A Role for Lengsin, a Recruited Enzyme, in Terminal Differentiation in the Vertebrate Lens

National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 04/2008; 283(10):6607-15. DOI: 10.1074/jbc.M709144200
Source: PubMed

ABSTRACT Lengsin is an eye lens-specific member of the glutamine synthetase (GS) superfamily. Lengsin has no GS activity, suggesting that it has a structural rather than catalytic role in lens. In situ hybridization and immunofluorescence showed that lengsin is expressed in terminally differentiating secondary lens fiber cells. Yeast two-hybrid (Y2H) and recombinant protein experiments showed that full-length lengsin can bind the 2B filament region of vimentin. In affinity chromatography, lengsin also bound the equivalent region of CP49 (BFSP2; phakinin), a related intermediate filament protein specific to the lens. Both the vimentin and CP49 2B fragments bound lengsin in surface plasmon resonance spectroscopy with fast association and slow dissociation kinetics. Lengsin expression correlates with a transition zone in maturing lens fiber cells in which cytoskeleton is reorganized. Lengsin and lens intermediate filament proteins co-localize at the plasma membrane in maturing fiber cells. This suggests that lengsin may act as a component of the cytoskeleton itself or as a chaperone for the reorganization of intermediate filament proteins during terminal differentiation in the lens.

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    • "), aA-crystallin (CRYAA) (Hawse et al. 2005), and aB-crystallin (Hawse et al. 2005); (F) actin-capping regulator encoding transcript tropomodulin 1 (TMOD1) (Nowak and Fowler 2012); (G) Lensgin (GLULD1/LGSN) (Wyatt et al. 2008); (H) cell-cycle regulator encoding transcript cyclin-dependent kinase 2 (CDK2) (Gao et al. 1999); (I) lens signaling encoding transcripts coiled-coil domain containing 80 (CCDC80/EQUARIN) (Song et al. 2012), EPH receptor type A2 (EPHA2) (Shi et al. 2012; Cheng et al. 2013), fibroblast growth factor receptor 2 (FGFR2) (Robinson 2006), and frizzled class receptor 3 (FZD3) (Dawes et al. 2013); and (J) lens DNA binding encoding transcripts paired box 6 (PAX6) (Cvekl and Piatigorsky 1996), heat shock transcription factor 4 (HSF4) (Fujimoto et al. 2004; Somasundaram and Bhat 2004), SRY (sex determining region Y)-box 2 (SOX2) (Kondoh et al. 2004), prospero homeobox 1 (PROX1) (Duncan et al. 2002), and GATA binding protein 3 (GATA3) (Maeda et al. 2009). "
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    ABSTRACT: The mature eye lens contains a surface layer of epithelial cells called the lens epithelium that require a functional mitochondrial population to maintain the homeostasis and transparency of the entire lens. The lens epithelium overlies a core of terminally differentiated fiber cells that must degrade their mitochondria to achieve lens transparency. These distinct mitochondrial populations make the lens a useful model system to identify those genes that regulate the balance between mitochondrial homeostasis and elimination. Here we used an RNA sequencing and bioinformatics approach to identify the transcript levels of all genes expressed by distinct regions of the lens epithelium and maturing fiber cells of the embryonic Gallus gallus (chicken) lens. Our analysis detected over 15,000 unique transcripts expressed by the embryonic chicken lens. Of these, over 3000 transcripts exhibited significant differences in expression between lens epithelial cells and fiber cells. Multiple transcripts coding for separate mitochondrial homeostatic and degradation mechanisms were identified to exhibit preferred patterns of expression in lens epithelial cells that require mitochondria relative to lens fiber cells that require mitochondrial elimination. These included differences in the expression levels of metabolic (DUT, PDK1, SNPH), autophagy (ATG3, ATG4B, BECN1, FYCO1, WIPI1), and mitophagy (BNIP3L/NIX, BNIP3, PARK2, p62/SQSTM1) transcripts between lens epithelial cells and lens fiber cells. These data provide a comprehensive window into all genes transcribed by the lens and those mitochondrial regulatory and degradation pathways that function to maintain mitochondrial populations in the lens epithelium and to eliminate mitochondria in maturing lens fiber cells.
    G3-Genes Genomes Genetics 06/2014; 4(8). DOI:10.1534/g3.114.012120 · 3.20 Impact Factor
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    • "Galectin-1 is an example of a protein with potential adhesion/growth regulatory functions in the eye [28], but has not been annotated as a plasma protein. Lengsin is an eye lens-specific member of the glutamine synthetase superfamily [29]. Opticin is a classical vitreous protein, where it is associated with humor collagen fibrils [30]. "
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    ABSTRACT: Mapping of proteins involved in normal eye functions is a prerequisite to identify pathological changes during eye disease processes. We therefore analysed the proteome of human vitreous by applying in-depth proteomic screening technologies. For ethical reasons human vitreous samples were obtained by vitrectomy from "surrogate normal patients" with epiretinal gliosis that is considered to constitute only negligible pathological vitreoretinal changes. We applied different protein prefractionation strategies including liquid phase isoelectric focussing, 1D SDS gel electrophoresis and a combination of both and compared the number of identified proteins obtained by the respective method. Liquid phase isoelectric focussing followed by SDS gel electrophoresis increased the number of identified proteins by a factor of five compared to the analysis of crude unseparated human vitreous. Depending on the prefractionation method proteins were subjected to trypsin digestion either in-gel or in solution and the resulting peptides were analysed on a UPLC system coupled online to an LTQ Orbitrap XL mass spectrometer. The obtained mass spectra were searched against the SwissProt database using the Mascot search engine. Bioinformatics tools were used to annotate known biological functions to the detected proteins. Following this strategy we examined the vitreous proteomes of three individuals and identified 1111 unique proteins. Besides structural, transport and binding proteins, we detected 261 proteins with known enzymatic activity, 51 proteases, 35 protease inhibitors, 35 members of complement and coagulation cascades, 15 peptide hormones, 5 growth factors, 11 cytokines, 47 receptors, 30 proteins of visual perception, 91 proteins involved in apoptosis regulation and 265 proteins with signalling activity. This highly complex mixture strikingly differs from the human plasma proteome. Thus human vitreous fluid seems to be a unique body fluid. 262 unique proteins were detected which are present in all three patient samples indicating that these might represent the constitutive protein pattern of human vitreous. The presented catalogue of human vitreous proteins will enhance our understanding of physiological processes in the eye and provides the groundwork for future studies on pathological vitreous proteome changes.
    Proteome Science 05/2013; 11(1):22. DOI:10.1186/1477-5956-11-22 · 1.73 Impact Factor
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    • "7B, C). Furthermore, Lengsin is expressed in the subpopulation of early differentiating fiber cells which are not yet denucleated (Fig. 7D; (Harding et al., 2008; Wyatt et al., 2008)). In uhrf1 and dnmt1 mutants, Lengsin is expressed in the disorganized cells that make up the mutant lens periphery (Figs. "
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    ABSTRACT: DNA methylation is one of the key mechanisms underlying the epigenetic regulation of gene expression. During DNA replication, the methylation pattern of the parent strand is maintained on the replicated strand through the action of Dnmt1 (DNA Methyltransferase 1). In mammals, Dnmt1 is recruited to hemimethylated replication foci by Uhrf1 (Ubiquitin-like, Containing PHD and RING Finger Domains 1). Here we show that Uhrf1 is required for DNA methylation in vivo during zebrafish embryogenesis. Due in part to the early embryonic lethality of Dnmt1 and Uhrf1 knockout mice, roles for these proteins during lens development have yet to be reported. We show that zebrafish mutants in uhrf1 and dnmt1 have defects in lens development and maintenance. uhrf1 and dnmt1 are expressed in the lens epithelium, and in the absence of Uhrf1 or of catalytically active Dnmt1, lens epithelial cells have altered gene expression and reduced proliferation in both mutant backgrounds. This is correlated with a wave of apoptosis in the epithelial layer, which is followed by apoptosis and unraveling of secondary lens fibers. Despite these disruptions in the lens fiber region, lens fibers express appropriate differentiation markers. The results of lens transplant experiments demonstrate that Uhrf1 and Dnmt1 functions are required lens-autonomously, but perhaps not cell-autonomously, during lens development in zebrafish. These data provide the first evidence that Uhrf1 and Dnmt1 function is required for vertebrate lens development and maintenance.
    Developmental Biology 02/2011; 350(1):50-63. DOI:10.1016/j.ydbio.2010.11.009 · 3.55 Impact Factor
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