Immunological detection of D-β-aspartate-containing protein in lens-derived cell lines

Alpha-crystallin is the major protein of the mammalian lens and its average molecular weight is approximately 800 kDa. It is composed of two kinds of structurally and functionally related polypeptides, αA-and αB-crystallin subunits, each with a molecular weight of 20 kDa Recently, we prepared a polyclonal antibody against peptide Gly-Leu-D-β-Asp-Ala-Thr-Gly-Leu-D-β-Asp-Ala-ThrGly-Leu-D-β-Asp-Ala-Thr (anti-peptide 3R antibody) that corresponded to three repeats of positions 149-153 in human αA-crystallin [11]. This antibody cross-reacted specifically with D-β-Asp-151-containing αA-crystallin. Because formation of D-Asp is accompanied by isomerization to form the β-Asp (isoaspartate) residue, three isomers of Asp residues, L-β-Asp, D-α-Asp and D-β-Asp isomers, are formed in the protein Cell culture systems are used widely for the analysis of cellular functions related to particular organ systems. For lens research, it is of particular interest to find conditions that reflect the situation within this organ. In order to establish whether the D-β-Asp-containing protein is present in cultured lens cells, we cultured two cell lines, αTN4-1 and N/N1003A, which are commonly used in lens research Conclusions: The results indicate that the N/N1003A cell line expressed a 50 kDa D-β-Asp-containing protein, which may share a common amino acid sequence with αA-and αB-crystallin

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