Cataract-Causing Mutant R188C of βB2 Crystallin With Low Structural Stability is Sensitive to Environmental Stresses and Prone to Aggregates Formation

Abstract

This study investigated a Chinese family with congenital posterior polar cataracts linked to the βB2-R188C mutation. βB2-crystallin, a key structural component of the lens, is crucial for maintaining lens transparency and stability. We examined the effects of the R188C mutation on βB2-crystallin's structural stability and resistance to environmental stressors using purified proteins and cellular models. The βB2-R188C mutant showed poor stability and a tendency to aggregate under physiological and pathological conditions. The mutation disrupted the oligomerization equilibrium, causing dissociation of dimers into monomers. Molecular dynamics simulations and spectroscopic experiments revealed abnormal protein folding induced by the R188C mutation, increasing susceptibility to environmental stressors. Aggregation was observed in both prokaryotic and eukaryotic models under normal conditions, with enhanced severity under environmental stressors. Notably, lanosterol treatment or αB-crystallin partially reversed aggregation. In summary, the R188C mutation promotes abnormal aggregation by destabilizing βB2-crystallin and disrupting oligomerization equilibrium, potentially leading to cataract formation. Targeting aggregate formation with small molecules like lanosterol or enhancing molecular chaperone activity offers a promising strategy for cataract prevention and treatment.

Keywords

aggregates / cataract / structural stability

References

[1] C. Schmitt and O. Hockwin, “The Mechanisms of Cataract Formation,” Journal of Inherited Metabolic Disease 13 (1990): 501-508.

[2] X. Chen, J. Xu, X. Chen, and K. Yao, “Cataract: Advances in Surgery and Whether Surgery Remains the Only Treatment in Future,” Advances in Ophthalmology Practice and Research 1 (2021): 100008.

[3] P. A. Asbell, I. Dualan, J. Mindel, D. Brocks, M. Ahmad, and S. Epstein, “Age-Related Cataract,” Lancet 365 (2005): 599-609.

[4] J. Li, X. Chen, Y. Yan, and K. Yao, “Molecular Genetics of Congenital Cataracts,” Experimental Eye Research 191 (2020): 107872.

[5] A. Shiels and J. F. Hejtmancik, “Mutations and Mechanisms in Congenital and Age-Related Cataracts,” Experimental Eye Research 156 (2017): 95-102.

[6] W. H. Chan, S. Biswas, J. L. Ashworth, and I. C. Lloyd, “Educational Paper: Congenital and Infantile Cataract: Aetiology and Management,” European Journal of Pediatrics 171 (2012): 625-630.

[7] H. Taylan Şekeroğlu and G. E. Utine, “Congenital Cataract and Its Genetics: The Era of Next-Generation Sequencing,” Turkish Journal of Ophthalmology 51 (2021): 107-113.

[8] K. L. Moreau and J. A. King, “Protein Misfolding and Aggregation in Cataract Disease and Prospects for Prevention,” Trends in Molecular Medicine 18 (2012): 273-282.

[9] G. Wistow, “The Human Crystallin Gene Families,” Human Genomics 6 (2012): 26.

[10] J. Horwitz, “Alpha-Crystallin,” Experimental Eye Research 76 (2003): 145-153.

[11] J. F. Hejtmancik, P. T. Wingfield, and Y. V. Sergeev, “Molecular Genetics of Age-Related Cataract,” Experimental Eye Research 79 (2004): 3-9.

[12] K. S. Ghosh and P. Chauhan, “Crystallins and Their Complexes,” in Macromolecular Protein Complexes II: Structure and Function (Springer, 2019), 439-460.

[13] A. Mishra, B. Krishnan, S. S. Srivastava, and Y. Sharma, “Microbial Βγ-Crystallins,” Progress in Biophysics and Molecular Biology 115 (2014): 42-51.

[14] V. P. Vendra, G. Agarwal, S. Chandani, V. Talla, N. Srinivasan, and D. Balasubramanian, “Structural Integrity of the Greek Key Motif in Βγ-Crystallins Is Vital for Central Eye Lens Transparency,” PLoS ONE 8 (2013): e70336.

[15] V. P. Vendra, I. Khan, S. Chandani, A. Muniyandi, and D. Balasubramanian, “Gamma Crystallins of the Human Eye Lens,” Biochimica Et Biophysica Acta 1860 (2016): 333-343.

[16] M. Michiel, E. Duprat, F. Skouri-Panet, et al., “Aggregation of Deamidated Human βB2-Crystallin and Incomplete Rescue by α-Crystallin Chaperone,” Experimental Eye Research 90 (2010): 688-698.

[17] M. G. Sharaf, S. Cetinel, V. Semenchenko, K. F. Damji, L. D. Unsworth, and C. Montemagno, “Peptides for Targeting βB2-crystallin Fibrils,” Experimental Eye Research 165 (2017): 109-117.

[18] J. L. Velasco-Bolom and L. Domínguez, “Protein-Protein Association Properties of Human βB2-Crystallins,” Proteins(Early View), https://doi.org/10.1002/prot.26547.

[19] Y. B. Xi, X. J. Chen, W. J. Zhao, and Y. B. Yan, “Congenital Cataract-Causing Mutation G129C in γC-Crystallin Promotes the Accumulation of Two Distinct Unfolding Intermediates That Form Highly Toxic Aggregates,” Journal of Molecular Biology 427 (2015): 2765-2781.

[20] L. B. Qi, L. D. Hu, H. Liu, H. Y. Li, X. Y. Leng, and Y. B. Yan, “Cataract-causing Mutation S228P Promotes βB1-crystallin Aggregation and Degradation by Separating Two Interacting Loops in C-terminal Domain,” Protein Cell 7 (2016): 501.

[21] Y. Yu, Y. Qiao, Y. Ye, J. Li, and K. Yao, “Identification and Characterization of Six β-Crystallin Gene Mutations Associated With Congenital Cataract in Chinese Families,” Molecular Genetics and Genomic Medicine 9, no. 3 (2021): e1617.

[22] N. Lin, H. Song, Y. Zhang, et al., “Truncation mutations of CRYGD gene in congenital cataracts cause protein aggregation by disrupting the structural stability of γD-crystallin,” International Journal of Biological Macromolecules 277 (2024):134292.

[23] A. Shiels and J. F. Hejtmancik, “Biology of Inherited Cataracts and Opportunities for Treatment,” Annual Review of Vision Science 5 (2019): 123-149.

[24] L. Zhao, X. J. Chen, J. Zhu, et al., “Lanosterol Reverses Protein Aggregation in Cataracts,” Nature 523 (2015): 607-611.

[25] A. Shiels, T. M. Bennett, and J. F. Hejtmancik, “Cat-Map: Putting Cataract on the Map,” Molecular Vision 16 (2010): 2007-2015.

[26] O. Messina-Baas and S. A. Cuevas-Covarrubias, “Inherited Congenital Cataract: A Guide to Suspect the Genetic Etiology in the Cataract Genesis,” Molecular Syndromology 8 (2017): 58-78.

[27] D. Gill, R. Klose, F. L. Munier, et al., “Genetic Heterogeneity of the Coppock-Like Cataract: A Mutation in CRYBB2 on Chromosome 22q11.2,” Investigative Ophthalmology and Visual Science 41 (2000): 159-165.

[28] B. Bax, R. Lapatto, V. Nalini, et al., “X-Ray Analysis of βB2-Crystallin and Evolution of Oligomeric Lens Proteins,” Nature 347 (1990): 776-780.

[29] P. Russell and C. Chambers, “Interaction of an Altered β-Crystallin With Other Proteins in the Philly Mouse Lens,” Experimental Eye Research 50 (1990): 683-687.

[30] V. P. R. Vendra, G. Agarwal, S. Chandani, V. Talla, N. Srinivasan, and D. Balasubramanian, “Structural Integrity of the Greek Key Motif in Βγ-Crystallins is Vital for Central Eye Lens Transparency,” PLoS One 8 (2013): e70336.

[31] S. L. Flaugh, M. S. Kosinski-Collins, and J. King, “Interdomain Side-Chain Interactions in Human γD Crystallin Influencing Folding and Stability,” Protein Science 14 (2005): 2030-2043.

[32] J. Wu, S. Chen, J. Xu, et al., “Insight into Pathogenic Mechanism Underlying the Hereditary Cataract Caused by βB2-G149V Mutation,” Biomolecules 13 (2023): 864.

[33] J. Xu, Y. Zhang, J. Liu, et al., “Heteromeric formation with βA3 protects the low thermal stability of βB1-L116P,” British Journal of Ophthalmology 107 (2023): 12.

[34] K. Zhang, W.-J. Zhao, X.-Y. Leng, S. Wang, K. Yao, and Y.-B. Yan, “The Importance of the Last Strand at the C-Terminus in βB2-Crystallin Stability and Assembly,” Biochimica Et Biophysica Acta 1842, no. 1 (2014): 44-55.

[35] M. A. Smith, O. A. Bateman, R. Jaenicke, and C. Slingsby, “Mutation of Interfaces in Domain-Swapped Human βB2-Crystallin,” Protein Science: A Publication of the Protein Society 16 (2007): 615-625.

[36] E. J. Yearley, P. D. Godfrin, T. Perevozchikova, et al., “Observation of Small Cluster Formation in Concentrated Monoclonal Antibody Solutions and Its Implications to Solution Viscosity,” Biophysical Journal 106 (2014): 1763-1770.

[37] R. Esfandiary, A. Parupudi, J. Casas-Finet, D. Gadre, and H. Sathish, “Mechanism of Reversible Self-Association of a Monoclonal Antibody: Role of Electrostatic and Hydrophobic Interactions,” Journal of Pharmaceutical Sciences 104 (2015): 577-586.

[38] H. Bloemendal, W. de Jong, R. Jaenicke, N. H. Lubsen, C. Slingsby, and A. Tardieu, “Ageing and Vision: Structure, Stability and Function of Lens Crystallins,” Progress in Biophysics and Molecular Biology 86 (2004): 407-485.

[39] M. A. Smith, O. A. Bateman, R. Jaenicke, and C. Slingsby, “Mutation of Interfaces in Domain-Swapped Human βB2-Crystallin,” Protein Science 16 (2007): 615-625.

[40] J.-L. Velasco-Bolom and L. Domínguez, “Exploring the Folding Process of Human βB2-Crystallin Using Multiscale Molecular Dynamics and the Markov State Model,” Physical Chemistry Chemical Physics 22 (2020): 26753-26763.

[41] Y.-B. Xi, K. Zhang, A.-B. Dai, S.-R. Ji, K. Yao, and Y.-B. Yan, “Cataract-Linked Mutation R188H Promotes βB2-Crystallin Aggregation and Fibrillization During Acid Denaturation,” Biochemical and Biophysical Research Communications 447 (2014): 244-249.

[42] T. R. Silvers and J. K. Myers, “Osmolyte Effects on the Self-Association of Concanavalin A: Testing Theoretical Models,” Biochemistry 52 (2013): 9367-9374.

[43] J. Liu, C. A. Blasie, S. Shi, S. B. Joshi, C. R. Middaugh, and D. B. Volkin, “Characterization and Stabilization of Recombinant Human Protein Pentraxin (rhPTX-2),” Journal of Pharmaceutical Sciences 102 (2013): 175299.

[44] R. Esfandiary, D. B. Hayes, A. Parupudi, et al., “A Systematic Multitechnique Approach for Detection and Characterization of Reversible Self-Association During Formulation Development of Therapeutic Antibodies,” Journal of Pharmaceutical Sciences 102 (2013): 3089-3099.

[45] J. Xu, H. Wang, A. Wang, et al., “βB2 W151R Mutant is Prone to Degradation, Aggregation and Exposes the Hydrophobic Side Chains in the Fourth Greek Key Motif,” Biochimica Et Biophysica Acta Molecular Basis of Disease 1867 (2021): 166018.

[46] S. R. Singh, J. Zhang, C. O'Dell, et al., “Effect of Polysorbate 80 Quality on Photostability of a Monoclonal Antibody,” AAPS Pharmscitech 13 (2012): 422-430.

[47] A. Sreedhara, J. Yin, M. Joyce, et al., “Effect of Ambient Light on IgG1 Monoclonal Antibodies During Drug Product Processing and Development,” European Journal of Pharmaceutics and Biopharmaceutics: Official Journal of Arbeitsgemeinschaft Fur Pharmazeutische Verfahrenstechnik Ev 100 (2016): 38-46.

[48] R. Torosantucci, C. Schöneich, and W. Jiskoot, “Oxidation of Therapeutic Proteins and Peptides: Structural and Biological Consequences,” Pharmaceutical Research 31 (2014): 541-553.

[49] M. F. Lou, “Redox Regulation in the Lens,” Progress in Retinal and Eye Research 22 (2003): 657-682.

[50] W. Wang and C. J. Roberts, “Non-Arrhenius Protein Aggregation,” AAPS Journal 15, no. 3 (2013): 840-851.

[51] M. Sleutel, A. E. Van Driessche, W. Pan, E. K. Reichel, D. Maes, and P. G. Vekilov, “Does Solution Viscosity Scale the Rate of Aggregation of Folded Proteins?,” Journal of Physical Chemistry Letters 3 (2012): 1258-12563.

[52] H. Feng, Q. Zhao, N. Zhao, et al., “A Cell-Permeable Photosensitizer for Selective Proximity Labeling and Crosslinking of Aggregated Proteome,” Advanced Science 11 (2024): 18.

[53] A. Dall'Agnese, M. M. Zheng, S. Moreno, et al., “Proteolethargy is a Pathogenic Mechanism in Chronic Disease,” Cell 188 (2024): 207-221.e30.

[54] S. Kinger, Y. A. Jagtap, A. R. Dubey, et al., “Lanosterol Elevates Cytoprotective Response Through Induced-Proteasomal Degradation of Aberrant Proteins,” Biochimica Et Biophysica Acta Molecular Cell Research 1871 (2024): 119631.

[55] H. Kang, Z. Yang, and R. Zhou, “Non-Arrhenius Protein Aggregation,” Journal of the American Chemical Society 140 (2018): 8479-8486.

[56] W. Chen, X. Chen, Z. Hu, et al., “Correction: Association of pol Diversity With Antiretroviral Treatment Outcomes Among HIV-Infected African Children,” PLoS ONE 8 (2013): e81213.

[57] A. Barati, L. Rezaei Somee, M. B. Shahsavani, et al., “Insights Into the Dual Nature of αB-Crystallin Chaperone Activity From the p.P39L Mutant at the N-terminal Region,” Scientific Reports 14 (2024):7353.

[58] S. Yamamoto, A. Yamashita, N. Arakaki, H. Nemoto, and T. Yamazaki, “Prevention of Aberrant Protein Aggregation by Anchoring the Molecular Chaperone αB-Crystallin to the Endoplasmic Reticulum,” Biochemical and Biophysical Research Communications 455 (2014): 241-245.

[59] A. Lu, P. Duan, J. Xie, et al., “Recent Progress and Research Trend of Anti-Cataract Pharmacology Therapy: A Bibliometric Analysis and Literature Review,” European Journal of Pharmacology 934 (2022): 175299.

[60] D. Lyu, L. Zhang, Z. Qin, et al., “Modeling Congenital Cataract In Vitro Using Patient-Specific Induced Pluripotent Stem Cells,” NPJ Regenerative Medicine 6 (2021): 60.

[61] H. Lin, H. Ouyang, J. Zhu, et al., “Lens Regeneration Using Endogenous Stem Cells With Gain of Visual Function,” Nature 531 (2016): 323-328.

[62] X. Chen, H. Wang, H. Chen, et al., “Lens Regeneration In Situ Using hESCs-Derived Cells—Similar to Natural Lens,” Iscience 26 (2023): 106921.

RIGHTS & PERMISSIONS

2025 The Author(s). Exploration published by Henan University and John Wiley & Sons Australia, Ltd.