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Imbalances in the eye lens proteome are linked to cataract formation

Abstract

The prevalent model for cataract formation in the eye lens posits that damaged crystallin proteins form light-scattering aggregates. The α-crystallins are thought to counteract this process as chaperones by sequestering misfolded crystallin proteins. In this scenario, chaperone pool depletion would result in lens opacification. Here we analyze lenses from different mouse strains that develop early-onset cataract due to point mutations in α-, β-, or γ-crystallin proteins. We find that these mutant crystallins are unstable in vitro; in the lens, their levels are substantially reduced, and they do not accumulate in the water-insoluble fraction. Instead, all the other crystallin proteins, including the α-crystallins, are found to precipitate. The changes in protein composition and spatial organization of the crystallins observed in the mutant lenses suggest that the imbalance in the lenticular proteome and altered crystallin interactions are the bases for cataract formation, rather than the aggregation propensity of the mutant crystallins.

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Fig. 1: Effects of aging on the eye lens of wild-type mice.
Fig. 2: Cataract-associated mutant crystallins are conformationally destabilized.
Fig. 3: Mutant crystallins do not accumulate in the high-molecular-weight or water-insoluble fraction of lens extracts.
Fig. 4: SAXS reveals structural changes in the mutant eye lens.
Fig. 5: Imbalances in the murine eye lens proteome drive cataract formation.

Data availability

All data supporting the conclusions are available with the article. Raw data, including polyacrylamide gels, are available upon request to the authors.

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Acknowledgements

We thank D. Catici for experimental help; E. Bürkle, M. Stadler, G.-M. Feind and F. Rührnößl for their technical assistance; as well as F. Tippel and M. Schwieder from NanoTemper Technologies GmbH for the kind support with our nanoDSF measurements. This work was supported by a grant from the DFG (SFB 1035) to S.W., M.H. and J.B. This research was further supported by the Austrian Science Foundation (P28854, I3792, DK-MCD W1226 to T.M.), the Austrian Research Promotion Agency (FFG: 864690, 870454), the Integrative Metabolism Research Center Graz, the Austrian infrastructure program 2016/2017, the Styrian government (Zukunftsfonds) and BioTechMed/Graz. N.C.H.L. acknowledges funding from the Alexander von Humboldt Foundation. We thank H. Ehmann, J. Gautsch, P. Kotnik and A. Scheiflinger-Latal from Anton Paar Graz for their technical support with SAXS experiments on intact eye lenses and the production of a sample holder for eye lenses, as well as M. Kriechbaum for SAXS support. We thank F.-X. Schmid for comments on the manuscript and discussions.

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P.W.N.S, N.C.H.L, C.D., O.P., O.V.A., M.H., S.W., J.G, T.M. and J.B. designed the study, interpreted data and wrote the manuscript. P.W.N.S, N.C.H.L, C.P., K.C.B, B.R., B.B., F.P., J.P. and T.M. performed the experiments.

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Correspondence to Johannes Buchner.

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Peer review information Nature Structural & Molecular Biology thanks Roy Quinlan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Inês Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Characterization of the proteome of wild-type mice.

(a) Graphs show different measurements from C3HeB/FeJ (squares) and C57Bl/6 J (circles) mice. Top, body weight (filled symbols) and lens wet weight (open symbols). Middle and bottom, amounts of extractable water-soluble and -insoluble protein per lens wet weight was determined by the Bradford protein assay, using BSA as a standard. Data are given as mean and s.d. per lens pair (left and right eye) of one animal. Please see Supplementary Table 2 and 3 for exact sample size. (b) 2-DE map of the water-soluble fraction of lenses from newborn wild-type mice. For densitometric analysis (n = 2 individual animals), only the low molecular crystallin region was considered (see excerpt). The fraction of α- (gold), β- (orange) and γ-crystallins (blue) was determined up to 12 months of age, for the water-soluble (solid bars) and -insoluble (shaded bars) fraction. Please see Supplementary Data 1 for numerical values. Scheimpflug images of C57Bl/6 J lenses at 3, 6 and 12 months are shown as a reference for wild-type behavior.

Extended Data Fig. 2 Changes in the proportion of crystallin isoforms during aging in wild-type and mutant mice.

Graphs show relative proportions of single crystallin isoforms, plotted against age, in the water-soluble or -insoluble fractions from whole lens extracts (C3HeB/FeJ (black squares), αA mutant (red circles), γD mutant (red triangles) and C57Bl/6 J (black squares), βA2 mutant (red circles)). Data are shown as mean and s.d. for n = 2 individual animals. Data behind graphs are in Supplementary Data 1. The sum of the isoform proportions equals 1 and the analyses were performed for the water-soluble and water-insoluble fraction.

Extended Data Fig. 3 Characterization of the protein complexes found in the lens extracts from wild-type mice.

(a) Top, SEC-HPLC profiles of water-soluble proteins in whole lens extracts for both wild-type strains. The elution profiles of samples from two mice (pair of lenses, each) are shown (as solid and traced lines). Bottom, the integrated signal was plotted against the age to follow fractional changes over time; fractions are color-coded as shown on top. Data are means and s.d. from the 2 replicates shown on top. (b) Top, 2-DE and EM analyses to check for the protein composition of isolated HMW and αL fraction. Scale bar: 50 nm. Bottom, crystallin proportions were determined by densitometric analysis of the 2-DE gels (light grey: αA-crystallin, dark grey: αB-crystallin, orange: β-crystallins, blue: γ-crystallins). (c) SV-AUC profiles showing analyses of αL particles from two mice each, revealing a slight increase in their sedimentation coefficient (s(20,w)) during aging (grey and black lines). Error bars result from averaging consecutive scans during SV-AUC experiments. Apparent maxima of the g(s*) distribution show the most populated particle species. (d) Scattering profiles and Guinier plots of lens extracts from C3HeB/FeJ wild-type mice at different ages: 1 (black), 3 (grey) and 9 (red) months.

Extended Data Fig. 4 2-DE of crystallin fractions from wild-type and mutant mice.

After separation by SEC-HPLC, the individual fractions were checked for their protein composition using 2-DE. Note that the type of ampholyte used (Pharmalyte) caused a staining artifact but that did not influence IEF or second dimension separation of the proteins, as shown at the bottom right. For densitometric analysis, the background signal of 2-DE gels was subtracted using the rolling ball background correction with 100 px in ImageJ. For each individual fraction, one 2-DE experiment was performed.

Extended Data Fig. 5 Cataract-associated crystallin mutants are thermodynamically destabilized.

(a) Far-UV CD spectroscopy analyses of secondary structure analysis of recombinant wild-type (black) and mutant (red) crystallins. Due to low solubility, no spectra were recorded for βA2-S47P. (b) NanoDSF measurements, recording the optical density to detect protein aggregation. The V124E mutation did not change the aggregation propensity of αA-crystallin, but decreased the chaperone activity towards the model substrate L-MDH (inset). Data shown as mean and s.d. from n = 3 independent samples, obtained with recombinant protein from the same batch. Wild-type crystallin (black) and crystallin mutant (red). Experiments were carried out in PBS, but for the βA2 mutant. Due to the low stability of βA2 S47P, Tris buffer was used and L-arginine was added for the measurements. The measurement with wild-type βA2 in PBS are shown in black and in grey for the Tris/Arginine buffer as a reference (c) SEC-MALS/ -HPLC and SV-AUC (inset) were employed to characterize the quaternary structure of wild-type (black) and mutant (red) crystallins. Note: Due to the low stability of βA2-S47P, quantitative unfolding of the protein occurs during SV-AUC runs. Data shown resembles a representative distribution from three independent samples, obtained with recombinant protein from the same batch. Error bars result from averaging consecutive scans during SV-AUC experiments. Apparent maxima of the g(s*) distribution show the most populated particle species.

Extended Data Fig. 6 Characterization of the proteome of mutant mice.

(a) Left, body weight (filled symbols) and lens wet weight (open symbols), per pair of eye lenses from one individual: αA mutant (squares), βA2 mutant (circles) and γD mutant (triangles). Middle and right, amount of water-soluble and -insoluble protein was determined by the Bradford protein assay using BSA as a standard. Data are given as mean ± SD of biological replicates (animals or pairs of eye lenses; please see Supplementary Table 2 and 3 for exact sample size). (b) Top, 2-DE map of the water-soluble fraction of lenses from newborn wild-type mice. Bottom, densitometric analysis; only the low molecular crystallin region was considered (see excerpt). The fraction of α- (gold), β- (orange) and γ-crystallins (blue) was determined up to 12 months of age, for the water-soluble (solid bars) and -insoluble (shaded bars) fraction. Please see Supplementary Data 1 for numerical values.

Extended Data Fig. 7 Comparison of extraction efficiency using different buffers.

2-DE maps showing comparison of the eye lens proteome pattern obtained for the water-insoluble fraction of the three mutants studied, solubilized in the presence of 6 M urea or 7 M urea plus 2 M thiourea. One experiment was performed per mutant, age and extraction buffer.

Extended Data Fig. 8 Protein distribution in whole lens extracts.

(a) The proportion of α-, β- and γ-crystallins was plotted as X/Y/Z-coordinates for the water-soluble and -insoluble fraction, to illustrate the distribution of crystallins in whole lens extracts. Data shown as mean and s.d. for n = 2; data from Supplementary Data 1. The sum of the three crystallin families equals 100 % with α-crystallins representing the Y-coordinate, β-crystallins the X-coordinate and γ-crystallins the Z-coordinate. The composition is changed in the mutants and EMORY mice when cataract is present. At 1 month, when the lens of EMORY mice is still clear, the crystallin composition is very similar to the wild-type composition. During aging, the γ-crystallins gradually decrease (Z-axis). (C57Bl/6 J (black circles), C3HeB/FeJ (black squares), αA mutant (red squares), βA2 mutant (red circles), γD mutant (red triangles), βB2 mutant (green circles), and EMORY (blue circles)). (b) The ratio of α-/β-crystallin content is changed in mutants for both fractions (C3HeB/FeJ (black squares), C57Bl/6 J (black circles), αA mutant (red squares), γD mutant (red circles), and βA2 mutant (red triangles)). Data shown are mean and s.d. for n = 2 animals (pairs of eye lenses).

Supplementary information

Supplementary Information

Supplementary Fig. 1, Supplementary Tables 1–5 and Supplementary Note 1.

Reporting Summary

Supplementary Data 1

Input data for lens extract analyses used in Fig. 1, Fig. 3, ED Fig. 1, ED Fig. 2, ED Fig. 3 and ED Fig. 6

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Schmid, P.W.N., Lim, N.C.H., Peters, C. et al. Imbalances in the eye lens proteome are linked to cataract formation. Nat Struct Mol Biol 28, 143–151 (2021). https://doi.org/10.1038/s41594-020-00543-9

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