Truncation Of Gap-junction Forming Protein, Connexin50During Lens Development And Its Corresponding Biological Functions

The gap junction-forming connexin (Cx)50is gradually truncated during lens development. Premature cleavage of lens connexins is thought to be associated with cataract formation. We have previously shown that Cx50is likely to be cleaved by caspase-3like protease during chick lens development. Here, using HPLC-electrospray tandem mass spectrometry we mapped two cleavage sites at the C-terminus of Cx50after Glu-368and Asp-379and identified caspase-3and caspase-1as the responsible proteases, respectively. The activity of caspase-1,like caspase-3, detected in the outer cortex increased during lens development, which coincided with the accumulation of the truncated fragments of Cx50in the core region of the lens.The truncated Cx50fragments present in older lenses were reproduced in the younger lens after treatment with UV radiation; this cleavage could be partially blocked by caspase-1/3-specific inhibitors. Interestingly, as compared to full-length Cx50, caspase-truncated Cx50showed a dramatic decrease in gap junction coupling and a loss of hemichannel function. Furthermore, expression of caspase-truncated Cx50fragments increased cell viability against UV radiation as compared to full-length Cx50. Together, these results suggest that both caspase-1and-3are responsible for the cleavage at the C-terminus of Cx50during lens development. The reduction of gap junction coupling and closure of hemichannels formed by truncated Cx50are likely to adaptively protect cells against elevated oxidative stress associated with lens aging.In the first chapter, we mapped two truncation sites, Glu-368and Asp-379, in the COOH-terminus of chicken Cx50using HPLC ESI-Tandem Mass Spectrometry and discovered the corresponding protease. We prepared full-length Cx50and GST fused COOH terminal peptide, which was subjected to in vitro enzymatic digestion. Results from these studies confirmed that Cx50is the direct substrate for Caspase-1and Caspase-3and the amino acid residues Glu-368and Asp-379are crucial for the cleavage.In the second chapter, we determined the localization of Caspase-1and-3in the lens tissue and discovered the internal association with the accumulation of truncated Cx50in the lens core region. Furthermore, we reproduced the truncation of Cx50in the younger lens with the treatment of UV-radiation, which normally showed in the older lens. More interestingly, as compared to full-length Cx50, caspase-truncated Cx50showed a dramatic decrease in gap junction coupling and a loss of hemichannel function and expression of caspase-truncated Cx50fragments increased cell viability against UV radiation.At the last chapter, the study of the truncation of Cx50also extended to the mammalian system with the discovering of truncation of mouse Cx50. Though the specific cleavage site and corresponding biological function are not clear yet, Caspase-1was proved to be involved in this truncation. More interestingly, this cleavage probably takes place in the intracellular loop region of mouse Cx50, which differs from the truncation in chicken Cx50.In a summary, we discovered mechanism of the truncation of Cx50during lens development and identified the specific truncation sites. Furthermore, a new protection system against oxidative stress in the lens fiber was introduced, which could be essential for the study on cataract in the future.