AKAP2 anchors PKA with aquaporin-0 to support ocular lens transparency.
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ABSTRACT:
SUBMITTER: Gold MG
PROVIDER: S-EPMC3272850 | biostudies-literature | 2012 Jan
REPOSITORIES: biostudies-literature
Publications
AKAP2 anchors PKA with aquaporin-0 to support ocular lens transparency.
Gold Matthew G MG Reichow Steve L SL O'Neill Susan E SE Weisbrod Chad R CR Langeberg Lorene K LK Bruce James E JE Gonen Tamir T Scott John D JD
EMBO molecular medicine 20111116 1
A decline in ocular lens transparency known as cataract afflicts 90% of individuals by the age 70. Chronic deterioration of lens tissue occurs as a pathophysiological consequence of defective water and nutrient circulation through channel and transporter proteins. A key component is the aquaporin-0 (AQP0) water channel whose permeability is tightly regulated in healthy lenses. Using a variety of cellular and biochemical approaches we have discovered that products of the A-kinase anchoring protei ...[more]
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Project description:PurposeAquaporin 0 (AQP0) is the major intrinsic protein in the lens and is essential for establishing proper fiber cell structure and organization. Cytoskeletal proteins that directly interact with the C terminus of AQP0 are identified herein.MethodsThe water-insoluble fraction of lens fiber cells was chemically cross-linked, and cross-linked peptides with the C terminus of AQP0 were identified by mass spectrometry. Coimmunoprecipitation and AQP0 C-terminal peptide pulldown experiments were used to confirm the protein-protein interaction.ResultsUnexpectedly, AQP0 was found to directly associate with ezrin/radixin/moesin (ERM) family members, proteins that are involved in linkage of actin filaments to the plasma membrane. Cross-linked peptides were detected between AQP0 and degenerate sequences of ezrin and radixin; however, AQP0 interaction with ezrin is believed to play a more significant function in the lens because of its higher level of expression and observed ezrin-specific cross-linking. The interaction was found to occur between the C terminus of AQP0 and subdomains F1 and F3 of ERM proteins. The interaction between AQP0 and ezrin was confirmed by coimmunoprecipitation and AQP0 C-terminal peptide pulldown experiments.ConclusionsConsidering the important known functions of the cellular actin cytoskeleton in fiber cell differentiation, the interaction of AQP0 and ERM proteins may play an important role in fiber cell morphology, elongation, and organization.
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Project description:Aquaporin-0 (AQP0), the major integral membrane protein in lens fiber cells, becomes highly modified with increasing age. The functional consequences of these modifications are being revealed, and the next step is to determine how these modifications affect the ocular lens, which is directly related to their abundances and spatial distributions. The aim of this study was to utilize matrix-assisted laser desorption ionization (MALDI) direct tissue profiling methods, which produce spatially-resolved protein profiles, to map and quantify AQP0 post-translational modifications (PTMs). Direct tissue profiling was performed using frozen, equatorial human lens sections of various ages prepared by conditions optimized for MALDI mass spectrometry profiling of membrane proteins. Modified forms of AQP0 were identified and further investigated using liquid chromatography tandem mass spectrometry (LC-MS/MS). The distributions of unmodified, truncated, and oleoylated forms of AQP0 were examined with a maximum spatial resolution of 500 ?m. Direct tissue profiling of intact human lens sections provided high quality, spatially-resolved, relative quantitative information of AQP0 and its modified forms indicating that 50% of AQP0 is truncated at a fiber cell age of 24 ± 1 year in all lenses examined. Furthermore, direct tissue profiling also revealed previously unidentified AQP0 modifications including N-terminal acetylation and carbamylation. N-terminal acetylation appears to provide a protective effect against N-terminal truncation.
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Project description:PurposeTo spatially map human lens Aquaporin-0 (AQP0) protein modifications, including lipidation, truncation, and deamidation, from birth through middle age using matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS).MethodsHuman lens sections were water-washed to facilitate detection of membrane protein AQP0. We acquired MALDI images from eight human lenses ranging in age from 2 months to 63 years. In situ tryptic digestion was used to generate peptides of AQP0 and peptide images were acquired on a 15T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Peptide extracts were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and database searched to identify peptides observed in MALDI imaging experiments.ResultsUnmodified, truncated, and fatty acid-acylated forms of AQP0 were detected in protein imaging experiments. Full-length AQP0 was fatty acid acylated in the core and cortex of young (2- and 4-month) lenses. Acylated and unmodified AQP0 were C-terminally truncated in older lens cores. Deamidated tryptic peptides (+0.9847 Da) were mass resolved from unmodified peptides by FTICR MS. Peptide images revealed differential localization of un-, singly-, and doubly-deamidated AQP0 C-terminal peptide (239-263). Deamidation was present at 4 months and increases with age. Liquid chromatography-MS/MS results indicated N246 undergoes deamidation more rapidly than N259.ConclusionsResults indicated AQP0 fatty acid acylation and deamidation occur during early development. Progressive age-related AQP0 processing, including deamidation and truncation, was mapped in human lenses as a function of age. The localization of these modified AQP0 forms suggests where AQP0 functions may change throughout lens development and aging.
| S-EPMC4675204 | biostudies-literature