Side-chain conformers to allow conversion from normal to isoaspartate in age-related proteins and peptides.

Dß (or D-iso)- and Lß- (or iso)- aspartyl (Asp) residues are accumulated in aged lens crystallins and amyloid beta (Aß) proteins, respectively, as a result of spontaneous, nonenzymatic isomerization of normal Lα-Asp. To explore why such uncommon Asp isomers are accumulated, the stability of Lα-, Lß-, and Dß-Asp was compared in view of the staggered side-chain conformers. By using cylindrin (KVKVLGD7VIEV) from αB-crystallin and Aß17-25 (L17VFF20AED23)VG25) containing Asp isomers, the vicinal spin-spin coupling constants of Asp Hα-Hß1 and Hα-Hß2 were quantified by high-resolution solution 1H NMR. It was found that the trans conformer was extremely preferred in Dß-Asp7 side-chain of cylindrin. In Aß17-25, the side chain of Lß-Asp23 was likely to adopt trans conformer, while gauche conformers were rather rich in Lα-Asp23. In gauche conformers, the close distance between Asp carboxylate carbon (CCOO-) and backbone nitrogen (N) next to Asp is advantageous to the intramolecular cyclization to form succinimide intermediate, followed by the conversion from α- to ß-Asp. The cyclization is limited in the trans conformer because of the long distance between CCOO- and N, to keep Dß- or Lß-Asp stable. This would be the reason for the site specificity of Asp isomerization in proteins. The higher population of trans conformer in Asp side chain, the less isomerization of Asp as shown as Asp76 in αA-crystallin. The stability and less reactivity of normal Asp and its isomers are the potential factors to determine whether or not the abnormal accumulation is permitted in aged crystallins and Aß.

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.

Glycosaminoglycan Binding and Non-Endocytic Membrane Translocation of Cell-Permeable Octaarginine Monitored by Real-Time In-Cell NMR Spectroscopy.

Glycosaminoglycans (GAGs), which are covalently-linked membrane proteins at the cell surface have recently been suggested to involve in not only endocytic cellular uptake but also non-endocytic direct cell membrane translocation of arginine-rich cell-penetrating peptides (CPPs). However, in-situ comprehensive observation and the quantitative analysis of the direct membrane translocation processes are challenging, and the mechanism therefore remains still unresolved. In this work, real-time in-cell NMR spectroscopy was applied to investigate the direct membrane translocation of octaarginine (R8) into living cells. By introducing 4-trifluoromethyl-l-phenylalanine to the N terminus of R8, the non-endocytic membrane translocation of 19F-labeled R8 (19F-R8) into a human myeloid leukemia cell line was observed at 4 °C with a time resolution in the order of minutes. 19F NMR successfully detected real-time R8 translocation: the binding to anionic GAGs at the cell surface, followed by the penetration into the cell membrane, and the entry into cytosol across the membrane. The NMR concentration analysis enabled quantification of how much of R8 was staying in the respective translocation processes with time in situ. Taken together, our in-cell NMR results provide the physicochemical rationale for spontaneous penetration of CPPs in cell membranes.

Kinetics of the competitive reactions of isomerization and peptide bond cleavage at l-α- and d-ß-aspartyl residues in an αA-crystallin fragment.

d-ß-aspartyl (Asp) residue has been found in a living body such as aged lens crystallin, although l-α-amino acids are constituents in natural proteins. Isomerization from l-α- to d-ß-Asp probably modulates structures to affect biochemical reactions. At Asp residue, isomerization and peptide bond cleavage compete with each other. To gain insight into how fast each reaction proceeds, the analysis requires the consideration of both pathways simultaneously and independently. No information has been provided, however, about these competitive processes because each reaction has been studied separately. The contribution of Asp isomers to the respective pathways has still been veiled. In this work, the two competitive reactions, isomerization and spontaneous peptide bond cleavage at Asp residue, were simultaneously observed and compared in an αA-crystallin fragment, S51 LFRTVLD58 SG60 containing l-α- and d-ß-Asp58 isomers. The kinetics showed that the formation of l- and d-succinimide (Suc) intermediate, as a first step of isomerization, was comparable at l-α- and d-ß-Asp. Although l-Suc was converted to l-ß-Asp, d-Suc was liable to return to the original d-ß-Asp, the reverse reaction marked enough to consider d-ß-Asp as apparently stable. d-ß-Asp was also resistant to the peptide bond cleavage. Such apparent less reactivity is probably the reason for gradual and abnormal accumulation of d-ß-Asp in a living body under physiological conditions. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

D-ß-aspartyl residue exhibiting uncommon high resistance to spontaneous peptide bond cleavage.

Although L-amino acids were selected as main constituents of peptides and proteins during chemical evolution, D-aspartyl (Asp) residue is found in a variety of living tissues. In particular, D-ß-Asp is thought to be stable than any other Asp isomers, and this could be a reason for gradual accumulation in abnormal proteins and peptides to modify their structures and functions. It is predicted that D-ß-Asp shows high resistance to biomolecular reactions. For instance, less reactivity of D-ß-Asp is expected to bond cleavage, although such information has not been provided yet. In this work, the spontaneous peptide bond cleavage was compared between Asp isomers, by applying real-time solution-state NMR to eye lens αΑ-crystallin 51-60 fragment, S(51)LFRTVLD(58)SG(60) and αΒ-crystallin 61-67 analog, F(61)D(62)TGLSG(67) consisting of L-α- and D-ß-Asp 58 and 62, respectively. Kinetic analysis showed how tough the uncommon D-ß-Asp residue was against the peptide bond cleavage as compared to natural L-α-Asp. Differences in pKa and conformation between L-α- and D-ß-Asp side chains were plausible factors to determine reactivity of Asp isomers. The present study, for the first time, provides a rationale to explain less reactivity of D-ß-Asp to allow abnormal accumulation.

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.

Staggered side-chain conformers of aspartyl residues prerequisite to transformation from L-α- to D-ß-aspartate 58 in human-lens αA-crystallin fragment.

D-ß-aspartyl (Asp) residues are found in aged human-lens αA-crystallin. To explore why the uncommon D-ß-Asp is accumulated, the stability of L-α-, D-α-, and D-ß-Asp residues is compared in view of the staggered side-chain conformers. By using αA-crystallin fragment, T(55)VLD(58)SGISEVR(65), composed of Asp58 isomers, the vicinal spin-spin coupling constants of Asp58 Hα-Hß1 and Hα-Hß2 are quantified by high-resolution solution NMR. The trans conformer is most preferred in the D-ß-Asp side-chain, whereas gauche+ and gauche- are abundant in L-α- and D-α-Asp. The close distance between Asp58 carboxylate carbon (CCOO-) and Ser59 nitrogen (N) in gauche+ and gauche- is advantageous to the intramolecular cyclization to form succinimide intermediate, followed by the transformation from α- to ß-Asp. The cyclization is not allowed in the trans conformer because of the long distance between CCOO- and N, to keep D-ß-Asp stable. The change from gauche to trans conformer in D-ß-Asp is exothermic and enthalpy-driven.

Uptake of sevoflurane limited by the presence of cholesterol in the lipid bilayer membrane: a multinuclear nuclear magnetic resonance study.

The effect of cholesterol on the uptake of a fluorinated general anesthetic, sevoflurane (SF, fluoromethyl 2,2,2-trifluoro-1-[trifluoromethyl]ethyl ether) was studied by multinuclear, high-resolution nuclear magnetic resonance (NMR) spectroscopy in combination with a pulsed-field gradient technique. Using large unilamellar vesicles of egg phosphatidylcholine/egg phosphatidylglycerol/cholesterol as model fluid cell membranes, the (19)F and (1)H NMR chemical shifts, longitudinal relaxation times (T1), and diffusion coefficients (D(eff)) were systematically analyzed to quantify the modulation of SF uptake to the lipid membrane by cholesterol. All NMR parameters (chemical shift, T1, and D(eff)) showed that SF uptake is limited by the presence of cholesterol in the membrane. It was found that SF uptake at 40 mol% cholesterol is limited to 50%-60% of the partitioning fraction in the absence of cholesterol in the membrane. This finding is attributed to the loss of motional freedom in the rigid membrane environment, as demonstrated by the gradual slowdown of lipid mobility D(eff) with increase in cholesterol concentration from 0 mol% to 40 mol%.

Kinetics of isomerization and inversion of aspartate 58 of αA-crystallin peptide mimics under physiological conditions.

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-ß, D-α and L-ß-Asp residues through succinimide intermediates. Of these Asp isomers, large amounts of D-ß- and L-ß-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 (T(55)VLD(58)SGISEVR(65)) and its isomers in which L-α-Asp at position 58 was replaced with L-ß-, D-ß- 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-ß-Asp peptide to L-succinimidyl peptide. The rate constant for hydrolysis from L-succinimidyl peptide to L-ß-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-ß-Asp peptide and for hydrolysis from D-succinimidyl peptide to D-ß-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.

Characterization of an antibody that recognizes peptides containing D-ß-aspartyl residues.

PURPOSE: Biologically uncommon D-ß-aspartyl (D-ß-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-ß-Asp-Ala-Thr-Gly-Leu-D-ß-Asp-Ala-Thr-Gly-Leu-D-ß-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-ß-Asp-containing peptides but did not react with L-α-Asp-, L-ß-Asp-, or D-α-Asp-containing peptides. However, it remains unclear whether the antibody recognizes the amino acid sequences surrounding the D-ß-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 (I(146)QTGLDATHAER(157)) and T6 (T(55)VLDAGISEVR(65)) 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-ß-Asp residues and does not react with other configurations of Asp such as the L-α, L-ß, 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-ß-Asp-Ala to detect D-ß-Asp containing proteins in living tissue. CONCLUSIONS: The anti peptide 3R antibody is a powerful and easy tool for detection of D-ß-Asp containing proteins in living tissues from patients with age-related diseases. However, to detect the D-ß-Asp containing proteins in the living tissues using the anti-peptide 3R antibody, the protein must contain the sequence Leu-D-ß-Asp-Ala.

UV B-irradiation enhances the racemization and isomerizaiton of aspartyl residues and production of Nε-carboxymethyl lysine (CML) in keratin of skin.

UV-B irradiation is one of the risk factors in age-related diseases. We have reported that biologically uncommon D-ß-Asp residues accumulate in proteins from sun-exposed elderly human skin. A previous study also reported that carboxymethyl lysine (CML; one of the advanced glycation end products (AGEs)) which is produced by the oxidation of glucose and peroxidation of lipid, also increases upon UV B irradiation. The formation of D-ß-Asp and CML were reported as the alteration of proteins in UV B irradiated skin, independently. In this study, in order to clarify the relationship between the formation of D-ß-Asp and CML, immunohistochemical analysis using anti-D-ß-Asp containing peptide antibodies and anti-CML antibodies was performed in UV B irradiated mice. Immunohistochemical analyses clearly indicated that an anti-D-ß-Asp containing peptide antibody and anti-CML antibody reacted at a common area in UV B irradiated skin. Western blot analyses of the proteins isolated from UV B irradiated skin demonstrated that proteins of 50-70 kDa were immunoreactive towards antibodies for both D-ß-Asp containing peptide and CML. These proteins were identified by proteomic analysis as members of the keratin families including keratin-1, keratin-6B, keratin-10, and keratin-14.