Dataset Information

Identification of a direct Aquaporin-0 binding site in the lens-specific cytoskeletal protein filensin.

ABSTRACT: An interaction between the C-terminus of aquaporin-0 (AQP0) and lens beaded filament protein filensin has been reported previously; however, the region of filensin that is involved in the interaction has not been determined. This study is designed to identify the region of filensin that interacts with AQP0. Chemical crosslinking coupled with mass spectrometry was used to identify the site of interaction. The protein complex was crosslinked with zero-length crosslinker: 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide Hydrochloride (EDC). The crosslinked membrane fraction was digested by trypsin and crosslinked peptides were identified by liquid chromatography-tandem mass spectrometry. A crosslinked peptide between bovine filensin 450-465 (VKGPKEPEPPADLYTK) and bovine AQP0 239-259 (GSRPSESNGQPEVTGEPVELK) was detected. AQP0/filensin crosslinking was not detected in superficial young fiber cells, but increased with fiber cell age in the lens cortex. AQP0/filensin crosslinking and filensin truncation were observed in the same regions of the lens. This crosslinked peptide can be detected in 75 kDa gel band confirming that AQP0/filensin crosslinking can occur between AQP0 and the filensin C-terminal fragment. These results suggest that the AQP0 C-terminus directly interacts with the region of filensin that is adjacent to the major truncation site and the polybasic cluster of residues in the filensin C-terminal tail. This interaction occurs in a specific region of the lens and could only occur between AQP0 and filensin C-terminal fragment in vivo. This interaction supports the dual roles of filensin in the lens; roles that could be important during lens development.

SUBMITTER: Wang Z

PROVIDER: S-EPMC5471609 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Identification of a direct Aquaporin-0 binding site in the lens-specific cytoskeletal protein filensin.

Wang Zhen Z Schey Kevin L KL

Experimental eye research 20170301

An interaction between the C-terminus of aquaporin-0 (AQP0) and lens beaded filament protein filensin has been reported previously; however, the region of filensin that is involved in the interaction has not been determined. This study is designed to identify the region of filensin that interacts with AQP0. Chemical crosslinking coupled with mass spectrometry was used to identify the site of interaction. The protein complex was crosslinked with zero-length crosslinker: 1-Ethyl-3-[3-dimethylamino ...[more]

PMID: 28259670

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Project description:The PITX3 bicoid-type homeodomain transcription factor plays an important role in lens development in vertebrates. PITX3 deficiency results in a spectrum of phenotypes from isolated cataracts to microphthalmia in humans, and lens degeneration in mice and zebrafish. While identification of downstream targets of PITX3 is vital for understanding the mechanisms of normal ocular development and human disease, these targets remain largely unknown. To isolate genes that are directly regulated by PITX3, we performed a search for genomic sequences that contain evolutionarily conserved bicoid/PITX3 binding sites and are located in the proximity of known genes. Two bicoid sites that are conserved from zebrafish to human were identified within the human promoter of the major intrinsic protein of lens fiber, MIP/AQP0. MIP/AQP0 deficiency was previously shown to be associated with lens defects in humans and mice. We demonstrate by both chromatin immunoprecipitation and electrophoretic mobility shift assay that PITX3 binds to MIP/AQP0 promoter region in vivo and is able to interact with both bicoid sites in vitro. In addition, we show that wild-type PITX3 is able to activate the MIP/AQP0 promoter via interaction with the proximal bicoid site in cotransfection experiments and that the introduction of mutations disrupting binding to this site abolishes this activation. Furthermore, mutant forms of PITX3 fail to produce the same levels of transactivation as wild-type when cotransfected with the MIP/AQP0 reporter. Finally, knockdown of pitx3 in zebrafish affects formation of a DNA-protein complex associated with mip1 promoter sequences; and examination of expression in pitx3 morphant and control zebrafish revealed a delay in and reduction of mip1 expression in pitx3-deficient embryos. Therefore, our data suggest that PITX3 is involved in direct regulation of MIP/AQP0 expression and that the alteration of MIP/AQP0 expression is likely to contribute to the lens phenotype in cataract patients with PITX3 mutations.

Project description:Maintenance of proper biomechanics of the eye lens is important for its structural integrity and for the process of accommodation to focus near and far objects. Several studies have shown that specialized cytoskeletal systems such as the beaded filament (BF) and spectrin-actin networks contribute to mammalian lens biomechanics; mutations or deletion in these proteins alters lens biomechanics. Aquaporin 0 (AQP0), which constitutes ∼45% of the total membrane proteins of lens fiber cells, has been shown to function as a water channel and a structural cell-to-cell adhesion (CTCA) protein. Our recent ex vivo study on AQP0 knockout (AQP0 KO) mouse lenses showed the CTCA function of AQP0 could be crucial for establishing the refractive index gradient. However, biomechanical studies on the role of AQP0 are lacking. The present investigation used wild type (WT), AQP5 KO (AQP5(-/-)), AQP0 KO (heterozygous KO: AQP0(+/-); homozygous KO: AQP0(-/-); all in C57BL/6J) and WT-FVB/N mouse lenses to learn more about the role of fiber cell AQPs in lens biomechanics. Electron microscopic images exhibited decreases in lens fiber cell compaction and increases in extracellular space due to deletion of even one allele of AQP0. Biomechanical assay revealed that loss of one or both alleles of AQP0 caused a significant reduction in the compressive load-bearing capacity of the lenses compared to WT lenses. Conversely, loss of AQP5 did not alter the lens load-bearing ability. Compressive load-bearing at the suture area of AQP0(+/-) lenses showed easy separation while WT lens suture remained intact. These data from KO mouse lenses in conjunction with previous studies on lens-specific BF proteins (CP49 and filensin) suggest that AQP0 and BF proteins could act co-operatively in establishing normal lens biomechanics. We hypothesize that AQP0, with its prolific expression at the fiber cell membrane, could provide anchorage for cytoskeletal structures like BFs and together they help to confer fiber cell shape, architecture and integrity. To our knowledge, this is the first report identifying the involvement of an aquaporin in lens biomechanics. Since accommodation is required in human lenses for proper focusing, alteration in the adhesion and/or water channel functions of AQP0 could contribute to presbyopia.

Dataset Information

Identification of a direct Aquaporin-0 binding site in the lens-specific cytoskeletal protein filensin.

Publications

Identification of a direct Aquaporin-0 binding site in the lens-specific cytoskeletal protein filensin.

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