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Heteromeric formation with βA3 protects the low thermal stability of βB1-L116P
  1. Jingjie Xu1,
  2. Ying Zhang1,
  3. Jian Liu1,2,
  4. Lidan Hu1,2,
  5. Chenqi Luo3,
  6. Ke Yao1,
  7. Xiangjun Chen1,2
  1. 1Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
  2. 2National Clinical Research Center for Child Health, Hangzhou, Zhejiang Province, China
  3. 3Eye Center, Second Hospital Affiliated to Medical College of Zhejiang University, Hangzhou, Zhejiang, China
  1. Correspondence to Dr Xiangjun Chen, Zhejiang University School of Medicine, Hangzhou 310020, China; chenxiangjun{at}zju.edu.cn; Professor Ke Yao; xlren{at}zju.edu.cn

Abstract

Background/aims Congenital cataract is the leading cause of visual disability and blindness in childhood. βB1-crystallin (CRYBB1) comprises about 1/10th of crystallin structural proteins, forming heteromers to maintain lens transparency. We previously reported a CRYBB1 mutation (c.347T>C, p.L116P) affecting 16 patients in a congenital nuclear cataract family. In this study, we investigate the underlying pathogenic mechanism of βB1-L116P.

Methods Protein isolation, size-exclusion chromatography, spectroscopy, Uncle stability screens and molecular dynamics simulations were used to assess βA3- and βB1-crystallin thermal stability, structural properties and heteromer formation.

Results Cells that overexpressed βB1-L116P tended to form aggregates and precipitations under heat-shock stress. Thermal denaturation and time-dependent turbidity experiments showed that thermal stability was significantly impaired. Moreover, protein instability appeared to increase with elevated concentrations detected by the Uncle system. Additionally, βA3 had a relative protective effect on βB1-L116P after heteromers were formed, although βA3 was relatively unstable and was usually protected by basic β-crystallins. Molecular dynamic simulations revealed that L116P mutation altered the hydrophobic residues at the surface around the mutant site, providing solvents more access to the internal and hydrophobic parts of the protein.

Conclusions Decreased βB1-crystallin thermal stability in the presence of the cataract-related L116P mutation contributes significantly to congenital cataract formation. Moreover, its formation of heteromers with βA3 protects against the low thermal stability of βB1-L116P.

  • Lens and zonules
  • Genetics

Data availability statement

All data relevant to the study are included in the article or uploaded as online supplemental information.

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Data availability statement

All data relevant to the study are included in the article or uploaded as online supplemental information.

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Footnotes

  • JX, YZ and JL are joint first authors.

  • JX, YZ and JL contributed equally.

  • Contributors XC and KY conceived, designed and supervised the research. JX, JL, YZ and CL performed the experiments. XC, JX, JL, YZ, and LH performed data analyses. XC, JX, JL, YZ and KY wrote the manuscript. XC is guarantor.

  • Funding This study was supported by the National Natural Science Foundation of China (No. 31872724, No. 82070939 and No. 81900837) and the Natural Science Foundation of Zhejiang Province for Distinguished Young Scholar (No. LR21H120001).

  • Competing interests None declared.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.