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Identification and Functional Assessment of Age-Dependent Truncations to Cx46 and Cx50 in the Human Lens
Nefeli Slavi1, Zhen Wang2, Lucas Harvey1
1Department of Biological and Vision Sciences and the Graduate Center for Vision Research, SUNY College of Optometry, New York, New York, United States.
Purpose:
Many proteins in the lens undergo extensive posttranslational modifications (PTMs) with age, leading to alterations in their function. The extent to which lens gap junction proteins, Cx46 and Cx50, accumulate PTMs with aging is not known. In this study, we identified truncations in Cx46 and Cx50 in the human lens using mass spectrometry. We also examined the effect of truncations on channel function using electrophysiological measurements.
Methods:
Human lenses were dissected into cortex, outer nucleus, and nucleus regions, and fiber cell membranes were subjected to trypsin digestion. Tryptic peptides were analyzed by liquid chromatography (LC)-electrospray tandem mass spectrometry (ESI/MS/MS). Effects of truncations on channel conductance, permeability, and gating were assessed in transfected cells.
Results:
Cleavage sites were identified in the C-terminus, the cytoplasmic loop, and the N-terminus of Cx46 and Cx50. Levels of C-terminal truncations, which were found at residues 238 to 251 in Cx46 and at residues 238 to 253 and 274 to 284 in Cx50, were similar in different lens regions. In contrast, levels of truncations in cytoplasmic loop and N-terminal domains of Cx46 and Cx50 increased dramatically from outer cortex to nucleus. Most of the C-terminally truncated proteins were functional, whereas truncations in the cytoplasmic loop did not result in the formation of functional channels.
Conclusions:
Accumulation of cytoplasmic loop and N-terminal truncations in the core might lead to decreases in coupling with age. This reduction is expected to lead to an increase in intracellular calcium and a decrease in levels of glutathione in the nucleus. These changes may ultimately lead to age-related nuclear cataracts.
Insights
Aging human lenses show protein truncations in Cx46 and Cx50 gap junctions. Cytoplasmic loop and N-terminal truncations impair channel function, potentially causing age-related cataracts.
Area of Science:
- Ophthalmology
- Cell Biology
- Biochemistry
Background:
- Proteins in the aging human lens undergo posttranslational modifications (PTMs), altering function.
- The accumulation of PTMs in lens gap junction proteins connexin 46 (Cx46) and connexin 50 (Cx50) with age is not well understood.
Purpose of the Study:
- To identify age-related truncations in human lens Cx46 and Cx50 using mass spectrometry.
- To investigate the functional consequences of these truncations on channel activity.
Main Methods:
- Human lenses were dissected, and fiber cell membranes were analyzed using liquid chromatography-electrospray tandem mass spectrometry (LC-ESI/MS/MS).
- Electrophysiological measurements in transfected cells assessed the effects of identified truncations on channel conductance, permeability, and gating.
Main Results:
- Mass spectrometry identified cleavage sites in the C-terminus, cytoplasmic loop, and N-terminus of Cx46 and Cx50.
- While C-terminal truncations generally retained function, truncations in the cytoplasmic loop domains of Cx46 and Cx50 resulted in non-functional channels.
- Truncations in the cytoplasmic loop and N-terminal domains increased from the lens cortex to the nucleus.
Conclusions:
- Accumulation of cytoplasmic loop and N-terminal truncations in the lens nucleus may decrease gap junction coupling with age.
- This reduction in coupling could lead to increased intracellular calcium and decreased glutathione, contributing to age-related nuclear cataracts.
