Eye lens β-crystallins are predicted by native ion mobility-mass spectrometry and computations to form compact higher-ordered heterooligomers

Eye lens crystallin proteins maintain the refractive properties of the lens but are not replaced after denucleation. Rolland et al. use native ion mobility-mass spectrometry, kinetics experiments, and computations to reveal that b-crystallins form heterodimers. These likely assemble into compact heterooligomers that enable the very high protein concentrations found in lens tissue

Deamidation Alters the Structure and Decreases the Stability of Human Lens βΑ3-Crystallin

According to the World Health Organization, cataracts account for half of the blindness in the world, with the majority occurring in developing countries. A cataract is a clouding of the lens of the eye due to light scattering of precipitated lens proteins or aberrant cellular debris. The major proteins in the lens are crystallins, and they are extensively deamidated during aging and cataracts. Deamidation has been detected at the domain and monomer interfaces of several crystallins during aging. The purpose of this study was to determine the effects of two potential deamidation sites at the predicted interface of the βA3-crystallin dimer on its structure and stability. The glutamine residues at the reported in vivodeamidation sites of Q180 in the C-terminal domain and at the homologous site Q85 in the N-terminal domain were substituted with glutamic acid residues by site-directed mutagenesis. Far-UV and near-UV circular dichroism spectroscopy indicated that there were subtle differences in the secondary structure and more notable differences in the tertiary structure of the mutant proteins compared to that of the wild type βA3-crystallin. The Q85E/Q180E mutant also was more susceptible to enzymatic digestion, suggesting increased solvent accessibility. These structural changes in the deamidated mutants led to decreased stability during unfolding in urea and increased precipitation during heat denaturation. When simulating deamidation at both residues, there was a further decrease in stability and loss of cooperativity. However, multiangle-light scattering and quasi-elastic light scattering experiments showed that dimer formation was not disrupted, nor did higher-order oligomers form. These results suggest that introducing charges at the predicted domain interface in the βA3 homodimer may contribute to the insolubilization of lens crystallins or favor other, more stable, crystallin subunit interactions

Aggregation of deamidated human bB2-crystallin and incomplete rescue by a-crystallin chaperone

International audienceAging of the lens is accompanied by extensive deamidation of the lens specific proteins, the crystallins. Deamidated crystallins are increased in the insoluble proteins and may contribute to cataracts. Deami- dation has been shown in vitro to alter the structure and decrease the stability of human lens bB1, bB2 and bA3-crystallin. Of particular interest, bB2 mutants were constructed to mimic the effect of in vivo dea- midations at the interacting interface between domains, at Q70 in the N terminal domain and at Q162, its C-terminal homologue. The double mutant was also constructed. We previously reported that deamida- tion at the critical interface sites decreased stability, while preserving the dimeric 3D structure. In the present study, dynamic light scattering, differential scanning calorimetry and small angle X-ray scattering were used to investigate the effect of deamidation on stability, thermal unfolding and aggregation. The bovine bLb fraction was used for comparative analysis. The chaperone requirements of the various samples were determined using bovine a-crystallins as the chaperone. Deamidation at both interface Gln residues or at Q70, but not Q162, significantly lowered the temperature for unfolding and aggregation, which was rapidly followed by precipitation. This deamidation-induced aggregation and precipitation was not completely prevented by a-crystallin chaperone. A potential mechanism for cataract formation in vivo involving accumulation of deamidated b-crystallin aggregates is discussed

A Nonsense Mutation in CRYBB1 Associated with Autosomal Dominant Cataract Linked to Human Chromosome 22q

Autosomal dominant cataract is a clinically and genetically heterogeneous lens disorder that usually presents as a sight-threatening trait in childhood. Here we have mapped dominant pulverulent cataract to the β-crystallin gene cluster on chromosome 22q11.2. Suggestive evidence of linkage was detected at markers D22S1167 (LOD score [Z] 2.09 at recombination fraction [θ] 0) and D22S1154 (Z=1.39 at θ=0), which closely flank the genes for βB1-crystallin (CRYBB1) and βA4-crystallin (CRYBA4). Sequencing failed to detect any nucleotide changes in CRYBA4; however, a G→T transversion in exon 6 of CRYBB1 was found to cosegregate with cataract in the family. This single-nucleotide change was predicted to introduce a translation stop codon at glycine 220 (G220X). Expression of recombinant human βB1-crystallin in bacteria showed that the truncated G220X mutant was significantly less soluble than wild type. This study has identified the first CRYBB1 mutation associated with autosomal dominant cataract in humans

Decreased heat stability and increased chaperone requirement of modified human βB1-crystallins

Purpose: To determine how deamidation and partial loss of the N- and C-terminal extensions alter the heat stability of βB1-crystallin. Methods: Human lens βB1, a deamidated βB1, Q204E, and αA-crystallins were expressed. Truncated βB1 was generated by proteolytic removal of part of its terminal extensions. The aggregation and precipitation of these proteins due to heating was monitored by circular dichroism and light scattering. The effect of heat on the stability of both monomers and oligomers was investigated. The flexibility of the extensions in wild type and deamidated βB1 was assessed by 1H NMR spectroscopy. Results: With heat, deamidated βB1 precipitated more readily than wild type βB1. Similar effects were obtained for either monomers or oligomers. Flexibility of the N-terminal extension in deamidated βB1 was significantly reduced compared to the wild type protein. Truncation of the extensions further increased the rate of heat-induced precipitation of deamidated βB1. The presence of the molecular chaperone, αA-crystallin, prevented precipitation of modified βB1s. More αA was needed to chaperone the truncated and deamidated βB1 than deamidated βB1 or truncated βB1. Conclusions: Deamidation and truncation of βB1 led to destabilization of the protein and decreased stability to heat. Decreased stability of lens crystallins may contribute to their insolubilization and cataract formation