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1.  Kinetics of Isomerization and Inversion of Aspartate 58 of αA-Crystallin Peptide Mimics under Physiological Conditions 
PLoS ONE  2013;8(3):e58515.
Although proteins consist exclusively of L-amino acids, we have reported that aspartyl (Asp) 58 and Asp 151 residues of αA-crystallin of eye lenses from elderly cataract donors are highly inverted and isomerized to D-β, D-α and L-β-Asp residues through succinimide intermediates. Of these Asp isomers, large amounts of D-β- and L-β-isomers are present but the amount of D-α-isomer is not significant. The difference in abundance of the Asp isomers in the protein may be due to the rate constants for the formation of the isomers. However, the kinetics have not been well defined. Therefore, in this study, we synthesized a peptide corresponding to human αA-crystallin residues 55 to 65 (T55VLD58SGISEVR65) and its isomers in which L-α-Asp at position 58 was replaced with L-β-, D-β- and D-α-Asp and determined the rate of isomerization and inversion of Asp residues under physiological conditions (37°C, pH7.4). The rate constant for dehydration from L-α-Asp peptide to L-succinimidyl peptide was 3 times higher than the rate constant for dehydration from L-β-Asp peptide to L-succinimidyl peptide. The rate constant for hydrolysis from L-succinimidyl peptide to L-β-Asp peptide was about 5 times higher than the rate constant for hydrolysis from L-succinimidyl peptide to L-α-Asp peptide. The rate constant for dehydration from L-α-Asp peptide to L-succinimidyl peptide was 2 times higher than the rate constant for dehydration from D-α-Asp peptide to D-succinimidyl peptide. The rate constants for hydrolysis from L-succinimidyl peptide to L-β-Asp peptide and for hydrolysis from D-succinimidyl peptide to D-β-Asp peptide were almost equal. Using these rate constants, we calculated the change in the abundance ratios of the 4 Asp isomers during a human lifespan. This result is consistent with the fact that isomerized Asp residues accumulate in proteins during the ageing process.
doi:10.1371/journal.pone.0058515
PMCID: PMC3591338  PMID: 23505525
2.  Chronic Exposure to Low Frequency Noise at Moderate Levels Causes Impaired Balance in Mice 
PLoS ONE  2012;7(6):e39807.
We are routinely exposed to low frequency noise (LFN; below 0.5 kHz) at moderate levels of 60–70 dB sound pressure level (SPL) generated from various sources in occupational and daily environments. LFN has been reported to affect balance in humans. However, there is limited information about the influence of chronic exposure to LFN at moderate levels for balance. In this study, we investigated whether chronic exposure to LFN at a moderate level of 70 dB SPL affects the vestibule, which is one of the organs responsible for balance in mice. Wild-type ICR mice were exposed for 1 month to LFN (0.1 kHz) and high frequency noise (HFN; 16 kHz) at 70 dB SPL at a distance of approximately 10–20 cm. Behavior analyses including rotarod, beam-crossing and footprint analyses showed impairments of balance in LFN-exposed mice but not in non-exposed mice or HFN-exposed mice. Immunohistochemical analysis showed a decreased number of vestibular hair cells and increased levels of oxidative stress in LFN-exposed mice compared to those in non-exposed mice. Our results suggest that chronic exposure to LFN at moderate levels causes impaired balance involving morphological impairments of the vestibule with enhanced levels of oxidative stress. Thus, the results of this study indicate the importance of considering the risk of chronic exposure to LFN at a moderate level for imbalance.
doi:10.1371/journal.pone.0039807
PMCID: PMC3387207  PMID: 22768129
3.  Co-localisation of advanced glycation end products and d-β-aspartic acid-containing proteins in gelatinous drop-like corneal dystrophy 
The British Journal of Ophthalmology  2012;96(8):1127-1131.
Purpose
Gelatinous drop-like corneal dystrophy (GDLD), also known as familial subepithelial corneal amyloidosis, is an autosomal recessive disorder that causes progressive corneal opacity due to accumulation of amyloid fibrils in the corneal stroma. Genetic analyses have revealed that a mutation in membrane component chromosome 1 surface marker 1 gene is responsible for GDLD. However, the mechanism of amyloid formation in the corneal stroma remains unclear. The present study attempted to reveal the role of advanced glycation end products (AGE) and d-amino acids in amyloid formation in GDLD.
Methods
Informed consent was obtained from five patients with GDLD, three patients with bullous keratopathy and three patients with interstitial keratitis and all the specimens were analysed. Localisation of amyloid fibrils was analysed using Congo-red and thioflavin T staining. In addition, the localisation of AGE (Nɛ-carboxy(methyl)-l-lysine, pyrraline and pentosidine) and d-β-aspartic acid-containing proteins, a major form of d-amino acid-containing proteins, was analysed immunohistochemically.
Results
In all GDLD specimens, strong immunoreactivity to AGE and d-β-aspartic acid-containing proteins was detected in the subepithelial amyloid-rich region. In contrast, amyloid fibrils, AGE, or d-amino acid-containing proteins were slightly detected in the corneal stroma of patients with bullous keratopathy and interstitial keratitis.
Conclusions
Abnormally accumulated proteins rich in AGE and d-β-aspartic acid co-localise in the amyloid lesions in GDLD. These results indicate that non-enzymatic post-translational modifications of proteins, including AGE formation and isomerisation of aspartyl residues, will be the cause as well as the result of amyloid fibril formations in GDLD.
doi:10.1136/bjophthalmol-2012-301728
PMCID: PMC3404710  PMID: 22694960
Advanced glycation end products; biochemistry; cornead-amino acids; d-β-aspartic acid; familial subepithelial corneal amyloidosis; GDLD; gelatinous drop-like corneal dystrophy; M1S1; Nɛ-(carboxy)methyl-l-lysin; optics and refraction; pathology; pentosidine; physiology; pyrraline; treatment surgery; tumour-associated calcium signal transducer 2 (TACSTD2)
4.  Characterization of an antibody that recognizes peptides containing D-β-aspartyl residues 
Molecular Vision  2012;18:996-1003.
Purpose
Biologically uncommon D-β-aspartyl (D-β-Asp) residues have been detected in proteins from various human tissues from elderly donors and are connected with cataract, age-related macular degeneration, Alzheimer disease and UV-irradiated skin. In a previous study, we prepared a highly specific antibody against the peptide Gly-Leu-D-β-Asp-Ala-Thr-Gly-Leu-D-β-Asp-Ala-Thr-Gly-Leu-D-β-Asp-Ala-Thr (designated peptide 3R) that corresponds to three repeats of positions 149–153 of human lens αA-crystallin. This antibody clearly distinguishes between the different configurations of the Asp residue in that it reacted strongly with the D-β-Asp-containing peptides but did not react with L-α-Asp-, L-β-Asp-, or D-α-Asp-containing peptides. However, it remains unclear whether the antibody recognizes the amino acid sequences surrounding the D-β-Asp residue. The purpose of the present study is to elucidate the sequence dependency of the epitope of the antigen.
Methods
To clarify the properties of the anti-peptide 3R antibody, we used F-moc (9-fluorenylmethoxycarbonyl) solid phase chemistry to synthesize various peptides and analogs based on the peptides T18 (I146QTGLDATHAER157) and T6 (T55VLDAGISEVR65) which correspond to amino acid sequences 146–157 and 55–65, respectively of human αA-crystallin. The specificity of antibody was confirmed by ELISA (enzyme-linked immunosorbent assay) using these peptides.
Results
The anti peptide 3R antibody specifically recognized D-β-Asp residues and does not react with other configurations of Asp such as the L-α, L-β, D-α isomers in peptides. When the Ala in the peptide was replaced by other amino acid residues, the antibody did not react with the antigen. The antibody requires the sequence Leu-D-β-Asp-Ala to detect D-β-Asp containing proteins in living tissue.
Conclusions
The anti peptide 3R antibody is a powerful and easy tool for detection of D-β-Asp containing proteins in living tissues from patients with age-related diseases. However, to detect the D-β-Asp containing proteins in the living tissues using the anti-peptide 3R antibody, the protein must contain the sequence Leu-D-β-Asp-Ala.
PMCID: PMC3339034  PMID: 22550393
5.  Structural features of isomerizable aspartyl residues in human α-crystallins 
Molecular Vision  2012;18:1823-1827.
Purpose
The aspartyl (Asp) residues 58 and 151 in αA-crystallin, and Asp36 and Asp62 in αB-crystallin in human lenses are known to be highly isomerized with aging. We investigate structural environments of these isomerizable aspartyl residues in α-crystallins of human lenses.
Methods
To perform limited proteolysis experiments of purified human αA- and αB-crystallins, endoproteinase Asp-N (EC 3.4.24.33), which selectively cleaves the peptide bonds at the amino side of aspartyl and cysteic acid residues, was employed. By proteolysis approach coupled with the time-of-flight mass spectrometry (TOF-MS) method, we determined the cleavage points along protein sequences.
Results
Proteolysis by endoproteinase Asp-N occurred preferentially at the site of isomerizable aspartyl residues in αA- and αB-crystallins.
Conclusions
It is found that isomerizable aspartyl residues in α-crystallins in human lenses were located not only in the solvent accessible area but also at regions displaying inherent conformational flexibility.
PMCID: PMC3398496  PMID: 22815635
6.  Localization of D-β-Aspartyl Residue-Containing Proteins in Various Tissues 
Prior to the emergence of life, it is believed that only l-amino acids were selected for formation of protein and that d-amino acids were eliminated on the primitive Earth. Whilst homochirality is essential for life, the occurrence of proteins containing d-β-aspartyl (Asp) residues in various tissues from elderly subjects has been reported recently. Here, we demonstrate the presence of a d-β-Asp-containing protein in the cardiac muscle of heart, blood vessels of the lung, chief cells of the stomach, longitudinal and circular muscle of the stomach, small intestine and large intestine. Since the d-β-Asp residue occurs through a succinimide intermediate, this isomer may potentially be generated in proteins more easily than initially thought. Formation of the d-β-Asp residue in proteins may be related to stress.
doi:10.3390/ijms10051999
PMCID: PMC2695262  PMID: 19564934
d-β-Aspartyl residue; racemization; immunohistochemistry; isomerization; stomach; heart; lung; oxidative stress

Results 1-6 (6)