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Miduturu Srinivas, Nefeli Slavi, Zhen Wang, Kevin L Schey; Post-translational Modifications of Gap Junction Proteins in the Human Lens. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3558.
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© ARVO (1962-2015); The Authors (2016-present)
A number of proteins in the lens undergo extensive post-translational modifications (PTMs) with age, leading to alterations in their function. The extent to which gap junction proteins expressed in the lens, Cx46 and Cx50, accumulate PTMs with aging is not known. In this study, we identified truncations in connexin proteins in the human lens using mass spectrometric analysis. In addition, we examined the effects of truncations on channel function.
Human lenses were dissected into outer cortex, inner cortex, and nucleus, and subjected to enzymatic digestion. Enzyme-digested peptides were analyzed by liquid chromatography (LC)-electrospray tandem mass spectrometry (ESI/MS/MS). The functional effects of different PTMs on conductance, permeability and gating were assessed using electrophysiological measurements in cells expressing truncated Cx46 and Cx50 channel proteins.
Truncation sites were identified in the C-terminus, the cytoplasmic loop and the N-terminus of Cx46 and Cx50. In the C-terminus, truncations were found at residues 238-251 in Cx46 and at residues 238-253 and 274-284 in Cx50. Levels of these C-terminal truncations were similar in the different regions of the lens. In contrast, levels of cytoplasmic loop truncations in Cx46 and Cx50 found by mass spectrometry increased dramatically from outer cortex to nucleus. For example, truncation at residue 129 in Cx46 and at residue 119 in Cx50 occurred predominantly in the nucleus. Electrophysiological studies indicated that all the C-terminal truncations were functional. Gating properties and single channel conductance of channels formed by Cx46 and by Cx50 truncated at residues 251 and 253, respectively, were similar to those formed by full-length connexins. In contrast to C-terminal truncations, we found that truncations in the cytoplasmic loop of both Cx46 and Cx50 prevented the formation of functional channels.
The accumulation of cytoplasmic loop truncations in the core might explain the large age-dependent reduction in the coupling conductance found in functional studies (Gao et al., 2013, IOVS, 54:7174). Because the delivery of anti-oxidant molecules such as glutathione to the nucleus is dependent on gap junction channels, the decrease in coupling conductance due to PTMs might underlie the age-dependent decrease in glutathione levels in the lens core, ultimately leading to cataracts.
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