Cataract-causing mutations L45P and Y46D impair the thermal stability of γC-crystallin
Introduction
Cataract, a major ocular disease, impairs vision among the elderly and considered the leading cause of blindness globally [1,2]. The causes of human lens cataractogenesis human lens are ageing, ultraviolet irradiation, internal ocular disease, systematic disease and inherited mutations [3]. Aggregation and deposition of crystallins are common features of cataracts [4,5]. Transparency of human lens depends on the ability of crystallins to maintain right fold structures and short-range order since crystallins at high concentrations (400–1000 mg/mL) accommodate up to 90% of soluble proteins in the human lens [6].
Globally, congenital cataract accounts for about 10% of blindness among children. About one-half of genetic disorders, which lead to congenital cataracts, are crystallins gene mutations [7]. For the vertebrate, crystallins are categorized into α-, β-, and γ-crystallins, having similar molecular weights of polypeptides (20–30 kDa) but different oligomeric status [8,9]. α-crystallins form oligomers (10–50-mers) that belong to the small heat shock protein family and act as chaperones in maintaining the stability of other lens structure proteins such as β/γ-crystallins [[10], [11], [12]]. β/γ-crystallins are the predominant structural proteins in human lens with similar tertiary structures composing of four Greek key motifs. β-crystallins potentially form homomers or heteromers ranging from dimer to octamer, while γ-crystallins are monomers [8].
In previous studies on the mechanism by which crystallins mutations caused congenital cataracts, thermal stability was applied to determine the stability differences between mutants and wild type (WT) [[13], [14], [15], [16], [17], [18], [19]] and to evaluate protein stability in different environments [[20], [21], [22]]. In most studies on the thermal stability of crystallins, their protein concentrations samples range from 0.2 to 5 mg/mL [14,19,22]. However, in human lens, concentrations of crystallins reach up to hundreds of mg/mL [6].
Herein, L45P (134 T > C) and Y46D (136 T > G) mutations of γC-crystallin were sourced from two Chinese families with nuclear congenital cataract in Second Affiliated Hospital of Zhejiang University, College of Medicine. Based on existing reports, the L45P mutation of γC-crystallin was reported in the UK with nuclear lamellar congenital cataract [23]. Elsewhere, the Y46D mutation was reported in a Chinese family with nuclear congenital cataract [24]. In our study, we evaluated the thermal stability of mutants and WT of γC-crystallin from low to high concentrations up to 200 mg/mL. We further investigated the effects of αA-crystallin on the thermal aggregation of L45P and Y46D mutants.
Section snippets
Reagents
The DNA polymerase, restriction endonucleases, and DNA ligase were sourced from Yeasen Biotech. The imidazole, SDS, and ANS were purchased from Sigma-Aldrich. The kanamycin and IPTG were obtained from Sangon Biotech. The Escherichia coli (E. coli) TOP10 and E. coli Rosetta (DE3) were purchased from Tiangen Biotech. The pET28a plasmid was obtained from Invitrogen. All other reagents were local products of analytical grade.
Site-directed mutagenesis and plasmid construction
The coding sequence of the human γC-crystallin WT was cloned from human
L45P and Y46D mutants formed aggregates at physiological temperature
Both SDS-PAGE and SEC were used to determine the WT and mutant protein samples having more than 98% purity (Fig. S1). The purified protein samples were incubated in a metal bath at 37 °C (around physiological temperature) to monitor stability around the body temperature. γC-crystallin WT was transparent at 37 °C whereas both L45P and Y46D mutants formed an apparent aggregates (Fig. 1A). Although L45P mutant formed visible precipitate, the Y46D mutant had increased turbidity at 10 mg/mL.
Discussion
Currently, there are no studies that have explored the effect of mutations on the thermal stability of γC-crystallin. In the present study, we showed that both L45P and Y46D mutations impaired the thermal stability of γC-crystallin at low and high concentrations with similar thermal denaturation process. L45P and Y46D mutants formed apparent aggregates. Notably, L45P mutant was more unstable than Y46D mutant at 37 °C. L45P and Y46D mutations affected the two-stage unfolding process of WT. The
Author contributions
Chenxi Fu and Jingjie Xu acquired and analyzed the data and drafted the manuscript. Chenxi Fu, Jingjie Xu, and Xiaoxia Yang performed experiments and revised the manuscript. Dr. Ke Yao and Dr. Xiangjun Chen conceived and designed this study, interpreted and analyzed the data, and revised the manuscript. All the authors have read and approved the final manuscript.
Funding
This word was supported by National Natural Science Foundation of China (grant numbers: No.31872724, No.81900837, No.81870641, and No. 82070939).
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors thank Prof. Yongbin Yan (Tsinghua University) and Prof. Jingyuan Li (Zhejiang University) for helpful suggestions.
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