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