Takumi Takata

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, Lβ, Dβ, 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 Lβ-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.

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-β-, L-β- 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 βA3-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-, β-, 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 β/γ-crystallin superfamily comprises oligomeric β-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 β-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 βA3-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 βA3-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 βA3-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.

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.

Quantitative analysis of isomeric (l-α-, l-β-, D-α-, D-β-) aspartyl residues in proteins from elderly donors.

Homochirality is essential for life. For a long time, it was considered that d-amino acids were excluded from living systems. In the past 30 years, however, d-amino acids have been found in living organisms in the form of free amino acids, peptides and proteins, owing to advances in the analysis of optical isomers of amino acids. Free D-amino acids and D-amino-acid-containing peptides have been shown to have important physiological functions. The amount of D-aspartate (Asp) residues in protein spontaneously increases in metabolically inert tissues such as the eye and brain during aging, and may be related to cataract formation and the development of Alzheimer disease, suggesting that D-Asp might be a molecular marker of aging and age-related disorders. The presence of D-Asp in living organisms is thought to result from the isomerization of L-Asp residues in some proteins. Furthermore, the isomerization of Asp does not occur uniformly but only at specific sites. Therefore, it is necessary to determine the sites of isomeric Asp in these proteins in order to elucidate the mechanism of spontaneous Asp isomerization during aging. Herein, we summarize the localization and mechanism of D-amino acids in proteins of living tissues, and the effects of D-amino acid formation in proteins. Furthermore, we describe methods for the analysis of protein-bound D-amino acids including a conventional enantioseparation method based on HPLC and a new convenient method based on LC-MS that can identify the specific sites of D-Asp in proteins.

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 βγ‐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 β‐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.

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 lβ-, dα-, and dβ-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 lβ-, dα-, and dβ-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 lβ-, dα-, and dβ-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.

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-β-Asp, D-α-Asp, or D-β-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-β-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.

d-Amino Acid Residues in Proteins Related to Aging and Age-Related Diseases and a New Analysis of the Isomers in Proteins

Homochirality is essential for the development and maintenance of life. Until relatively recently, the homochirality of amino acids in living systems was believed to be maintained with the exception of the presence of d-amino acids in the cell wall of microorganisms. However, d-amino acids were recently found in various higher organisms in proteins and peptides and as free amino acids. In proteins, d-aspartate (Asp) residues have been detected in various tissues such as the eye lens, teeth, bone, aorta, ligament, brain, and skin of elderly individuals, and thus d-amino acids can no longer be considered as uncommon in living organisms. The presence of d-amino acids may change the higher-order structure of proteins, and this may be the cause of age-related diseases including cataract and Alzheimer’s disease. d-Asp in aged tissues of living organisms is thought to result from the spontaneous racemization of the Asp residues. The racemization of Asp residues in proteins does not occur uniformly but does so at specific residues on the basis of the sequence context or structural considerations. Therefore, it is necessary to determine the nature of Asp residues at specific sites within particular proteins. However, the detection of d-amino acids in proteins to date has been complex and difficult. This review deals with 1) the presence of d-aspartate (Asp) residues in protein of living tissues, 2) the mechanism of d-Asp formation in protein under physiological conditions, 3) the influence of d-Asp on protein structure and function, and 4) recent advances in d-amino acid analysis in protein.