Journal of Molecular Biology
Volume 431, Issue 3, 1 February 2019, Pages 483-497
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The Structure and Stability of the Disulfide-Linked γS-Crystallin Dimer Provide Insight into Oxidation Products Associated with Lens Cataract Formation

https://doi.org/10.1016/j.jmb.2018.12.005Get rights and content

Highlights

  • Oxidized γS is dimeric with one inter- (C24) and two intramolecular disulfides (C22–C26).

  • The dimeric structure is extended and is not perturbed relative to the monomer.

  • γS dimer is stable at glutathione concentrations akin to aged and cataractous lenses.

  • γS dimer exhibits non-cooperative unfolding and increased aggregation propensity.

  • Oxidation of γS C22, C24, and C26 has been identified in age-related cataract lenses.

Abstract

The reducing environment in the eye lens diminishes with age, leading to significant oxidative stress. Oxidation of lens crystallin proteins is the major contributor to their destabilization and deleterious aggregation that scatters visible light, obscures vision, and ultimately leads to cataract. However, the molecular basis for oxidation-induced aggregation is unknown. Using X-ray crystallography and small-angle X-ray scattering, we describe the structure of a disulfide-linked dimer of human γS-crystallin that was obtained via oxidation of C24. The γS-crystallin dimer is stable at glutathione concentrations comparable to those in aged and cataractous lenses. Moreover, dimerization of γS-crystallin significantly increases the protein’s propensity to form large insoluble aggregates owing to non-cooperative domain unfolding, as is observed in crystallin variants associated with early-onset cataract. These findings provide insight into how oxidative modification of crystallins contributes to cataract and imply that early-onset and age-related forms of the disease share comparable development pathways.

Introduction

The eye lens contains a high concentration of crystallin proteins arranged in a well-ordered, short-range array that allows for lens transparency and the refraction of light onto the retina, thus ensuring proper vision [1], [2], [3]. In mammals, the crystallins comprise three types (α, β, and γ), of which there are several isoforms. The α-crystallins are members of the small heat-shock protein family, whereas the β- and γ-crystallins are a structurally homologous superfamily of proteins that are not related to small heat-shock proteins [4], [5], [6]. β- and γ-crystallins have a monomeric mass of approximately 20 kDa and consist of two domains, each containing two Greek-key β-sheet motifs, which are adjoined by a short linking peptide [6], [7], [8], [9].

There is little protein turnover in the eye lens, and thus, the crystallins are long-lived proteins that must maintain their structural integrity throughout life to preserve lens transparency [10], [11], [12]. Cataract occurs due to a loss of crystallin protein stability and the subsequent propensity of crystallins to partially unfold, leading to aggregation and precipitation [6], [13]. While cataract acquired during early life commonly stems from destabilizing, inheritable mutations in crystallin proteins, age-related cataract is thought to originate from cumulative post-translational modifications (PTMs) [14], [15]. Oxidation is a prevalent crystallin PTM in both aged and cataractous lenses [16], [17] that increases the aggregation propensity of some crystallins in vitro [18], [19], [20]. Cysteine residues are the principal site of protein oxidation [16], [21], and disulfide-linked crystallins are a major component of the insoluble fraction of cataractous lenses [22], [23]. The key factor in preventing crystallin oxidation is the cellular reductant glutathione, the levels of which diminish with age, to the extent that it is severely depleted in cataractous lenses [24], [25], [26].

γS-crystallin (γS) is one of the major crystallins in the human lens [27], and its abundance increases with age due to postnatal expression [28]. Human γS in cataractous lenses is oxidized at specific cysteine residues [17], including S-methylation [29], S-glutathionylation [30], and intermolecular disulfide bond formation [23]. Indeed, γS forms disulfide-linked dimers in vitro [31]. Disulfide-linked dimerization similarly occurs for the R14C mutant of γD-crystallin, leading to increased aggregation propensity and hereditary juvenile-onset cataract [32]. In light of this and the enhanced oxidative conditions in the aging lens, a detailed understanding of the structural and physiological implications of disulfide-linked dimerization of wild-type γS is needed. Herein, we isolated disulfide-linked, dimeric human γS and determined its structure by X-ray crystallography and small-angle X-ray scattering (SAXS). The significance of the disulfide bonding arrangement of the three clustered cysteine residues at positions 22, 24, and 26 is discussed. Furthermore, we provide biophysical and biochemical evidence for the role of the γS dimer in age-related cataract and the potential molecular mechanisms underlying this role.

Section snippets

Structure of human γS disulfide-linked dimer

Human γS monomer was expressed heterologously in Escherichia coli and purified using anion exchange and size exclusion chromatography (SEC). Previously, γS was reported to undergo time-dependent dimer formation under ambient, oxidative conditions at slightly elevated pH, for example, pH 8 [31]. In the present study, γS monomer was readily converted to dimer at physiological pH (i.e., pH 7) by concentrating the monomer to a minimum of 20 mg mL−1 and leaving the protein at 4 °C for 1 week.

Discussion

The evidence for cysteine oxidation, including disulfide crosslinks, between crystallin proteins in aging and cataractous lenses is extensive [16], [17], [21], [22], [23], [47]. Previous studies have shown that disulfide bonds can form in vitro in γ-crystallins [18], [31], [32], [48], but most studies do not demonstrate the ability for these disulfides to be viable in a reducing environment commensurate with that of the lens. Human lens GSH concentrations decrease with age from approximately 6

Production of γS monomer and dimer

A pET43.1 plasmid encoding recombinant human γS (178 amino acids; UniProt P22914) was purchased from Genscript and expressed in BL21(DE3) E. coli cells. Cells were cultured initially at 37 °C for 4–5 h. Expression was then induced with 500 μM IPTG, and the cell culture was incubated overnight at 30 °C. Cells were pelleted, resuspended in DEAE column buffer (20 mM Tris–HCl, pH 8.0), and lysed using sonication. The cell lysate was loaded onto an anion-exchange column (HiPrep DEAE FF 16/10; GE

Acknowledgments

We thank Prof. Roger Truscott and Ms. Jen Xiang for insightful discussions. We acknowledge Dr. Paul Carr for helpful discussions related to protein crystallography. The Centre for Advanced Microscopy at ANU is acknowledged for their assistance with TEM. We are grateful to Dr. Adam Carroll and the team at the Joint Mass Spectrometry Facility, ANU, for help and guidance in performing and designing mass spectrometry experiments. We are grateful to Dr. Robert Knott at the Australian Nuclear Science

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    • Deamidation of the human eye lens protein γS-crystallin accelerates oxidative aging

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      Solvent-exposed cysteines would appear to be disadvantageous for a long-lived protein. Indeed, the dimer of γS, formed by the intermolecular disulfide linkage of C25 and C25 of two γS monomers, is less stable and more aggregation-prone than the monomer (Thorn et al., 2019). The functional advantage of these solvent-accessible cysteines in γS is still under investigation, although they have been demonstrated to play a critical role in metal interactions.

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      The natural glutathione redox buffer in the lens becomes depleted with age, especially so in the course of cataract development, to the extent that γ-crystallin concentration exceeds glutathione concentration in the aged lens (Friedburg and Manthey, 1973; Giblin, 2000). We (Serebryany et al., 2018; Thorn et al., 2019) and others (Kaiser et al., 2019; Quinlan and Hogg, 2018; Roskamp et al., 2020; Skouri-Panet et al., 2001; Srikanthan et al., 2004) have suggested that these sequence-proximal protein thiols, which can form disulfides without changing the overall protein structure, may supplement and eventually inherit glutathione's redox buffer function. However, formation of disulfides between Cys residues that are distal in the sequence and structure has been noted in both γ- and β-crystallins (Hanson et al., 1998; Serebryany et al., 2016b; Takemoto, 1997a,b).

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      PyMOL, a molecular modeling program (http://www.pymol.org/), was used for analyzing the MD simulations [18]. The structure of the γS-crystallin dimer was constructed based on the human γS-crystallin dimer template (6FD8) [19] by PyMOL. Then, the conditions, a water box with dissolved 150 mM NaCl, were simulated by GROMACS.

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    D.C.T. and A.B.G. contributed equally to this work.

    1

    Current address: P.D. Mabbitt, MRC Protein Phosphorylation and Ubiquitination Unit, University of Dundee, Dundee, UK.

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