Functional Analysis of Mouse G6pc1 Mutations Using a Novel <i>In Situ</i> Assay for Glucose-6-Phosphatase Activity and the Effect of Mutations in Conserved Human G6PC1/G6PC2 Amino Acids on G6PC2 Protein Expression

<div><p>Elevated fasting blood glucose (FBG) has been associated with increased risk for development of type 2 diabetes. Single nucleotide polymorphisms (SNPs) in <i>G6PC2</i> are the most important common determinants of variations in FBG in humans. Studies using <i>G6pc2</i> knockout mice suggest that G6pc2 regulates the glucose sensitivity of insulin secretion. <i>G6PC2</i> and the related <i>G6PC1</i> and <i>G6PC3</i> genes encode glucose-6-phosphatase catalytic subunits. This study describes a functional analysis of 22 non-synonymous <i>G6PC2</i> SNPs, that alter amino acids that are conserved in human G6PC1, mouse G6pc1 and mouse G6pc2, with the goal of identifying variants that potentially affect G6PC2 activity/expression. Published data suggest strong conservation of catalytically important amino acids between all four proteins and the related G6PC3 isoform. Because human G6PC2 has very low glucose-6-phosphatase activity we used an indirect approach, examining the effect of these SNPs on mouse G6pc1 activity. Using a novel <i>in situ</i> functional assay for glucose-6-phosphatase activity we demonstrate that the amino acid changes associated with the human <i>G6PC2</i> rs144254880 (Arg79Gln), rs149663725 (Gly114Arg) and rs2232326 (Ser324Pro) SNPs reduce mouse G6pc1 enzyme activity without affecting protein expression. The Arg79Gln variant alters an amino acid mutation of which, in G6PC1, has previously been shown to cause glycogen storage disease type 1a. We also demonstrate that the rs368382511 (Gly8Glu), rs138726309 (His177Tyr), rs2232323 (Tyr207Ser) rs374055555 (Arg293Trp), rs2232326 (Ser324Pro), rs137857125 (Pro313Leu) and rs2232327 (Pro340Leu) SNPs confer decreased G6PC2 protein expression. In summary, these studies identify multiple <i>G6PC2</i> variants that have the potential to be associated with altered FBG in humans.</p></div

Analysis of the Effect of Amino Acid Changes on Mouse G6pc1 Protein Expression.

<p>832/13 cells were transiently transfected, as described in Materials and Methods, with expression vectors encoding either wild type (WT) mouse (m) G6pc1 or G6pc1 variants in which the indicated amino acid (AA) had been changed as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162439#pone.0162439.t001" target="_blank">Table 1</a>. Following transfection, cells were incubated for 18–20 hr in serum-containing medium. Cells were then harvested and protein expression assayed as described in Materials and Methods. In some instances these AA changes did not affect G6pc1 protein expression (<b>Panels A</b>-<b>C</b>). In other cases they resulted in reduced expression (<b>Panel D</b>); these data were quantitated by scanning with the results in <b>Panel E</b> showing the mean ± S.E.M. of 4 experiments. For simplicity and comparison with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162439#pone.0162439.g008" target="_blank">Fig 8</a>, these AAs are numbered based on the position of the equivalent conserved AA in human G6PC2 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162439#pone.0162439.g001" target="_blank">Fig 1</a>).</p

Analysis of the Effect of Human <i>G6PC2</i> Codon Variation on Protein Expression.

<p>832/13 cells were transiently transfected, as described in Materials and Methods, with expression vectors encoding either wild type (WT) mouse (m) G6pc2, human (h) G6PC2 or G6PC2 variants in which the codon used to encode the indicated AAs had been optimized as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162439#pone.0162439.s003" target="_blank">S2 Table</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162439#pone.0162439.s004" target="_blank">S3 Table</a>. Following transfection, cells were incubated for 18–20 hr in serum-containing medium. Cells were then harvested and protein expression assayed as described in Materials and Methods. In some instances codon optimization resulted in a switch to the same codon used to encode the equivalent AA in mouse G6pc2 (<b>Panel A</b>; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162439#pone.0162439.s003" target="_blank">S2 Table</a>). In other cases this resulted in a switch to a codon that was distinct from the codon used to encode the equivalent AA in mouse G6pc2 (<b>Panel B</b>; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162439#pone.0162439.s004" target="_blank">S3 Table</a>). Representative blots are shown.</p

Overexpression of G6pc1 Suppresses Glucose-Stimulated Fusion Gene Expression in 832/13 Cells.

<p><b>Panel A:</b> Schematic illustrating how the extent of glucose cycling catalyzed by glucokinase and G6pc1 determines intracellular G6P levels and hence activation of fusion gene expression through ChREBP and CREB. <b>Panel B:</b> Western blot showing that wild type (WT) and catalytically dead (D) G6pc1 are expressed at similar levels following expression in 832/13 cells. A representative blot is shown. <b>Panel C:</b> 832/13 cells were transiently co-transfected, as described in Materials and Methods, with the -206/+1 <i>Pklr</i>-luciferase or -7248/+62 <i>G6pc1</i>-luciferase fusion genes (2 μg), an expression vectors encoding <i>Renilla</i> luciferase (0.5 μg) and the indicated amounts of expression vectors encoding either wild type (WT) or catalytically dead (D) G6pc1. The total DNA added was kept constant using the empty pcDNA3 vector. Following transfection, cells were incubated for 18–20 hr in serum-free medium in the presence of 30 mM glucose. Cells were then harvested and luciferase activity assayed as described in Materials and Methods. Results were calculated as the ratio of firefly:<i>Renilla</i> luciferase activity and are presented as a percentage relative to that in 30 mM glucose-treated cells transfected with the empty pcDNA3 vector (2 μg). Results represent the mean ± S.E.M. of 3 experiments using independent preparations of both fusion gene constructs in which each experimental condition was assayed in triplicate. <b>Panel D:</b> 832/13 cells were transiently co-transfected, as described in Materials and Methods, with the -206/+1 <i>Pklr</i>-luciferase or -7248/+62 <i>G6pc1</i>-luciferase fusion genes (2 μg), an expression vectors encoding <i>Renilla</i> luciferase (0.5 μg) and 2 μg of expression vectors encoding either wild type (WT) or catalytically dead (D) G6pc1. Following transfection, cells were incubated for 18–20 hr in serum-free medium in the presence of the indicated glucose concentrations. Cells were then harvested and luciferase activity assayed as described in Materials and Methods. Results were calculated as the ratio of firefly:<i>Renilla</i> luciferase activity and are presented as a percentage relative to that in 30 mM glucose-treated cells in the presence of catalytically dead G6pc1. Results represent the mean ± S.E.M. of 3 experiments using independent preparations of both fusion gene constructs in which each experimental condition was assayed in triplicate.</p

Conservation of Amino Acids Between Human 12, Mouse G6pc2, Human G6PC1 and Mouse G6pc1.

<p>Sequence alignment showing the conservation of AAs between human (h) G6PC2, mouse (m) G6pc2, human G6PC1 and mouse G6pc1. Residues highlighted in green represent AAs mutation of which in G6PC1 causes GSD type 1a [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162439#pone.0162439.ref040" target="_blank">40</a>]. Residues highlighted in pink represent AAs that are changed by human <i>G6PC2</i> SNPs that were identified using the UCSC Genome Browser (<a href="https://genome.ucsc.edu/" target="_blank">https://genome.ucsc.edu/</a>) and HumSAVR (<a href="http://omictools.com/humsavar-tool" target="_blank">http://omictools.com/humsavar-tool</a>) databases. Residues highlighted in yellow represent conserved AAs in human G6PC2, mouse G6pc2, human G6PC1 and mouse G6pc1 that are changed by a human <i>G6PC2</i> SNP and where mutation in G6PC1 can cause GSD type 1a. Identities are indicated by filled circles and similarities by vertical bars.</p

Analysis of Human G6PC2:Mouse G6pc2 Chimeric Protein Expression.

<p>832/13 cells were transiently transfected, as described in Materials and Methods, with expression vectors encoding either wild type mouse (m) G6pc2, human (h) G6PC2 or the indicated chimeric proteins (<b>Panel A</b>). Following transfection, cells were incubated for 18–20 hr in serum-containing medium. Cells were then harvested and protein expression assayed as described in Materials and Methods (<b>Panel B</b>). A representative blot is shown.</p

Analysis of the Effect of Amino Acids Changed by Human <i>G6PC2</i> SNPs on Human G6PC2 and Mouse G6pc1 Protein Expression and Activity.

<p>Amino acids (AAs) changed by human <i>G6PC2</i> SNPs that are conserved in human G6PC2, mouse G6pc2, human G6PC1 and mouse G6pc1 were identified using the UCSC Genome Browser (<a href="https://genome.ucsc.edu/" target="_blank">https://genome.ucsc.edu/</a>) and HumSAVR (<a href="http://omictools.com/humsavar-tool" target="_blank">http://omictools.com/humsavar-tool</a>) databases. The G6PC2 domain affected by each AA change was predicted by comparison with the proposed structure of G6PC1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162439#pone.0162439.ref041" target="_blank">41</a>]. The Table shows the effect of these SNPs on G6pc1 enzyme activity based on comparison with wild type (WT) G6pc1 as assessed using a novel <i>in situ</i> enzyme assay (Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162439#pone.0162439.g002" target="_blank">2</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162439#pone.0162439.g003" target="_blank">3</a>). This assay measures the ability of G6pc1 to suppress glucose-stimulated fusion gene expression (Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162439#pone.0162439.g002" target="_blank">2</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162439#pone.0162439.g003" target="_blank">3</a>). Results for each variant represent the mean ± S.E.M. of 3 experiments using two independent preparations of each expression vector construct in which each experimental condition was assayed in triplicate. While all of these <i>G6PC2</i> SNPs change AAs that are conserved in G6PC1, mutation of some of these AAs in G6PC1 causes GSD type 1a [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162439#pone.0162439.ref040" target="_blank">40</a>]. In each case the AA associated with GSD type 1a is shown in parentheses. In each case the <i>G6PC2</i> SNP changes the AA to one distinct from that associated with GSD type 1a. For simplicity and comparisons between human G6PC2 and mouse G6pc1 the AAs in mouse G6pc1 are numbered based on the position of the equivalent conserved AA in human G6PC2 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162439#pone.0162439.g001" target="_blank">Fig 1</a>). N.D., not determined; N.C., no change. ^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162439#t001fn001" target="_blank">**</a>}, these residues have been associated with variations in FBG in healthy individuals who do not have diabetes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162439#pone.0162439.ref050" target="_blank">50</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162439#pone.0162439.ref052" target="_blank">52</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162439#pone.0162439.ref053" target="_blank">53</a>].</p

Analysis of the Effect of Human <i>G6PC2</i> SNPs on Human G6PC2 Protein Expression.

<p>COS 7 cells were transiently transfected, as described in Materials and Methods, with expression vectors encoding either wild type (WT) human (h) G6PC2 or G6PC2 variants in which the indicated amino acid (AA) had been changed as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162439#pone.0162439.t001" target="_blank">Table 1</a>. Following transfection, cells were incubated for 18–20 hr in serum-containing medium. Cells were then harvested and protein expression assayed as described in Materials and Methods. The variants shown either reduced (<b>Panels A-D</b>) or had little effect (<b>Panel E</b>) on human G6PC2 protein expression. Data were quantitated by scanning with the results in <b>Panels B</b> and <b>D</b> showing the mean ± S.E.M. of 4 experiments. Representative blots are shown.</p