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Glycation decreases calmodulin binding to lens transmembrane protein, MIP
1Departments of Biochemistry and Molecular Biology, and Ophthalmology, Medical College of Georgia, Augusta, GA 30912-2100, USA. smruthin@mail.mcg.edu
Abstract:
Channels of the major intrinsic protein (MIP) of the lens transport water, thus playing an important role in lens fiber cell homeostasis. Calmodulin (CAM) interacts with MIP and possibly regulates MIP channel permeability. Protein glycation has been implicated in lens opacification. We previously identified sites of glycation of MIP, which are in close proximity to the putative CAM binding site. This study is aimed to show the effect of in vitro and in vivo glycation on CAM binding to MIP. Our results show that MIP and MP20 are the major CAM binding proteins of the lens membrane. In vitro incubation of lens membranes with 1 M glucose decreased CAM binding by 38% (P<0.001). Similarly, there was a progressive decrease in CAM binding to diabetic lens membranes compared to age-matched controls (up to 30% decrease, P<0.01). Mutation of K228 and K238 as well as a triple K mutation (K228N, K238N, K259N) of MIP resulted in a decrease in CAM binding. Thus, post-translational protein modifications of MIP influence CAM binding. Since CAM is the ubiquitous Ca(2+) receptor, decreases in CAM binding to the target protein will affect the Ca(2+)-mediated cellular processes leading to lens opacification in diabetic and aging lenses.
Insights
Protein glycation, a process linked to cataracts, reduces calmodulin (CAM) binding to the major intrinsic protein (MIP) in the eye lens. This decreased binding impacts calcium signaling, potentially contributing to lens opacification.
Area of Science:
- Ocular biology
- Protein biochemistry
- Cellular homeostasis
Background:
- Major intrinsic protein (MIP) channels are crucial for lens water transport and cell homeostasis.
- Calmodulin (CAM) interacts with MIP, potentially regulating its channel activity.
- Protein glycation is implicated in lens opacification, with identified glycation sites near the CAM binding site on MIP.
Purpose of the Study:
- To investigate the impact of in vitro and in vivo protein glycation on CAM binding to MIP.
- To determine if post-translational modifications of MIP affect CAM binding.
Main Methods:
- In vitro incubation of lens membranes with glucose.
- Analysis of CAM binding in diabetic versus age-matched control lens membranes.
- Site-directed mutagenesis of specific lysine residues (K228, K238, K259) in MIP to assess their role in CAM binding.
Main Results:
- MIP and MP20 were identified as the primary CAM-binding proteins in the lens membrane.
- In vitro glycation with 1 M glucose reduced CAM binding to MIP by 38%.
- Diabetic lens membranes showed a progressive decrease in CAM binding (up to 30%) compared to controls.
- Mutations at K228 and K238, and a triple K mutation, decreased CAM binding to MIP.
Conclusions:
- Post-translational modifications, specifically glycation and specific lysine mutations of MIP, significantly influence CAM binding.
- Reduced CAM binding to MIP may disrupt calcium-mediated cellular processes.
- This disruption in calcium signaling is a potential mechanism contributing to lens opacification in diabetic and aging lenses.