (A) UV absorbance at 280 nm; (B) Immunoblots represent the peak fractions of α-crystallin, βH-crystallin and βL- +γ-crystallin obtained from chromatographic separation of wild type, αA-R49C heterozygous and αA-R49C homozygous lens water-soluble proteins, respectively. Immunoblot analysis of the chromatography peak fractions with antibodies to αA-crystallin, total β-crystallin and total γ-crystallin was performed. Note the distinct loss of immunoreactivity to γ-crystallin antibody with peak 4 (γ-crystallin) in the αA-R49C homozygous lens proteins. Note also that the concentration of the water-soluble α-crystallin fraction (αA + αB) was the highest in the wild type, and decreased gradually in the αA-R49C heterozygous and homozygous lenses. (C) Immunoblot analysis of the α-crystallin fraction of wild type, αA-R49C heterozygous and αA-R49C homozygous lenses with antibodies to β- and γ-crystallin. Note the gradual increase in β- and γ-crystallin immunoreactivity in the heterozygous and homozygous α-crystallin fractions. Note also that β- and γ-crystallin immunoreactivity was absent in the wild type α-crystallin fraction.

Figure 5. Bis-ANS fluorescence in wild type and αA-R49C mutant lenses

Fluorescence of bis-ANS (5 µM) was measured using an excitation wavelength of 360 nm. The emission was measured from 400 to 600 nm. Emission spectrum of bis-ANS in buffer (dashed gray line); 0.1 mg/ml α-crystallin fraction from wild type mouse lenses (black dashed line); α-crystallin fraction from αA-R49C heterozygous mouse lenses (dotted line); and α-crystallin fraction from αA-R49C homozygous mouse lenses (solid gray line). Note the fluorescence emission maximum of bis-ANS at 480 nm upon binding to α-crystallin. Note also the prominent enhancement in fluorescence intensity of bis-ANS bound to α-crystallin fraction isolated from αA-R49C mutant lenses.

Figure 6. Thermal stability of lens proteins from wild type and αA-R49C mutant lenses

The thermal stability of α-crystallin fraction was determined by time-dependent change in light scattering as monitored at 360 nm at 65°C. Wild type (dashed line); αA-R49C heterozygous (dotted line); αA-R49C homozygous (solid gray line). By 10 minutes of incubation α-crystallin fraction of the αA-R49C lenses began to aggregate and precipitate out of solution. In contrast, wild type α-crystallin showed significantly lower light scattering.