Anti-chaperone [eszett]A3/A1102-117 peptide interacting sites in human aB-crystallin

Purpose: Our previous work identified 23 low molecular weight (<3.5 kDa) crystallin peptides in the urea-soluble fractions of normal young, normal aged, and aged cataract human lenses. We found that one of these crystallin fragments, [beta]A3/ A1102-117 peptide (SDAYHIERLMSFRPIC), that are present in aged and cataract lens, increased the scattering of light by [beta]- and [gamma]-crystallins and alcohol dehydrogenase (ADH) and also reduced the chaperone-like activity of [alpha][beta]-crystallin. The present study was performed to identify the interacting sites of the [beta]A3/A1102-117 peptide in [alpha]B-crystallin. Methods: [beta]A3/A1102-117 peptide was first derivatized with sulfo-succinimidyl-2-[6-(biotinamido)-2-{pazidobenzamido}- hexanoamido] ethyl-1-3 dithio propionate (sulfo-SBED), a photoactivable, heterotrifunctional biotincontaining cross-linker. The biotin-derivatized peptide was then incubated with [alpha]B-crystallin at 37 [degrees]C for 2 h to allow complex formation followed by photolysis to facilitate the transfer of the biotin label from the peptide to [alpha]B-crystallin. Label transfer was confirmed by western blot, and the labeled [alpha]B-crystallin was digested with trypsin. Tryptic peptides from [alpha]B-crystallin carrying the biotin label were purified by avidin affinity chromatography, and [beta]A3/A1102-117 peptide interacting sites in [alpha]B-crystallin were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and nanospray quadrupole time-of-flight mass spectrometry (QqTOF MS/MS). Results: We found that the [beta]A3/A1102-117 peptide interacted with [alpha]B-crystallin regions 70LEKDR74, 83HFSPEELKVK92, 91VKVLGDVIEVHGK103, 93VLGDVIEVHGKHEER107, and 121KYR123, which are part of the [alpha]-crystallin domain, and were previously shown to be part of the functional chaperone site in [alpha]B-crystallin. The [beta]A3/A1102-117 peptide also interacted with regions at the COOH-terminal extension of [alpha]B-crystallin, 150KQVSGPER157, 164EEKPAVTAAPK174, and 164EEKPAVTAAPKK175. When two of the hydrophobic residues of [beta]A3/A1102-117 peptide were replaced with hydrophilic residues, the resulting substituted peptide, SDADHGERLMSFRPIC, did not show the anti-chaperone property. Conclusions: This study confirmed the interactions between a low molecular weight peptide derived from [beta]A3/A1- crystallin found in aged and cataract lenses and [alpha]B-crystallin. The binding of [beta]A3/A1102-117 peptide to the chaperone site and the COOH-terminal extension of [alpha]B-crystallin may explain its anti-chaperone property

Significance of interactions of low molecular weight crystallin fragments in lens aging and cataract formation

Analysis of aged and cataract lenses shows the presence of increased amounts of crystallin fragments in the high molecular weight aggregates of water-soluble and water-insoluble fractions. However, the significance of accumulation and interaction of low molecular weight crystallin fragments in aging and cataract development is not clearly understood. In this study, 23 low molecular mass (<3.5-kDa) peptides in the urea-soluble fractions of young, aged, and aged cataract human lenses were identified by mass spectroscopy. Two peptides, B-(1-18) (MDIAIHHPWIRRPFFPFH) and A3/A1-(59-74)(SD(N)AYHIERLMSFRPIC), present in aged and cataract lens but not young lens, and a third peptide, S-(167-178) (SPAVQSFRRIVE) present in all three lens groups were synthesized to study the effects of interaction of these peptides with intact -, -, and -crystallins and alcohol dehydrogenase, a protein used in aggregation studies. Interaction of B-(1-18) and A3/A1-(59-74) peptides increased the scattering of light by - and -crystallin and alcohol dehydrogenase. The ability of -crystallin subunits to function as molecular chaperones was significantly reduced by interaction with B-(1-18) and A3/A1-(59-74) peptides, whereas S peptide had no effect on chaperone-like activity of -crystallin. The A3/A1-(59-74 peptide caused a 5.64-fold increase in B-crystallin oligomeric mass and partial precipitation. Replacing hydrophobic residues in B-(1-18) and A3/A1-(59-74) peptides abolished their ability to induce crystallin aggregation and light scattering. Our study suggests that interaction of crystallin-derived peptides with intact crystallins could be a key event in age-related protein aggregation in lens and cataractogenesis

αA-Crystallin Peptide 66SDRDKFVIFLDVKHF80 Accumulating in Aging Lens Impairs the Function of α-Crystallin and Induces Lens Protein Aggregation

The eye lens is composed of fiber cells that are filled with α-, β- and γ-crystallins. The primary function of crystallins is to maintain the clarity of the lens through ordered interactions as well as through the chaperone-like function of α-crystallin. With aging, the chaperone function of α-crystallin decreases, with the concomitant accumulation of water-insoluble, light-scattering oligomers and crystallin-derived peptides. The role of crystallin-derived peptides in age-related lens protein aggregation and insolubilization is not understood.We found that αA-crystallin-derived peptide, (66)SDRDKFVIFLDVKHF(80), which accumulates in the aging lens, can inhibit the chaperone activity of α-crystallin and cause aggregation and precipitation of lens crystallins. Age-related change in the concentration of αA-(66-80) peptide was estimated by mass spectrometry. The interaction of the peptide with native crystallin was studied by multi-angle light scattering and fluorescence methods. High molar ratios of peptide-to-crystallin were favourable for aggregation and precipitation. Time-lapse recordings showed that, in the presence of αA-(66-80) peptide, α-crystallin aggregates and functions as a nucleus for protein aggregation, attracting aggregation of additional α-, β- and γ-crystallins. Additionally, the αA-(66-80) peptide shares the principal properties of amyloid peptides, such as β-sheet structure and fibril formation.These results suggest that crystallin-derived peptides such as αA-(66-80), generated in vivo, can induce age-related lens changes by disrupting the structure and organization of crystallins, leading to their insolubilization. The accumulation of such peptides in aging lenses may explain a novel mechanism for age-related crystallin aggregation and cataractogenesis

Hydroimidazolone Modification of the Conserved Arg12 in Small Heat Shock Proteins: Studies on the Structure and Chaperone Function Using Mutant Mimics

Methylglyoxal (MGO) is an α-dicarbonyl compound present ubiquitously in the human body. MGO reacts with arginine residues in proteins and forms adducts such as hydroimidazolone and argpyrimidine in vivo. Previously, we showed that MGO-mediated modification of αA-crystallin increased its chaperone function. We identified MGO-modified arginine residues in αA-crystallin and found that replacing such arginine residues with alanine residues mimicked the effects of MGO on the chaperone function. Arginine 12 (R12) is a conserved amino acid residue in Hsp27 as well as αA- and αB-crystallin. When treated with MGO at or near physiological concentrations (2–10 µM), R12 was modified to hydroimidazolone in all three small heat shock proteins. In this study, we determined the effect of arginine substitution with alanine at position 12 (R12A to mimic MGO modification) on the structure and chaperone function of these proteins. Among the three proteins, the R12A mutation improved the chaperone function of only αA-crystallin. This enhancement in the chaperone function was accompanied by subtle changes in the tertiary structure, which increased the thermodynamic stability of αA-crystallin. This mutation induced the exposure of additional client protein binding sites on αA-crystallin. Altogether, our data suggest that MGO-modification of the conserved R12 in αA-crystallin to hydroimidazolone may play an important role in reducing protein aggregation in the lens during aging and cataract formation

Lens aging: Effects of crystallins

Photograph of the Sooner Theatre

αA-Crystallin-derived mini-chaperone modulates stability and function of cataract causing αAG98R-crystallin.

A substitution mutation in human αA-crystallin (αAG98R) is associated with autosomal dominant cataract. The recombinant mutant αAG98R protein exhibits altered structure, substrate-dependent chaperone activity, impaired oligomer stability and aggregation on prolonged incubation at 37 °C. Our previous studies have shown that αA-crystallin-derived mini-chaperone (DFVIFLDVKHFSPEDLTVK) functions like a molecular chaperone by suppressing the aggregation of denaturing proteins. The present study was undertaken to determine the effect of αA-crystallin-derived mini-chaperone on the stability and chaperone activity of αAG98R-crystallin.Recombinant αAG98R was incubated in presence and absence of mini-chaperone and analyzed by chromatographic and spectrometric methods. Transmission electron microscope was used to examine the effect of mini-chaperone on the aggregation propensity of mutant protein. Mini-chaperone containing photoactive benzoylphenylalanine was used to confirm the interaction of mini-chaperone with αAG98R. The rescuing of chaperone activity in mutantα-crystallin (αAG98R) by mini-chaperone was confirmed by chaperone assays. We found that the addition of the mini-chaperone during incubation of αAG98R protected the mutant crystallin from forming larger aggregates that precipitate with time. The mini-chaperone-stabilized αAG98R displayed chaperone activity comparable to that of wild-type αA-crystallin. The complexes formed between mini-αA-αAG98R complex and ADH were more stable than the complexes formed between αAG98R and ADH. Western-blotting and mass spectrometry confirmed the binding of mini-chaperone to mutant crystallin.These results demonstrate that mini-chaperone stabilizes the mutant αA-crystallin and modulates the chaperone activity of αAG98R. These findings aid in our understanding of how to design peptide chaperones that can be used to stabilize mutant αA-crystallins and preserve the chaperone function