The primary lens epithelial cell cultures used in this study were established from
TTase +/+ and
TTase −/− mouse lens epithelia and were validated to contain the lens-specific αA-crystallin protein.
33 Because the tissue of
TTase −/− mice, including the eye, expresses a truncated form of
TTase mRNA from the fusion of exon 1 and exon 3 sequences, it is conceivable that the same
TTase mRNA is also expressed in LECs isolated from these mice. The upregulation of truncated
TTase mRNA in the null eye probably reflects a compensatory upregulation of gene activity in response to loss of TTase function. The mouse TTase is 107 amino acids long, and its C-terminal 38 amino acids are encoded by exon 2. The exon 1–exon 3 fusion mRNA expressed from the targeted TTase allele would allow extension of the reading frame into the noncoding region of exon 3 to include 17 more amino acids, resulting in a protein that is 21 amino acids shorter (because of the deletion of exon 2) than in the wild-type protein. The active site of TTase protein (23-Cys-Pro-Tyr-Cys-26) is encoded by exon 1. However, 13 of 14 amino acids in the GSH-binding site, which are encoded by the exon 2 sequence, would be missing in the mutant protein. This may greatly affect the structure and function of the mutant TTase protein in the knockout mice. Toward this end, we examined the expression of TTase protein in various tissue and LECs from knockout mice using two different preparations of antibodies against the human TTase (both antibodies were made against the entire length of TTase protein). These studies show that no TTase protein could be detected in tissue or LECs of homozygous knockout mice
(Figs. 1B 2B) . We also could not detect any truncated TTase protein that might be expressed from the mutated
TTase gene, suggesting that if the mutant TTase protein were produced, because of its extreme short half-life, the protein would have been degraded in the process. Even if it were not degraded, the phenomenon of oversensitivity to oxidation exhibited by the
TTase −/− cells could not have been caused by the truncated TTase proteins because
TTase −/− cells could be normalized when pure TTase protein was delivered into the
TTase −/− cells. This strongly suggests that the abnormal property of the
TTase −/− cells is caused by a lack of TTase-1, not by possible toxicity from the truncated TTase protein in the lenses of
TTase knockout mice.