Deamidation frequently is invoked as an important driver of crystallin aggregation and cataract formation. Here, we characterized the structural and biophysical consequences of cumulative Asn to Asp changes in γD-crystallin. Using NMR spectroscopy, we demonstrate that N- or C-terminal domain-confined or fully Asn to Asp changed γD-crystallin exhibits essentially the same 1H-15N HSQC spectrum as the wild-type protein, implying that the overall structure is retained. Only a very small thermodynamic destabilization for the overall Asn to Asp γD-crystallin variants was noted by chaotropic unfolding, and assessment of the colloidal stability, by measuring diffusion interaction parameters, yielded no substantive differences in association propensities. Furthermore, using molecular dynamics simulations, no significant changes in dynamics for proteins with Asn to Asp or iso-Asp changes were detected. Our combined results demonstrate that substitution of all Asn by Asp residues, reflecting an extreme case of deamidation, did not affect the structure and biophysical properties of γD-crystallin. This suggests that these changes alone cannot be the major determinant in driving cataract formation.

Cataract disease is strongly associated with progressively accumulating oxidative damage to the extremely long-lived crystallin proteins of the lens. Cysteine oxidation affects crystallin folding, interactions, and light-scattering aggregation especially strongly due to the formation of disulfide bridges. Minimizing crystallin aggregation is crucial for lifelong lens transparency, so one might expect the ubiquitous lens crystallin superfamilies (α and βγ) to contain little cysteine. Yet, the Cys content of γ-crystallins is well above the average for human proteins. We review literature relevant to this longstanding puzzle and take advantage of expanding genomic databases and improved machine learning tools for protein structure prediction to investigate it further. We observe remarkably low Cys conservation in the βγ-crystallin superfamily; however, in γ-crystallin, the spatial positioning of Cys residues is clearly fine-tuned by evolution. We propose that the requirements of long-term lens transparency and high lens optical power impose competing evolutionary pressures on lens βγ-crystallins, leading to distinct adaptations: high Cys content in γ-crystallins but low in βB-crystallins. Aquatic species need more powerful lenses than terrestrial ones, which explains the high methionine content of many fish γ- (and even β-) crystallins. Finally, we discuss synergies between sulfur-containing and aromatic residues in crystallins and suggest future experimental directions.

Congenital cataract affects 1-15 per 10,000 newborns worldwide, and 20,000-40,000 children are born every year with developmental bilateral cataracts. Mutations in the crystallin genes are known to cause congenital cataracts. Crystallins, proteins present in the eye lens, are made up of four Greek key motifs separated into two domains. Greek key motifs play an important role in compact folding to provide the necessary refractive index and transparency. The present study was designed to understand the importance of the fourth Greek key motif in maintaining lens transparency by choosing a naturally reported Y134X mutant human γD- crystallin in a Danish infant and its relationship to lens opacification and cataract.
Human γD-crystallin complementary DNA (cDNA) was cloned into the pET-21a vector, and the Y134X mutant clone was generated by site-directed mutagenesis. Wild-type and mutant proteins were overexpressed in the BL21 DE3 pLysS cells of E. coli. Wild-type protein was purified from the soluble fraction using the ion exchange and gel filtration chromatography methods. Mutant protein was predominantly found in insoluble fraction and purified from inclusion bodies. The structure, stability, aggregational, and amyloid fibril formation properties of the mutant were compared to those of the wild type using the fluorescence and circular dichroism spectroscopy methods.
Loss of the fourth Greek key motif in human γD-crystallin affects the backbone conformation, alters the tryptophan micro-environment, and exposes a nonpolar hydrophobic core to the surface. Mutant is less stable and opens its Greek key motifs earlier with a concentration midpoint (CM) of unfolding curve of 1.5 M compared to the wild type human γD-crystallin (CM: 2.5 M). Mutant is capable of forming self-aggregates immediately in response to heating at 48.6 °C.
Loss of 39 amino acids in the fourth Greek key motif of human γD-crystallin affects the secondary and tertiary structures and exposes the hydrophobic residues to the solvent. These changes make the molecule less stable, resulting in the formation of light-scattering particles, which explains the importance of the fourth Greek key in the underlying mechanism of opacification and cataract.

Congenital cataract (CC), the most prevalent cause of childhood blindness and amblyopia, necessitates prompt and precise genetic diagnosis. The objective of this study is to identify the underlying genetic cause in a Swiss patient with isolated CC. Whole exome sequencing (WES) and copy number variation (CNV) analysis were conducted for variant identification in a patient born with a total binocular CC without a family history of CC. Sanger Sequencing was used to confirm the variant and segregation analysis was used to screen the non-affected parents. The first de novo missense mutation at c.391T>C was identified in exon 3 of CRYGC on chromosome 2 causing the substitution of a highly conserved Tryptophan to an Arginine located at p.Trp131Arg. Previous studies exhibit significant changes in the tertiary structure of the crystallin family in the following variant locus, making CRYGC prone to aggregation aggravated by photodamage resulting in cataract. The variant can be classified as pathogenic according to the American College of Medical Genetics and Genomics (ACMG) criteria (PP3 + PM1 + PM2 + PS2; scoring 10 points). The identification of this novel variant expands the existing knowledge on the range of variants found in the CRYGC gene and contributes to a better comprehension of cataract heterogeneity.

The anterior lens epithelium has the ability to differentiate into lens fibres throughout its life. The present study aims to identify and functionally characterize the adult stem cells in the human lens epithelium. Whole mounts of lens epithelium from donor eyes (normal/cataract) were immunostained for SOX2, gap junction protein alpha 1 (GJA1), PAX6, α, β and γ-crystallins, followed by a confocal analysis. The functional property of adult stem cells was analysed by their sphere forming ability using cultured lens epithelial cells from different zones. Based on marker expression, the lens epithelium was divided into four zones: the central zone, characterized by a small population of PAX6+, GJA1-, β-crystallin- and γ-crystallin- cells; the germinative zone, characterized by PAX6+, GJA1+, β-crystallin- and γ-crystallin-; the transitional zone, characterized by PAX6+, GJA1+, β-crystallin+ and γ-crystallin-; and the equatorial zone, characterized by PAX6+/-, GJA1+, β-crystallin+, and γ-crystallin+ cells. The putative lens epithelial stem cells identified as SOX2+ and GJA1 membrane expression negative cells were located only in the central zone (1.89 ± 0.84%). Compared to the other zones, a significant percentage of spheres were identified in the central zone (1.68 ± 1.04%), consistent with the location of the putative adult lens epithelial stem cells. In the cataractous lens, an absence of SOX2 expression and a significant reduction in sphere forming ability (0.33 ± 0.11%) were observed in the central zone. The above findings confirmed the presence of putative stem cells in the central zone of the adult human lens epithelium and indicated their probable association with cataract development.

Highly concentrated lens proteins, mostly β- and γ-crystallin, are responsible for maintaining the structure and refractivity of the eye lens. However, with aging and cataract formation, β- and γ-crystallin are associated with the lens membrane or other lens proteins forming high-molecular-weight proteins, which further associate with the lens membrane, leading to light scattering and cataract development. The mechanism by which β- and γ-crystallin are associated with the lens membrane is unknown. This work aims to study the interaction of β- and γ-crystallin with the phospholipid membrane with and without cholesterol (Chol) with the overall goal of understanding the role of phospholipid and Chol in β- and γ-crystallin association with the membrane. Small unilamellar vesicles made of Chol/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (Chol/POPC) membranes with varying Chol content were prepared using the rapid solvent exchange method followed by probe tip sonication and then dispensed on freshly cleaved mica disk to prepare a supported lipid membrane. The βL- and γ-crystallin from the cortex of the bovine lens was used to investigate the time-dependent association of βL- and γ-crystallin with the membrane by obtaining the topographical images using atomic force microscopy. Our study showed that βL-crystallin formed semi-transmembrane defects, whereas γ-crystallin formed transmembrane defects on the phospholipid membrane. The size of semi-transmembrane defects increases significantly with incubation time when βL-crystallin interacts with the membrane. In contrast, no significant increase in transmembrane defect size was observed in the case of γ-crystallin. Our result shows that Chol inhibits the formation of membrane defects when βL- and γ-crystallin interact with the Chol/POPC membrane, where the degree of inhibition depends upon the amount of Chol content in the membrane. At a Chol/POPC mixing ratio of 0.3, membrane defects were observed when both βL- and γ-crystallin interacted with the membrane. However, at a Chol/POPC mixing ratio of 1, no association of γ-crystallin with the membrane was observed, which resulted in a defect-free membrane, and the severity of the membrane defect was decreased when βL-crystallin interacted with the membrane. The semi-transmembrane or transmembrane defects formed by the interaction of βL- and γ-crystallin on phospholipid membrane might be responsible for light scattering and cataract formation. However, Chol suppressed the formation of such defects in the membrane, likely maintaining lens membrane homeostasis and protecting against cataract formation.

Aromatic residues forming tyrosine corners within Greek key motifs are critical for the folding, stability, and order of βγ-crystallins and thus lens transparency. To delineate how a double amino acid substitution in an N-terminal-domain tyrosine corner of the CRYGS mutant p.F10_Y11delinsLN causes juvenile autosomal dominant cortical lamellar cataracts, human γS-crystallin c-DNA was cloned into pET-20b (+) and a p.F10_Y11delinsLN mutant was generated via site-directed mutagenesis, overexpressed, and purified using ion-exchange and size-exclusion chromatography. Structure, stability, and aggregation properties in solution under thermal and chemical stress were determined using spectrofluorimetry and circular dichroism. In benign conditions, the p.F10_Y11delinsLN mutation does not affect the protein backbone but alters its tryptophan microenvironment slightly. The mutant is less stable to thermal and GuHCl-induced stress, undergoing a two-state transition with a midpoint of 60.4 °C (wild type 73.1 °C) under thermal stress and exhibiting a three-state transition with midpoints of 1.25 and 2.59 M GuHCl (wild type: two-state transition with Cm = 2.72 M GuHCl). The mutant self-aggregates upon heating at 60 °C, which is inhibited by α-crystallin and reducing agents. Thus, the F10_Y11delinsLN mutation in human γS-crystallin impairs the protein\'s tryptophan microenvironment, weakening its stability under thermal and chemical stress, resulting in self-aggregation, lens opacification, and cataract.

Liquid-liquid phase separation of protein solutions has regained heightened attention for its biological importance and pathogenic relevance. Coarse-grained models are limited when explaining residue-level effects on phase equilibrium. Here we report phase diagrams for γ-crystallins using atomistic modeling. The calculations were made possible by combining our FMAP method for computing chemical potentials and Brownian dynamics simulations for configurational sampling of dense protein solutions, yielding the binodal and critic temperature (Tc). We obtain a higher Tc for a known high-Tc γ-crystallin, γF, than for a low-Tc paralog, γB. The difference in Tc is corroborated by a gap in second virial coefficient. Decomposition of inter-protein interactions reveals one amino-acid substitution between γB and γF, from Ser to Trp at position 130, as the major contributor to the difference in Tc. This type of analysis enables us to link phase equilibrium to amino-acid sequence and to design mutations for altering phase equilibrium.

Congenital cataract is a leading cause of treatable childhood blindness and both clinically and genetically heterogeneous. Among the already characterized phenotypes, coralliform cataract is a rare special form of congenital cataracts. Although previous studies had shown that mutations in the γD-crystallin (CRYGD) can result in congenital coralliform cataracts, no conclusive genotype-phenotype correlation might be drawn. Here we aimed to identify the spectrum and frequency of CRYGD gene mutations in congenital coralliform cataracts of Chinese origin.
The medical records of 392 Chinese families with congenital cataracts were reviewed between January 2011 and December 2021. The families, clinically documented to have congenital coralliform cataracts, were screened for mutations in candidate CRYGD gene. The genomic DNA of all subjects was extracted from peripheral blood leukocytes. PCR amplified and direct sequencing were performed to identify the disease-causing mutation.
A total of 12 families with coralliform cataracts were recruited in this study in the past 10 years, accounting for 3.1% of the families with congenital cataracts. Of the 12 families, all affected individuals presented with bilateral non-progressive coralliform cataracts since birth, with the best-corrected Snellen visual acuities ranging from 20/200 to 20/25. A recurrent c.70 C > A (p. P24T) mutation in CRYGD was identified in 10 families (83.3%) with congenital cataract, which co-segregated with all affected individuals and was not observed in unaffected family members or ethnically matched normal controls.
The coralliform cataract is characterized by being bilateral, non-progressive and present at birth. A recurrent p.P24T CRYGD mutation occurs independently in 83.3% of the Chinese families with congenital coralliform cataracts and most likely represents a mutational hot spot, which underscore the relations between coralliform cataract and p.P24T CRYGD.

Eye lens α- and β-/γ-crystallin proteins are not replaced after fiber cell denucleation and maintain lens transparency and refractive properties. The exceptionally high (∼400-500 mg/mL) concentration of crystallins in mature lens tissue and multiple other factors impede precise characterization of β-crystallin interactions, oligomer composition, size, and topology. Native ion mobility-mass spectrometry is used here to probe β-crystallin association and provide insight into homo- and heterooligomerization kinetics for these proteins. These experiments include separation and characterization of higher-order β-crystallin oligomers and illustrate the unique advantages of native IM-MS. Recombinantly expressed βB1, βB2, and βA3 isoforms are found to have different homodimerization propensities, and only βA3 forms larger homooligomers. Heterodimerization of βB2 with βA3 occurs ∼3 times as fast as that of βB1 with βA3, and βB1 and βB2 heterodimerize less readily. Ion mobility experiments, molecular dynamics simulations, and PISA analysis together reveal that observed oligomers are consistent with predominantly compact, ring-like topologies.