Yuko Momose

The mechanisms of simultaneous stereoinversion, racemization, and isomerization at specific aspartyl residues of aged lens proteins

Proteins have been considered to consist exclusively of L-amino acids in living tissues. However, we found biologically uncommon D-aspartyl (Asp) residues at specific sites in alphaA- and alphaB-crystallin from the aged human lens (mean age: 80 years). In alphaB-crystallin, the Asp-36 and Asp-62 residues are highly racemized (D/L ratios: 0.92 for Asp-36; 0.54 for Asp-62). More interestingly, the configuration of the Asp-58 and Asp-151 residues in alphaA-crystallin is inverted to the D-isomer (D/L ratio: 3.1 for Asp-58, 5.7 for Asp-151). A D/L ratio > 1.0 is not considered to be due to racemization, but rather is thought to result from stereoconfiguration inversion. Our report was the first observation that inversion occurred in the configuration of amino acids in vivo during the natural aging process. We also found that these enantiomers were simultaneously isomerized to form beta-Asp residues. We propose that the mechanism of D- and beta-Asp formation in the protein depends on the primary structure and the presence of a chiral reaction field, which induces formation of D-Asp.

Changes of Secondary Structure Detected by Laser Raman Spectroscopy in Model Peptides of Human αA-Crystallin Due to Substitution of D-Aspartyl Residues for L-Aspartyl Residues

PURPOSE We have found that two aspartyl (Asp-151 and Asp-58) residues of alphaA-crystallin are inverted and isomerized to the biologically uncommon D-beta-Asp residues during aging. In order to elucidate the correlation between the formation of the D-beta-Asp isomer and the environment surrounding the Asp in the protein, we performed a Raman spectroscopic study using two synthetic peptides: T6 peptide containing Asp-58, and T18 peptide, containing Asp-151, which correspond to the tryptic peptides of human alphaA-crystallin. METHODS Both T6 (Thr-Val-Leu-Asp(58)-Ser-Gly-Ile-Ser-Glu-Val-Arg) and T18 (Ile-Gln-Thr-Gly-Leu-Asp(151)-ala-thr-his-ala-Glu-Arg) peptides were synthesized with four optical isomers which have L-alpha-, D-alpha, L-beta and D-beta-aspartyl residues. These peptides were subjected to Raman measurement. RESULTS The Raman spectrum of the L-alpha-Asp T18 peptide measured as dry powder revealed that the secondary structure of this peptide is mainly anti-parallel beta-sheet. The main structure of the D-beta-Asp T18 peptide when in dry powder form was altered to an alpha-helix and/or random structure. The main structure of L-alpha-Asp T18 peptide when measured in aqueous solution also converted to an alpha-helix and/or random structure. The conversion of L-alpha-to D-beta-Asp in T6 peptides when in dry powder form revealed no alteration of secondary beta-sheeted structure. CONCLUSION Raman spectroscopy clearly revealed a large conformational change in the secondary structure of T18 peptide caused by substitution of normal L-alpha-Asp to biologically uncommon Asp-isomers. This result indicates that the inversion of an amino acid in a protein greatly affects the secondary structure of the protein.

The racemization and isomerization of the ASP-151 residue in αA-crystallin is induced by ultraviolet B irradiation

Fuji.i, NI., Momose, yl. , and Takehana, M 2. 1Group of Field and Reaction, Precursory Research for Embryonic Science and Technology (PRESTO), Research and Development Corporation of Japan (JRDC), Nervous Informatics Laboratory, National Institute of Bioscience and Human Technology, Tsukuba, Ibaraki 305,Japan. FAX#: 81-298-54-6213 e-mail:nfujii~_ibh.go.jp 2Kyoritsu College of Pharmacy, Skibakohen, Minatoku Tokyo 105, Japan