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.

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 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.

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.

Alpha B- and ßA3-crystallins containing d-aspartic acids exist in a monomeric state.

Crystallin stability and subunit-subunit interaction are essential for eye lens transparency. There are three types of crystallins in lens, designated as α-, ß-, and γ-crystallins. Alpha-crystallin is a hetero-polymer of about 800kDa, consisting of 35-40 subunits of two different αA- and αB-subunits, each of 20kDa. The ß/γ-crystallin superfamily comprises oligomeric ß-crystallin (2-6 subunits) and monomeric γ-crystallin. Since lens proteins have very long half-lives, they undergo numerous post-translational modifications including racemization, isomerization, deamidation, oxidation, glycation, and truncation, which may decrease crystallin solubility and ultimately cause cataract formation. Racemization and isomerization of aspartyl (Asp) residues have been detected only in polymeric α- and oligomeric ß-crystallin, while the situation in monomeric γ-crystallin has not been studied. Here, we investigated the racemization and isomerization of Asp in the γ-crystallin fraction of elderly donors. The results show that Asp residues of γS-, γD- and γC-crystallins were not racemized and isomerized. However, strikingly, we found that a portion of αB-crystallin and ßA3-crystallin moved to the lower molecular weight fraction which is the same size of γ-crystallin. In those fractions, Asp-96 of αB-crystallin and Asp-37 of ßA3-crystallin were highly inverted, which do not occur in the native lens higher molecular weight fraction. Our results indicate the possibility that the inversion of Asp residues may induce dissociation of αB- and ßA3-crystallins from the polymeric and oligomeric states. This is the first report that stereoinversion of amino acids disturbs lens protein assembly in aged human lens.

Rapid survey of four Asp isomers in disease-related proteins by LC-MS combined with commercial enzymes.

Until relatively recently, it was considered that D-amino acids were excluded from living systems except for the cell wall of microorganisms. However, D-aspartate residues have now been detected in long-lived proteins from various tissues of elderly humans. Formation of D-aspartate in proteins induces aggregation and loss of function, leading to age-related disorders such as cataracts and Alzheimer disease. A recent study used LC-MS to analyze isomers of Asp residues in proteins precisely without complex purification of the proteins. However, to identify the four Asp isomers (Lα, Lß, Dß, and Dα) on the chromatogram, it was necessary to synthesize reference peptides containing the four different Asp isomers as standards. Here, we describe a method for rapidly and comprehensively identifying Asp isomers in proteins using a combination of LC-MS and commercial enzymes without synthesizing reference peptides. The protein sample is treated with trypsin, trypsin plus Asp-N, trypsin plus PIMT, trypsin plus paenidase, and the resulting peptides are applied to LC-MS. Because Asp-N hydrolyzes peptide bonds on the N-terminus of only Lα-Asp residues, it differentiates peptides containing Lα-Asp from those containing the other three isomers. Similarly, PIMT recognizes only peptides containing Lß-Asp residues, and paenidase internally cleaves the C-terminus of Dα-Asp residues. This approach was successfully applied to the analysis of all tryptic peptides in aged lens. The comprehensive quantitative data of Asp isomer formation in age-related proteins obtained via this method might be used as biomarkers of age-related disease.

Characterization of ßB2-crystallin tryptophan mutants reveals two different folding states in solution.

Conserved tryptophan residues are critical for the structure and the stability of ß/γ-crystallin in the lenses of vertebrates. During aging, in which the lenses are continuously exposed to ultraviolet irradiation and other environmental stresses, oxidation of tryptophan residues in ß/γ-crystallin is triggered and impacts the lens proteins to varying degrees. Kynurenine derivatives, formed by oxidation of tryptophan, accumulate, resulting in destabilization and insolubilization of ß/γ-crystallin, which correlates with age-related cataract formation. To understand the contribution of tryptophan modification on the structure and stability of human ßB2-crystallin, five tryptophan residues were mutated to phenylalanine considering its similarity in structure and hydrophilicity to kynurenine. Among all mutants, W59F and W151F altered the stability and homo-oligomerization of ßB2-crystallin-W59F promoted tetramerization whereas W151F blocked oligomerization. Most W59F dimers transformed into tetramer in a month, and the separated dimer and tetramer of W59F demonstrated different structures and hydrophobicity, implying that the biochemical properties of ßB2-crystallin vary over time. By using SAXS, we found that the dimer of ßB2-crystallin in solution resembled the lattice ßB1-crystallin dimer (face-en-face), whereas the tetramer of ßB2-crystallin in solution resembled its lattice tetramer (domain-swapped). Our results suggest that homo-oligomerization of ßB2-crystallin includes potential inter-subunit reactions, such as dissociation, unfolding, and re-formation of the dimers into a tetramer in solution. The W>F mutants are useful in studying different folding states of ßB2-crystallin in lens.

Characterization of different-sized human αA-crystallin homomers and implications to Asp151 isomerization.

Site-specific modifications of aspartate residues spontaneously occur in crystallin, the major protein in the lens. One of the primary modification sites is Asp151 in αA-crystallin. Isomerization and racemization alter the crystallin backbone structure, reducing its stability by inducing abnormal crystallin-crystallin interactions and ultimately leading to the insolubilization of crystallin complexes. These changes are considered significant factors in the formation of senile cataracts. However, the mechanisms driving spontaneous isomerization and racemization have not been experimentally demonstrated. In this study, we generated αA-crystallins with different homo-oligomeric sizes and/or containing an asparagine residue at position 151, which is more prone to isomerization and racemization. We characterized their structure, hydrophobicity, chaperone-like function, and heat stability, and examined their propensity for isomerization and racemization. The results show that the two differently sized αA-crystallin variants possessed similar secondary structures but exhibited different chaperone-like functions depending on their oligomeric sizes. The rate of isomerization and racemization of Asp151, as assessed by the deamidation of Asn151, was also found to depend on the oligomeric sizes of αA-crystallin. The predominant isomerization product via deamidation of Asn151 in the different-sized αA-crystallin variants was L-ß-Asp in vitro, while various modifications occurred around Asp151 in vivo. The disparity between the findings of this in vitro study and in vivo studies suggests that the isomerization of Asp151 in vivo may be more complex than what occurs in vitro.

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.

Solvent accessibility of betaB2-crystallin and local structural changes due to deamidation at the dimer interface.

In the lens of the eye the ordered arrangement of the major proteins, the crystallins, contributes to lens transparency. Members of the beta/gamma-crystallin family share common beta-sheet rich domains and hydrophobic regions at the monomer-monomer or domain-domain interfaces. Disruption of these interfaces, due to post-translational modifications, such as deamidation, decreases the stability of the crystallins. Previous experiments have failed to define the structural changes associated with this decreased stability. Using hydrogen/deuterium exchange with mass spectrometry (HDMS), deamidation-induced local structural changes in betaB2-crystallin were identified. Deamidation was mimicked by replacing glutamines with glutamic acids at homologous residues 70 and 162 in the monomer-monomer interface of the betaB2-crystallin dimer. The exchange-in of deuterium was determined from 15 s to 24 h and the global and local changes in solvent accessibility were measured. In the wild type betaB2-crystallin (WT), only about 20% of the backbone amide hydrogen was exchanged, suggesting an overall low accessibility of betaB2-crystallin in solution. This is consistent with a tightly packed domain structure observed in the crystal structure. Deuterium levels were initially greater in N-terminal domain (N-td) peptides than in homologous peptides in the C-terminal domain (C-td). The more rapid incorporation suggests a greater solvent accessibility of the N-td. In the betaB2-crystallin crystal structure, interface Gln are oriented towards their opposite domain. When deamidation was mimicked at Gln70 in the N-td, deuterium levels increased at the interface peptide in the C-td. A similar effect in the N-td was not observed when deamidation was mimicked at the homologous residue, Gln162, in the C-td. This difference in the mutants can be explained by deamidation at Gln70 disrupting the more compact C-td and increasing the solvent accessibility in the C-td interface peptides. When deamidation was mimicked at both interface Gln, deuterium incorporation increased in the C-td, similar to deamidation at Gln70 alone. In addition, deuterium incorporation was decreased in the N-td in an outside loop peptide adjacent to the mutation site. This decreased accessibility may be due to newly exposed charge groups facilitating ionic interactions or to peptides becoming more buried when other regions became more exposed. The highly sensitive HDMS methods used here detected local structural changes in solution that had not been previously identified and provide a mechanism for the associated decrease in stability due to deamidation. Changes in accessibility due to deamidation at the interface led to structural perturbations elsewhere in the protein. The cumulative effects of multiple deamidation sites perturbing the structure both locally and distant from the site of deamidation may contribute to aggregation and precipitation during aging and cataractogenesis in the lens.

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.

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.

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.

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.

Deamidation alters interactions of beta-crystallins in hetero-oligomers.

PURPOSE: Cataracts are a major cause of blindness worldwide. A potential mechanism for loss of visual acuity may be due to light scattering from disruption of normal protein-protein interactions. During aging, the lens accumulates extensively deamidated crystallins. We have previously reported that deamidation in the betaA3-crystallin (betaA3) dimer decreased the stability of the dimer in vitro. The purpose of the present study was to investigate if deamidation altered the interaction of betaA3 with other beta-crystallin subunits. METHODS: Deamidation was mimicked by replacing glutamines, Q85 and Q180, at the predicted interacting interface between betaA3 domains with glutamic acids by site-directed mutagenesis. Human recombinant wild type betaA3 or the doubly deamidated mutant betaA3 Q85E/Q180E (DM betaA3) were mixed with either betaB1- or betaB2-crystallin (betaB1 or betaB2) subunits. After incubation at increasing temperatures, hetero-oligomers were resolved from individual subunits and their molar masses determined by size exclusion chromatography with in line multiangle laser light scattering. Structural changes of hetero-oligomers were analyzed with fluorescence spectroscopy and blue-native PAGE. RESULTS: Molar masses of the hetero-oligomer complexes indicated betaA3 formed a polydispersed hetero-tetramer with betaB1 and a mondispersed hetero-dimer with betaB2. Deamidation at the interface in the betaA3 dimer decreased formation of the hetero-oligomer with betaB1 and further decreased formation of the hetero-dimer with betaB2. During thermal-induced denaturation of the deamidated betaA3 dimer, betaB1 but not betaB2 was able to prevent precipitation of betaA3. CONCLUSIONS: Deamidation decreased formation of hetero-oligomers between beta-crystallin subunits. An excess accumulation of deamidated beta-crystallins in vivo may disrupt normal protein-protein interactions and diminish the stabilizing effects between them, thus, contributing to the accumulation of insoluble beta-crystallins during aging and cataracts.

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.

Localization of D-beta-aspartic acid-containing proteins in human eyes.

PURPOSE: Biologically uncommon D-beta-aspartic acid (D-beta-Asp) has been detected in proteins from various human tissues in elderly donors. Previous studies have identified D-beta-Asp residues at four different specific sites in alpha-crystallin from aged human lenses and an increased amount of D-beta-Asp residues with age. D-beta-Asp is formed as a result of racemization and accumulates with age; therefore, it is thought to be a potential marker of aging. To reveal the role of the D-beta-Asp formation in the aging process of eyes, immunohistochemical localization of D-beta-Asp was investigated in ocular samples of various ages. METHODS: Polyclonal antibody to the D-beta-Asp-containing peptide was prepared. To confirm the specificity of the antibody, SDS-PAGE and Western blotting analyses of lens were performed. To detect the locality of the D-beta-Asp-containing protein, immunohistochemical staining using the antibody was carried out in ocular samples obtained from nine donors 18 to 88 years of age and two fetuses. RESULTS: The antibody to the D-beta-Asp-containing peptide reacted with the lens peptide of the aged donors around 20 kDa that was compatible with alpha-A crystallin. In addition, the binding of the antibody to the alpha-A crystallin was almost completely blocked with the addition of the excess D-beta-Asp-containing peptide. The antibody showed a negative reaction with any of the tissues in the eye of human fetuses and in donors younger than 18 years. In contrast, relatively strong immunoreactivity to the D-beta-Asp-containing peptides was seen in the nuclei of the lens, in nonpigmented ciliary epithelial cells, in drusen, and in the sclera of elderly donors. In addition, moderate to weak immunoreactivity was seen in the cortex of the lens, in the blood vessels of the retina, in the optic nerve head, and in the lamina cribrosa of elderly donors. Furthermore, the immunoreactions were almost completely blocked with the addition of the excess D-beta-Asp-containing peptide in the reaction mixture. CONCLUSIONS: The D-beta-Asp-containing proteins appeared in various ocular tissues with age. This study clearly demonstrated that the D-beta-Asp-containing proteins are more widespread in aged tissues than previously thought. The formation of D-beta-Asp in protein can cause major changes in its structure because different side chain orientations can induce an abnormal peptide backbone, and the main chain of the peptide can then become elongated by the beta linkage. Therefore, this modification can be the result of the partial unfolding of protein, leading to various age-related ocular diseases. In particular, D-beta-Asp would provide a new aspect of the molecular mechanisms of age-related macular degeneration because drusen is positive for D-beta-Asp.