Before the emergence of life, it is considered that energy for the synthesis of DL-amino acids came from various sources on the primitive earth, such as ultraviolet light, electric discharges, etc.q_Although the chemical and physical properties of L-amino acids and D-amino acids are extremely similar, only the L-amino acids were selected for polymerization and formation of peptides and proteins on the primitive earth. In this chemical evolution step, D-amino acids were eliminated and all living organisms are now composed predominantly of L-amino acids. Nobody knows why, where and how nature selected L-amino acids, or whether the selection of L-amino acids had a logical reason or was a chance occurrence. However, it is clear that only one of the enantiomers could be selected because polymers which consist of many diastereoisomers of amino acids would not be able to be folded into a proper structure like a current protein. Therefore, homochilarity is essential for life. Once the L-amino acid world was established, D-amino acids were excluded from living systems. For this reason, the presence of D-amino acids in living organisms has not been studied in the life sciences for a long time. However, D-amino acids were recently detected in various living organisms in the form of free amino acids, peptides, and proteins. Free D-amino acids were observed in mammalian tissues related to developmental stage, while some D-amino acid-containing peptides were found in opioid peptides. The presence of these D-amino acids in proteins was considered to be a result of racemization during aging. We study the presence of D-amino acids in various tissues and the mechanism of D-amino acid formation in proteins. Proteins consist exclusively of L-amino acids; nonetheless, D-aspartic acid (D-Asp) has been detected in various human tissue samples such as tooth, bone, aorta brain, and eye lenses from elderly individuals. The presence of D-Asp in aged tissues of the living body has been explained as a result of racemization of aspartyl residues in the protein over time inasmuch as the proteins in such tissues are metabolically inert.
Our study focused on various post-translational modifications such as deamidation, racemization, stereoinversion, isomerization, truncation, phosphorylation, oxidation and an increase in intramolecular disulfide bonding in inert protiens like lens and skin. These posttranslational modifications generate during aging process spontaneously and also are increased by UV, X-ray gamma-ray irradiations. We are studying this theme by the following techniques; amino acid sequence analysis, mass analysis (TOF), 2D electrophoresis, analysis of optical isomers of amino acid by RP-HPLC, peptide mapping, western blotting. Especially we are in frontline at analysis of racemization, stereoinversion, isomerization of lens protein. We discovered a biologically uncommon D--isomer at Asp-58 and Asp-151 in alphaA-crystallin, and also at Asp-36 and Asp-62 in alphaB-crystallin, from aged human lenses. The D-Asp formation was also accompanied by isomerization from the natural alpha-Asp to the biologically uncommon beta-Asp (isoaspartate). Therefore, four isomers, which are normal L-alpha-Asp, biologically uncommon L-beta-Asp, D-alpha-Asp, and D-beta-Asp are formed in alphaA-crystallins. The isomers increased with age, and the total amount of three isomers was more than that of normal L-alpha-Asp in the alphaA-crystallin of the human lenses of subjects in the 80 year range. We also cleared that the reaction is induced by a chiral field created by the higher order structure of the protein itself. The post-translational modifications may cause the formation of cataract by affecting the close-packing of the crystallins and/or by reducing the chaperone-like activity of alpha-crystallins. The reaction increases by aging, UV -irradiation and gamma-ray irradiation.
1) Localization of D-beta-Asp in tissues and cells
Recently, we succeeded in preparation of the antibody which recognizes specifically the D-beta-Asp-containing peptide. We found that some proteins in skin tissues and cell wall from aged human contained D-beta-Asp using the antibody. We have just started to analyze the D-beta-Asp containing proten. It will be useful indicator of aging protein. We believe that such studies on the relationship between the stereoconfiguration of amino acid and the higher order structure of the protein will open up a new field of protein chemisty and biochemistry, and shed light on the aging process, in this case, the relationship between D-amino acid formation and cataract. D-beta-Asp-containing peptide will be useful indicator of aging protein.
2) The Effect of gamma ray irradiation and ultraviolet ray irradiation on the chaperon activity of alpha-crystallin
The mammalian lens soluble proteins consist mainly of alpha, beta and gamma-crystallin. Recent studies have demonstrated that alpha-crystallin functions as a molecular chaperone, that is, alpha-crystallin inhibits the heat aggregation of beta, or gamma-crystallin, and retains the transparency of the lens. In the present study, we carrided out gamma-irradiation on alpha-crystallin, in order to clear the relaitonship between the loss of chaperon activity of and that of the secondary structure of alpha-crystallin.