Recombinant purified proteins are recognized by specific antibodies.

<p>The proteins were detected in both the bacterial lysates following induction (L) and after the purification process (P) (<b>A</b>). Following protein induction and purification, recombinant MIPS-160 and IMPase 1 were specifically recognized by the Anti-6x His tag antibody (<b>B</b>), while each protein was selectively recognized by antibodies raised against the human orthologues: Impa1 (<b>C</b>) or Isyna1 (<b>D</b>).</p

MIPS160 and IMPase 1 kinetics properties under different osmotic conditions.

<p>Kinetic properties were measured (in three separated experiments) under high osmolalities (450 mOsm), achieved by increase of either Na⁺ or K⁺ levels. The results are shown in absolute values and in fold of change compared to the values observed in the standard buffers (SB) with the basal osmolality (MAB = 124 mOsm; IAB = 220 mOsm). Five substrate concentrations were used to determine the kinetic properties (units: <i>K</i>_M, mM ± SE; V_max, nmol sec^-1 ± SE; <i>k</i>_<i>cat</i>, sec^-1 ± SE; <i>k</i>_<i>cat</i>/<i>K</i>_M, sec^-1·mM^-1 ± SE). Asterisks denote significant differences with the values observed in MAB or IAB (<i>standard buffers</i>, SB) from three separate experiments (<i>t</i>-test, <i>P</i> < 0.05).</p

Multiple sequence analysis of MIPS isoforms from different species.

<p>MIPS sequences from different species (including three splicing variants from human, Hs, three from rat, and two known alternative variants from Mozambique tilapia, MIPS-160) were aligned using T-Coffee. A unique portion of the MIPS-250 variant that is not shared by any other sequence is highlighted (in block 3 from top). Accession numbers used to build the MSA are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123212#pone.0123212.s006" target="_blank">S4 Table</a> in Supporting Information.</p

Direct Ionic Regulation of the Activity of <i>Myo</i>-Inositol Biosynthesis Enzymes in Mozambique Tilapia

<div><p><i>Myo</i>-inositol (Ins) is a major compatible osmolyte in many cells, including those of Mozambique tilapia (<i>Oreochromis mossambicus</i>). Ins biosynthesis is highly up-regulated in tilapia and other euryhaline fish exposed to hyperosmotic stress. In this study, enzymatic regulation of two enzymes of Ins biosynthesis, Ins phosphate synthase (MIPS) and inositol monophosphatase (IMPase), by direct ionic effects is analyzed. Specific MIPS and IMPase isoforms from Mozambique tilapia (MIPS-160 and IMPase 1) were selected based on experimental, phylogenetic, and structural evidence supporting their role for Ins biosynthesis during hyperosmotic stress. Recombinant tilapia IMPase 1 and MIPS-160 activity was assayed <i>in vitro</i> at ionic conditions that mimic changes in the intracellular milieu during hyperosmotic stress. The <i>in vitro</i> activities of MIPS-160 and IMPase 1 are highest at alkaline pH of 8.8. IMPase 1 catalytic efficiency is strongly increased during hyperosmolality (particularly for the substrate D-Ins-3-phosphate, Ins-3<i>P</i>), mainly as a result of [Na⁺] elevation. Furthermore, the substrate-specificity of IMPase 1 towards D-Ins-1-phosphate (Ins-1<i>P</i>) is lower than towards Ins-3<i>P</i>. Because MIPS catalysis results in Ins-3<i>P</i> this results represents additional evidence for IMPase 1 being the isoform that mediates Ins biosynthesis in tilapia. Our data collectively demonstrate that the Ins biosynthesis enzymes are activated under ionic conditions that cells are exposed to during hypertonicity, resulting in Ins accumulation, which, in turn, results in restoration of intracellular ion homeostasis. We propose that the unique and direct ionic regulation of the activities of Ins biosynthesis enzymes represents an efficient biochemical feedback loop for regulation of intracellular physiological ion homeostasis during hyperosmotic stress.</p></div

Enzymatic activity of purified IMPase 1 and MIPS-160.

<p>(<b>A</b>) MIPS-160 and IMPase 1 were incubated with their respective substrates at different pH, and reaction rate was measured for each point. Values are expressed relative to the highest activity observed (for both enzymes, at pH 8.8). (<b>B</b>) Enzymatic activity of both MIPS-160 (top) and IMPase 1 were determined with or without their known required cofactors (NAD⁺ and Mg²⁺, respectively). (<b>C</b>) Known MIPS and IMPase inhibitors (500 μM) significantly reduce the activity of MIPS-160 (100 μM 2-deoxy-G6-<i>P</i> [2dG6P]) and IMPase 1 (Ins-1<i>P</i> as substrate, 50 μM L690,330 or 5 mM LiCl as inhibitors).</p

Enzymatic activity rate for MIPS-160 and IMPase 1 is modified under different osmotic conditions.

<p>MIPS-160 (<b>A</b>) and IMPase 1 (<b>B</b>) activity changes when concentration of ions (NaCl or KCl) is increased. Relative rate (activity compared to the activity in the assay buffer unsupplemented with ions) is graphed for the different osmolalities (achieved by addition of enough NaCl or KCl to reach the final osmolality). For IMPase 1, two different substrates (Ins-1 and Ins-3<i>P</i>) were assayed. In all cases, substrate concentration was 500 μM. Activity is expressed as relative expression (fold of change) compared to the activity in the unsupplemented SB.</p

IMPase 1 and MIPS-160 3D structural models and conservation.

<p>(<b>A</b>) 3D models for both IMPase 1 (top) and MIPS-160 (bottom) were generated using the I-TASSER server. Models (colored from N- to C-end) are shown superimposed to the 3D structure with the best r.m.s.d value for each model (in white). (<b>B</b>) Conservation of amino acids at the structural level (generated by CONSURF). Using the IMPase 1 and MIPS-160 models, the most highly conserved amino acid residues (levels 9 to 7) are shown in the left panel, while the less conserved amino acids (levels 6 to 1) are shown in the right. Additionally, in (<b>A</b>) and (<b>B</b>), co-crystallized catalysis-relevant molecules are shown: <i>IMPase 1</i>, Ins monophosphate (red) and Mg²⁺ ions (orange); MIPS-160: NAD⁺ (yellow), 2-D-glucitol phosphate (red).</p

Phylogenetic tree of vertebrate IMPase proteins.

<p>(<b>A</b>) Full-length protein sequences of IMPase isoforms from <i>Homo sapiens</i> (Hs), <i>Bos taurus</i> (Bt), <i>Gallus gallus</i> (Gg), <i>Xenopus laevis</i> (Xl) and fish: <i>Anguilla anguilla</i> (Aa), <i>Danio rerio</i> (Dr), <i>Gasterosteus aculeatus</i> (Ga), <i>Oryzias latipes</i> (Ol), <i>O</i>. <i>mossambicus</i> (Om), <i>O</i>. <i>niloticus</i> (On), <i>Salmo salar</i> (Ss), <i>Tetraodon nigroviridis</i> (Tn), <i>Takifugu rubripes</i> (Tr) and <i>Xiphophorus maculatus</i> (Xm) were retrieved from the corresponding genomes. Results for best bi-directional BLAST are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123212#pone.0123212.s004" target="_blank">S2 Table</a> in Supporting Information, and accession numbers used to build the tree are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123212#pone.0123212.s005" target="_blank">S3 Table</a> in Supporting Information. The tree was built by maximum parsimony (bootstrap values for 500 replicates are shown). In light gray, the IMPase 1 clade is highlighted, while the dark gray clade corresponds to the IMPase 2 clade. Red dots denote that a sequence is orthologous to the human <i>Hs</i> 1.1 (by bidirectional best BLAST hit), while blue dots depict fish sequences that are orthologues to the Nile tilapia On 1.1 sequence.. The latter sequences fall in a separate subclade (blue branches). Labelling of the sequences correspond to the species followed by the numbers assigned in their corresponding annotation in databases, except for <i>O</i>. <i>mossambicus</i> IMPase 1 (underlined). (<b>B</b>) Analysis of primary sequence of orthologues to Hs 1.1 (red) and to On 1.1 (blue). Grand average of hydrophobicity (GRAVY) and overall contents of negatively charged amino acids (Glu and Asp) in each sequence are shown. Asterisks represent significant differences analyzed by two tailed t-test (***, <i>P</i><0.0001; * <i>P</i><0.05).</p

Stimulation of envelope cross-linking by the ionophore X537A.

<p>Shown is the degree of light scattering (A³⁴⁰) by cross-linked envelopes isolated after addition of SDS and DTT. (A) Confluent cultures of OmL cells with (X537A) and without (Con) overnight treatment with ionophore; compilation of 4 independent experiments. (B) HEK293FT cells not transfected (Con) or transfected with the full length coding region of Tgm1A or Tgm1B; two days after transfection, cultures were treated overnight with ionophore; compilation of two independent experiments. In each panel, differences between treated and control samples were judged significant (p<0.01) by ANOVA using STATA SE9 statistical software.</p

Relative levels of Tgm1A and Tgm1B mRNA in cultured lip cells.

<p>Values are presented for 3 independent samples (mean ± std dev) relative to those of the housekeeping gene, Talin1.</p