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

Eye lens α- and ß-/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 ß-crystallin interactions, oligomer composition, size, and topology. Native ion mobility-mass spectrometry is used here to probe ß-crystallin association and provide insight into homo- and heterooligomerization kinetics for these proteins. These experiments include separation and characterization of higher-order ß-crystallin oligomers and illustrate the unique advantages of native IM-MS. Recombinantly expressed ßB1, ßB2, and ßA3 isoforms are found to have different homodimerization propensities, and only ßA3 forms larger homooligomers. Heterodimerization of ßB2 with ßA3 occurs ∼3 times as fast as that of ßB1 with ßA3, and ßB1 and ßB2 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.

Characterization of K-binding factor involved in water-soluble complex of menaquinone-7 produced by Bacillus subtilis natto.

Vitamin Ks are expected to contribute bone and cardiovascular health. Especially, menaquinone-7 has a higher bioavailability and a longer half-life than other vitamin Ks in the human body. However, their low water-solubility limits their application. On the other hand, Bacillus subtilis natto produces a water-soluble complex, which comprises menaquinone-7 and peptides. The peptide named K-binding factor (KBF) has been reported as the main component of the complex. In the present, the structural characteristics of KBF were studied. Mass spectrometry showed significant peaks at m/z = 1050, while the previous PAGE suggested that molecular weight of KBF was ~ 3k. Amino acid analysis revealed that the 1k peptides were the various combinations of nine amino acids, among which Asx, Glx, Val, Leu and Met were found to be the most abundant. The peptides could serve as detergent properties. The 1k peptides could be isolated by reverse-phase high performance liquid chromatography. The bundle of three 1k detergent-like peptides would participate to the micelle structure containing menqauinone-7 inside. In conclusion, a basic unit of KBF would be the ~ 1k peptides, and the three basic unit assemble to the ~ 3k bundle, then the bundle form a water-soluble micelle including menqauinone-7 inside.

Isomerization of Asp is essential for assembly of amyloid-like fibrils of αA-crystallin-derived peptide.

Post-translational modifications are often detected in age-related diseases associated with protein misfolding such as cataracts from aged lenses. One of the major post-translational modifications is the isomerization of aspartate residues (L-isoAsp), which could be non-enzymatically and spontaneously occurring in proteins, resulting in various effects on the structure and function of proteins including short peptides. We have reported that the structure and function of an αA66-80 peptide, corresponding to the 66-80 (66SDRDKFVIFLDVKHF80) fragment of human lens αA-crystallin, was dramatically altered by the isomerization of aspartate residue (Asp) at position 76. In the current study, we observed amyloid-like fibrils of L-isoAsp containing αA66-80 using electron microscopy. The contribution of each amino acid for the peptide structure was further evaluated by circular dichroism (CD), bis-ANS, and thioflavin T fluorescence using 14 alanine substituents of αA66-80, including L-isoAsp at position 76. CD of 14 alanine substituents demonstrated random coiled structures except for the substituents of positively charged residues. Bis-ANS fluorescence of peptide with substitution of hydrophobic residue with alanine revealed decreased hydrophobicity of the peptide. Thioflavin T fluorescence also showed that the hydrophobicity around Asp76 of the peptide is important for the formation of amyloid-like fibrils. One of the substitutes, H79A (SDRDKFVIFL(L-isoD)VKAF) demonstrated an exact ß-sheet structure in CD and highly increased Thioflavin T fluorescence. This phenomenon was inhibited by the addition of protein-L-isoaspartate O-methyltransferase (PIMT), which is an enzyme that changes L-isoAsp into Asp. These interactions were observed even after the formation of amyloid-like fibrils. Thus, isomerization of Asp in peptide is key to form fibrils of αA-crystallin-derived peptide, and L-isoAsp on fibrils can be a candidate for disassembling amyloid-like fibrils of αA-crystallin-derived peptides.

Elucidation of the mechanism of subunit exchange in αB crystallin oligomers.

AlphaB crystallin (αB-crystallin) is a key protein for maintaining the long-term transparency of the eye lens. In the eye lens, αB-crystallin is a "dynamical" oligomer regulated by subunit exchange between the oligomers. To elucidate the unsettled mechanism of subunit exchange in αB-crystallin oligomers, the study was carried out at two different protein concentrations, 28.5 mg/mL (dense sample) and 0.45 mg/mL (dilute sample), through inverse contrast matching small-angle neutron scattering. Interestingly, the exchange rate of the dense sample was the same as that of the dilute sample. From analytical ultracentrifuge measurements, the coexistence of small molecular weight components and oligomers was detected, regardless of the protein concentration. The model proposed that subunit exchange could proceed through the assistance of monomers and other small oligomers; the key mechanism is attaching/detaching monomers and other small oligomers to/from oligomers. Moreover, this model successfully reproduced the experimental results for both dense and dilute solutions. It is concluded that the monomer and other small oligomers attaching/detaching mainly regulates the subunit exchange in αB-crystallin oligomer.

Effect of a Lens Protein in Low-Temperature Culture of Novel Immortalized Human Lens Epithelial Cells (iHLEC-NY2).

The prevalence of nuclear cataracts was observed to be significantly higher among residents of tropical and subtropical regions compared to those of temperate and subarctic regions. We hypothesized that elevated environmental temperatures may pose a risk of nuclear cataract development. The results of our in silico simulation revealed that in temperate and tropical regions, the human lens temperature ranges from 35.0 °C to 37.5 °C depending on the environmental temperature. The medium temperature changes during the replacement regularly in the cell culture experiment were carefully monitored using a sensor connected to a thermometer and showed a decrease of 1.9 °C, 3.0 °C, 1.7 °C, and 0.1 °C, after 5 min when setting the temperature of the heat plate device at 35.0 °C, 37.5 °C, 40.0 °C, and 42.5 °C, respectively. In the newly created immortalized human lens epithelial cell line clone NY2 (iHLEC-NY2), the amounts of RNA synthesis of αA crystallin, protein expression, and amyloid ß (Aß)1-40 secreted into the medium were increased at the culture temperature of 37.5 °C compared to 35.0 °C. In short-term culture experiments, the secretion of Aß1-40 observed in cataracts was increased at 37.5 °C compared to 35.0 °C, suggesting that the long-term exposure to a high-temperature environment may increase the risk of cataracts.

Simultaneous and Rapid Detection of Multiple Epimers and Isomers of Aspartyl Residues in Lens Proteins Using an LC-MS-MRM Method.

Traditionally, studies of post translational modifications (PTMs) by mass analysis have been limited to modifications such as deamidation and oxidation that have a mass shift. Although Asp isomerization is an important PTM, the selective detection of Asp isomers by mass spectrometry was originally thought to be impossible due to the identical mass of the isomers. The recent development of an LC-MS-based method has facilitated rapid and accurate quantitative analysis of Asp isomers in long-lived proteins; however, because the quantification is based on the extracted ion chromatogram acquired by an MS1 scan, this methodology is not always efficient for detecting extremely low-abundance peptides in complex biological samples. In this paper, we evaluated Asp isomer-containing peptides of αA-crystallin present in tryptic digests of human lens samples with different degrees of protein aggregation and different ages using LC coupled with multiple reaction monitoring (MRM). In a single analysis, the LC-MRM method enabled three tryptic peptides containing isomers of Asp58, Asp91/92, and Asp151 to be detected simultaneously. The extent of isomerization and epimerization of these specific Asp sites in αA-crystallin increased with the progress of α-crystallin aggregation. For the analysis of samples known to isomerize at specific Asp residues, MRM gives a more rapid, less laborious, and high-quality separation of Asp isomer-containing peptides relative to the previous MS1-based quantitative method.

Asp isomerization increases aggregation of α-crystallin and decreases its chaperone activity in human lens of various ages.

α-Crystallin, comprising 40-50 subunits of αA- and αB-subunits, is a long-lived major soluble chaperone protein in lens. During aging, α-crystallin forms aggregates of high molecular weight (HMW) protein and eventually becomes water-insoluble (WI). Isomerization of Asp in α-crystallin has been proposed as a trigger of protein aggregation, ultimately leading to cataract formation. Here, we have investigated the relationship between protein aggregation and Asp isomerization of αA-crystallin by a series of analyses of the soluble α-crystallin, HMW and WI fractions from human lens samples of different ages (10-76 years). Analytical ultracentrifugation showed that the HMW fraction had a peak sedimentation coefficient of 40 S and a wide distribution of values (10-450 S) for lens of all ages, whereas the α-crystallin had a much smaller peak sedimentation coefficient (10-20 S) and was less heterogeneous, regardless of lens age. Measurement of the ratio of isomers (Lα-, Lß-, Dα-, Dß-) at Asp58, Asp91/92 and Asp151 in αA-crystallin by liquid chromatography-mass spectrometry showed that the proportion of isomers at all three sites increased in order of aggregation level (α-crystallin < HMW < WI fractions). Among the abnormal isomers of Asp58 and Asp151, Dß-isomers were predominant with a very few exceptions. Notably, the chaperone activity of HMW protein was minimal for lens of all ages, whereas that of α-crystallin decreased with increasing lens age. Thus, abnormal aggregation caused by Asp isomerization might contribute to the loss of chaperone activity of α-crystallin in aged human lens.

Site-specific rapid deamidation and isomerization in human lens αA-crystallin in vitro.

Recent studies have suggested that the isomerization/racemization of aspartate residues in proteins increases in aged tissues. One such residue is Asp151 in lens-specific αA-crystallin. Although many isomerization/racemization sites have been reported in various proteins, the factors that lead to those modifications in proteins in vivo remain obscure. Therefore, an in vitro system is needed to assess the mechanisms of modifications of Asp under various conditions. Deamidation of Asn to Asp in proteins occurs more rapidly than isomerization/racemization of Asp, although the reaction passes through the same intermediate in both pathways. Here, therefore, we replaced Asp151 in human lens αA-crystallin with Asn by using site-directed mutagenesis. The recombinant protein was expressed in Escherichia coli and used to investigate the deamidation/isomerization/racemization of Asn151 after incubation at 50°C for various durations and under different pH. After incubation, the mutant αA-crystallin was subjected to enzymatic digestion followed by liquid chromatography-MS/MS to evaluate the ratio of modifications in Asn151-containing peptides. The Asp151Asn αA-crystallin mutant showed rapid deamidation to Asp with the formation of specific Asp isomers. In particular, deamidation increased greatly under basic conditions. By contrast, subunit-subunit interactions between αA-crystallin and αB-crystallin had little effect on the modification of Asn151. Our findings suggest that the Asp151Asn αA-crystallin mutant represents a good in vitro model protein to assess deamidation, isomerization, and the racemization intermediates. Furthermore, our in vitro results show a different trend from in vivo data, implying the presence of specific factors that induce racemization from L-Asp to D-Asp residues in vivo.

Negative charge at aspartate 151 is important for human lens αA-crystallin stability and chaperone function.

Aggregation of lens protein is a major cause of senile cataract. Lens crystallins contain many kinds of modification that accumulate over lifespan. In particular, isomerization of Asp 151 in αA-crystallin has been found in aged lenses; however, its significance is unknown. The purpose of this study was to determine the effects of isomerization of Asp 151 in αA-crystallin. Trypsin digestion followed by liquid chromatography-mass spectrometry analysis of the water-soluble high molecular weight (HMW) fraction from human lens samples showed that isomerization of Asp 151 in αA-crystallin is age-independent, and that 50% of isomerization occurs shortly after birth. However, the extent of Asp 151 isomerization varied with the size of αA-crystallin oligomer species separated from the HMW fraction from aged lens. To evaluate the effects of modification, Asp 151 of αA-crystallin was replaced by glycine, alanine, isoleucine, asparagine, glutamate, or lysine by site-directed mutagenesis. All substitutions except for glutamate decreased heat stability and chaperone function as compared with wild-type αA-crystallin. In particular, abnormal hydrophobicity and alteration of the charge state at Asp 151 caused loss of stability and chaperone activity of αA-crystallin; these properties were recovered to some extent when the mutant protein was mixed 1:1 with wild-type αA-crystallin. The results suggest that, by itself, age-independent isomerization of Asp 151 in αA-crystallin may not contribute to cataract formation. However, the long-term deleterious effect of Asp 151 isomerization on the structure and function of αA-crystallin might cooperatively contribute to the loss of transparency of aged human lens.

Asp 58 modulates lens αA-crystallin oligomer formation and chaperone function.

Senile cataract onset is caused by insolubilization of lens proteins. The lens crystallin protein family correctly orders the formation of homo- or hetero-oligomers in lens fiber cells. Because lens fiber cells do not divide, covalent post-translational modifications, such as isomerization of aspartate residues, accumulate with aging. Although many isomerization sites of αA-crystallin have been reported, their structural and functional contributions have never been identified. In this study, αA-crystallin was extracted from aged human lens and separated into each oligomeric state by size exclusion chromatography and electrophoresis. The novel combination methodology of in-solution/gel tryptic digestion with liquid chromatography equipped with mass spectrometry (LC-MS/MS) was used to evaluate the isomerization of Asp 58. The contributions of isomerization to assembly, solubility, and chaperone functions of αA-crystallin were estimated using a series of mutations of Asp 58 in αA-crystallin. The results indicated that the isomerization of Asp 58 depended on the oligomer size and age of the lens. The substitution of Asp 58 for hydrophobic residues increased αA-crystallin oligomer size and decreased solubility. All substitutions decreased the chaperone function of αA-crystallin for aggregates of bovine ßL-crystallin and alcohol dehydrogenase. The data indicated that Asp 58 in αA-crystallin was critical for intermolecular interactions in the lens. Our results also suggested that LC-MS/MS-based isomerization analyses of in-gel-digested products could be useful for investigating the isomerization of Asp residues in oligomeric states. This method could also be used to analyze d/l ratios of amino acid residues in soluble protein aggregates.

Identification of Isomeric Aspartate residues in ßB2-crystallin from Aged Human Lens.

Many post-translational modifications such as oxidation, deamidation and isomerization of amino acid residues occur in lens proteins with aging. One such modification, isomerization of aspartate in lens α-crystallin, has been well studied by amino acid enantiomer analysis and LC-MS/MS. LC-MS/MS can quickly and easily identify D- and L-amino acid-containing peptides without purification of lens protein mixtures. However, this method has a weak point in that isomeric peptides of major components are detected predominantly, while those from minor proteins such as ß- and γ-crystallins have not been fully determined. Therefore, the isomerization of amino acid residues in ß- and γ-crystallin families has been little studied. To solve those problems and detect the isomerization of Asp residues in lens ßB2-crystallin, the main component of the ß-crystallin family, here we have developed steps for sample fractionation before d/l analysis based on either LC-MS/MS or amino acid derivatization to diastereoisomers followed by RP-HPLC. To capture a small amount of peptide, a multiple reaction monitoring (MRM) method based on quadrupole MS/MS (Q-MS) was applied to the water-soluble fraction of whole lens. The d/l analysis based on both LC-MS/MS and diastereoisomer formation showed the presence of multiple isomerization sites, including Asp4, Asp83, Asp92 and Asp192, in ßB2-crystallin in aged lens. These isomerization sites were confirmed to exist in an age-dependent manner by Q-MS. Synthetic peptides of ßB2-crystallin containing different isomers of Asp showed differential elution profiles during RP-HPLC, indicating differences in the local structure or hydrophobicity of Asp-isomer-containing peptides. These results suggest that the isomerization sites are distributed on exposed regions of ßB2-crystallin and thus likely to have an impact on crystallin subunit-subunit interactions, induce abnormal crystallin aggregation, and contribute to senile cataract formation in aged lens.

D-Amino acids in protein: The mirror of life as a molecular index of aging.

Proteins are composed exclusively of l-amino acids. Among elderly individuals, however, d-aspartic acid (d-Asp) residues have been found in eye lens and brain, as well as in other tissues. The presence of d-Asp may change the higher-order structure of a protein, which in turn may have a role in age-related disorders such as cataract and Alzheimer's disease. d-Asp results from the spontaneous racemization of Asp residues in susceptible proteins. During aging, natural lα-Asp residues in proteins are non-enzymatically isomerized via a succinimidyl intermediate to l-ß-, d-α- and d-ß-isomers. This isomerization does not happen uniformly, but instead occurs at specific residues that are susceptible to isomerization due to their sequence or structural context. Thus, it is necessary to establish the nature of each individual Asp residue in susceptible proteins. Recently, a new method based on LC-MS/MS for the analysis of Asp isomerization at specific protein sites has been described. In this review, we first show that the homochirality of amino acids in proteins is not guaranteed throughout life. We then describe the development of a new method for protein-bound d-amino acid analysis, and discuss the negative influence that d-Asp has on protein structure and function.

Identification of á´ -amino acid-containing peptides in human serum.

Biologically uncommon d-aspartate (d-Asp) residues have been shown to accumulate in proteins associated with age-related human disorders, such as cataract and Alzheimer disease. Such d-Asp-containing proteins are unlikely to be broken down completely because metabolic enzymes recognize only proteins or peptides composed exclusively of l-amino acids. Therefore, undigested d-Asp-containing peptides may exist in blood and, if detectable, may be a useful biomarker for associated diseases. In this study, we investigated d-amino acid-containing peptides in adult human serum by a qualitative d-amino acid analysis based on a diastereomer method and LC-MS/MS method. As a result, two d-Asp-containing peptides were detected in serum, both derived from the fibrinogen ß-chain, a glycoprotein that helps in the formation of blood clots. One of the peptides was fibrinopeptide B, which prevents fibrinogen from forming polymers of fibrin, and the other was same peptide with C-terminal Arginine missing. To our knowledge, this is the first report of the presence of d-amino acid-containing peptides in serum and the approach described will provide a new direction on the serum proteome and fragmentome.

Ferulic Acid Suppresses Amyloid ß Production in the Human Lens Epithelial Cell Stimulated with Hydrogen Peroxide.

It is well known that oxidative stresses induce the production of amyloid ß (Aß) in the brain, lens, and retina, leading to age-related diseases. In the present study, we investigated the effects of ferulic acid on the Aß levels in H2O2-stimulated human lens epithelial (HLE) SRA 01/04 cells. Three types of Aß peptides (Aß1-40, Aß1-42, and Aß1-43) were measured by ELISA, and the levels of mRNA for the expressed proteins related to Aß production (APP, BACE1, and PS proteins) and degradation (ADAM10, NEP, and ECE1 proteins) were determined by quantitative real-time RT-PCR. H2O2 stimulation augmented gene expression of the proteins related to Aß production, resulting in the production of three types of Aß peptides. Treatment with 0.1 µM ferulic acid attenuated the augmentations of gene expression and production of the proteins related to the secretion of three types of Aß peptides in the H2O2-stimulated HLE cells. These results provided evidence of antioxidative functions of ferulic acid for lens epithelial cells.

Isomeric Replacement of a Single Aspartic Acid Induces a Marked Change in Protein Function: The Example of Ribonuclease A.

lα-Aspartic acid (Asp) residues in proteins are nonenzymatically isomerized to abnormal lß-, dα-, and dß-Asp isomers under physiological conditions. Such an isomerization of Asp residues is considered to be a trigger of protein denaturation because it either elongates the main chain or induces a different orientation of the side chain within the protein structure or both. However, previous studies have found no direct evidence of the effects of Asp isomers on protein function. Therefore, the production of Asp-isomer-containing proteins is required to verify the effects of Asp isomerization. Here, we describe the production of an Asp-isomer-containing protein using the expressed protein ligation. As a model protein, bovine pancreatic ribonuclease A (RNase A, EC 3.1.27.5), which catalyzes the cleavage of phosphodiester bonds in RNA, was used. In this study, lα-Asp at position 121 in RNase A was replaced by lß-, dα-, and dß-Asp. The objective aspartic acid at position 121 is located near the active site and related to RNA cleavage. The RNase A with lα-Asp at position 121 showed a normal activity. By contrast, the catalytic activity of lß-, dα-, and dß-Asp-containing RNase A was markedly decreased. This study represents the first synthesis and analysis of a protein containing four different Asp isomers.

New insight into the dynamical system of αB-crystallin oligomers.

α-Crystallin possesses a dynamic quaternary structure mediated by its subunit dynamics. Elucidation of a mechanism of subunit dynamics in homo-oligomers of αB-crystallin was tackled through deuteration-assisted small-angle neutron scattering (DA-SANS) and electrospray ionization (ESI) native mass spectrometry (nMS). The existence of subunit exchange was confirmed with DA-SANS, and monomers liberated from the oligomers were observed with nMS. With increasing temperature, an increase in both the exchange rate and monomer population was observed despite the absence of oligomer collapse. It is proposed that transiently liberated subunits, namely, "traveling subunits," play a role in subunit exchange. Moreover, we propose that protein function is regulated by these traveling subunits.

Aggregation of Trp > Glu point mutants of human gamma-D crystallin provides a model for hereditary or UV-induced cataract.

Numerous mutations and covalent modifications of the highly abundant, long-lived crystallins of the eye lens cause their aggregation leading to progressive opacification of the lens, cataract. The nature and biochemical mechanisms of the aggregation process are poorly understood, as neither amyloid nor native-state polymers are commonly found in opaque lenses. The ßγ-crystallin fold contains four highly conserved buried tryptophans, which can be oxidized to more hydrophilic products, such as kynurenine, upon UV-B irradiation. We mimicked this class of oxidative damage using Trp→Glu point mutants of human γD-crystallin. Such substitutions may represent a model of UV-induced photodamage-introduction of a charged group into the hydrophobic core generating "denaturation from within." The effects of Trp→Glu substitutions were highly position dependent. While each was destabilizing, only the two located in the bottom of the double Greek key fold-W42E and W130E-yielded robust aggregation of partially unfolded intermediates at 37°C and pH 7. The αB-crystallin chaperone suppressed aggregation of W130E, but not W42E, indicating distinct aggregation pathways from damage in the N-terminal vs C-terminal domain. The W130E aggregates had loosely fibrillar morphology, yet were nonamyloid, noncovalent, showed little surface hydrophobicity, and formed at least 20°C below the melting temperature of the native ß-sheets. These features are most consistent with domain-swapped polymerization. Aggregation of partially destabilized crystallins under physiological conditions, as occurs in this class of point mutants, could provide a simple in vitro model system for drug discovery and optimization.

Isomerization of aspartyl residues in crystallins and its influence upon cataract.

BACKGROUND: Age-related cataracts, which probably form due to insolubilization of lens proteins, can lead to loss of vision. Although the exact reason is unknown, lens protein aggregation may be triggered by increases in PTMs such as D-ß-, L-ß- and D-α-Asp isomers. These isomers have been observed in aged lens; however, there have been few quantitative and site-specific studies owing to the lack of a quick and precise method for distinguishing between D- and L-Asp in a peptide or protein. SCOPE OF REVIEW: We describe a new method for detecting peptides containing Asp isomers at individual sites in any protein by using an LC-MS/MS system combined with commercial enzymes that specifically react with different isomers. We also summarize current data on the effect of Asp isomerization on lens crystallins. MAJOR CONCLUSIONS: The new technique enabled the analysis of isomers of Asp residues in lens proteins precisely and quickly. An extensive proportion of Asp isomerization was observed at all Asp sites of crystallins in the insoluble fraction of aged lens. In addition, d-amino acid substitutions in crystallin-mimic peptides showed altered structural formation and function. These results indicate that isomerization of Asp residues affects the stability, structure and inter-subunit interaction of lens crystallins, which will induce crystallin aggregation and insolubilization, disrupt the associated functions, and ultimately contribute to the onset of senile cataract formation. GENERAL SIGNIFICANCE: The mechanism underlying the onset of age-related diseases may involve isomerization, whereby D-amino acids are incorporated in the L-amino acid world of life. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

Isomerization of Asp residues plays an important role in αA-crystallin dissociation.

Aged cataract formation is caused by the accumulative precipitation of lens proteins incorporating diverse post-translational modifications. α-Crystallin, a major structural and functional lens protein, consists of a large polymeric structure that is dissociated and insolubilized with accumulative post-translational modifications. One such modification, isomerization of Asp, was recently identified in αB-crystallin monomers derived from aged lens. However, the distributions of Asp isomers in each lens fraction remain unknown. Here, α-crystallin fractions from aged lens were separated into heteropolymeric and monomeric forms to determine the Asp isomerization ratios in each fraction. Lens of four different ages were homogenized and centrifuged, and the soluble fraction was applied to size-exclusion chromatography. The heteropolymeric α-crystallin and monomeric crystallin fractions were obtained and concentrated. After trypsin digestion, each fraction was independently applied to liquid chromatography equipped with mass spectrometry to extract α-crystallin-derived peptides containing Asp isomers. The results showed that Asp58, Asp84 and Asp151 of αA-crystallin were highly isomerized in the monomeric fraction, but not isomerized to the same level in the heteropolymeric fraction. Each type of Asp isomerization increased in an age-dependent manner, was site-specific and was similar to previous results from lens water-insoluble fractions. These results imply that isomerization of Asp residues leads to dissociation of αA-crystallin from the heteropolymeric state and induces insolubilization in aged lens. Taken together, our findings suggest that isomerization of Asp might disrupt the higher order polymeric state of α-crystallin, resulting in decreased solubility and function, ultimately contributing to lens protein impairment and cataract formation with aging.

Effect of Asp 96 isomerization on the properties of a lens αB-crystallin-derived short peptide.

One of the major reasons for age-related cataract formation is an accumulation of insoluble lens proteins. In particular, higher-order α-crystallin aggregates, comprising αA and αB subunits, are insolubilized by the build up of various post-translational modifications over time. Although we previously found an exceptional amount of Asp96 isomerization in αB-crystallin from aged human lens, the biological effect remains unknown. To approximate the effect of Asp 96 isomerization in αB-crystallin, here residues 93-103 of αB-crystallin were chemically synthesized as peptides in which l-α-Asp was replaced with l-ß-Asp, D-α-Asp, or D-ß-Asp. The resulting peptides were then compared in a biological assay. The results showed that isomerization of Asp 96 altered both the local structure of peptide and its stability against enzymatic digestion. In addition, the synthesized peptides decreased the insoluble fraction of heated α-crystallin. The D-ß-Asp-containing peptide further decreased heat-induced precipitation of α-crystallin, and a chaperone assay based on heated alcohol dehydrogenase implied differential interaction of the peptides with substrate depending on the Asp isomer present in each. Our results suggest that the formation of Asp isomers is likely to affect the higher-order oligomer structure of α-crystallin and thereby its chaperone functions in aged lens.