Journal: The Journal of Biological Chemistry

Article Title: Nonsynonymous single-nucleotide polymorphisms in the G6PC2 gene affect protein expression, enzyme activity, and fasting blood glucose

doi: 10.1016/j.jbc.2021.101534

Figure Lengend Snippet: Comparison of human G6PC1 and G6PC2 protein expression driven by the pcDNA3.1D and pJPA5 vectors. 832/13 cells were transiently transfected with either pcDNA3.1D or pJPA5 expression vectors encoding human G6PC1 or G6PC2 with a C terminal V5 His Tag. Following transfection, cells were incubated for 18–20 h in serum-containing media. Cells were subsequently harvested and protein expression assayed by Western blotting as described in . A, G6PC expression was assessed using an anti-V5 antibody and equal protein loading was confirmed using both Ponceaus staining and measurement of actin expression. B, the conditions used to assess G6PC2 and actin expression are semiquantitative. Representative blots are shown. G6PC, glucose-6-phosphatase catalytic subunit.

Article Snippet: A human G6PC1 cDNA (Accession number BC130478 ; IMAGE clone number 40146509) was purchased from Transomic technologies.

Techniques: Comparison, Expressing, Transfection, Incubation, Western Blot, Staining

Journal: The Journal of Biological Chemistry

Article Title: Nonsynonymous single-nucleotide polymorphisms in the G6PC2 gene affect protein expression, enzyme activity, and fasting blood glucose

doi: 10.1016/j.jbc.2021.101534

Figure Lengend Snippet: Conservation of amino acids between human G6PC2, mouse G6PC2, human G6PC1, and mouse G6PC1. Sequence alignment showing the conservation of AAs between human (h) and mouse (m) G6PC1 and G6PC2. Residues highlighted in yellow represent conserved AAs that are changed by a human G6PC2 SNP and where mutation in G6PC1 can cause GSD type 1a ( Table 1 ). Residues highlighted in pink represent conserved AAs that are changed by human G6PC2 SNPs but where mutation in G6PC1 has not been associated with GSD type 1a. Residues highlighted in green represent nonconserved AAs that are changed by human G6PC2 SNPs. Identities are indicated by filled circles and similarities by vertical bars. Gray boxes represent putative transmembrane domains determined using the TMHMM algorithm ( https://services.healthtech.dtu.dk/ ) . The predictions made by this program differ from earlier models ( , ). The figure shows the predicted domains for human G6PC1. G6PC1 and G6PC2 have an extended sequence motif (KXXXXXXRP-(X12-54)-PSGH-(X31-54)-SRXXXXXHXXXD) ( boxed ), similar to that found in bacterial vanadate-sensitive haloperoxidases and mammalian phosphatidic acid phosphatases, which constitutes the active site of these enzymes . G6PC1 and G6PC2 also share a C terminal ER retention sequence (KK; boxed ) . Adapted from Ref. . G6PC, glucose-6-phosphatase catalytic subunit; GSD1a, glycogen storage disease type 1a; SNP, single-nucleotide polymorphism.

Article Snippet: A human G6PC1 cDNA (Accession number BC130478 ; IMAGE clone number 40146509) was purchased from Transomic technologies.

Techniques: Sequencing, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Nonsynonymous single-nucleotide polymorphisms in the G6PC2 gene affect protein expression, enzyme activity, and fasting blood glucose

doi: 10.1016/j.jbc.2021.101534

Figure Lengend Snippet: Analysis of the effect of human G6PC2 SNPs that affect conserved amino acids associated with GSD1a in the context of human G6PC1

Article Snippet: A human G6PC1 cDNA (Accession number BC130478 ; IMAGE clone number 40146509) was purchased from Transomic technologies.

Techniques: Mutagenesis, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Nonsynonymous single-nucleotide polymorphisms in the G6PC2 gene affect protein expression, enzyme activity, and fasting blood glucose

doi: 10.1016/j.jbc.2021.101534

Figure Lengend Snippet: Analysis of the effect of human G6PC2 SNPs that affect conserved amino acids associated with GSD1a in the context of human G6PC1, on G6PC2 protein expression. Human G6PC2 SNPs were identified that affect AAs conserved between mouse and human G6PC1 and mouse and human G6PC2 where a mutation is known to cause GSD1a in the context of human G6PC1 ( Table 1 ). The effect of these G6PC2 SNPs on G6PC2 protein expression was determined as described in Fig. 3 . Protein expression was quantified as described in . Results show mean data −/+ SD, n = 4–10. ∗ p < 0.05 vs. WT. G6PC, glucose-6-phosphatase catalytic subunit; SNP, single-nucleotide polymorphism.

Article Snippet: A human G6PC1 cDNA (Accession number BC130478 ; IMAGE clone number 40146509) was purchased from Transomic technologies.

Techniques: Expressing, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Nonsynonymous single-nucleotide polymorphisms in the G6PC2 gene affect protein expression, enzyme activity, and fasting blood glucose

doi: 10.1016/j.jbc.2021.101534

Figure Lengend Snippet: Analysis of the effect of human G6PC2 SNPs that affect amino acids that are not associated with GSD1a in the context of human G6PC1, on G6PC2 protein expression. Human G6PC2 SNPs were identified that affect AAs conserved between mouse and human G6PC1 and mouse and human G6PC2, which are not associated with GSD1a in the context of human G6PC1, as well as amino acids that are not conserved in G6PC1 ( Table 2 ). The effect of these G6PC2 SNPs on G6PC2 protein expression was determined as described in Fig. 3 . Protein expression was quantified as described in . Results show mean data −/+ SD, n = 4 to 10. ∗ p < 0.05 vs. WT. G6PC, glucose-6-phosphatase catalytic subunit; SNP, single-nucleotide polymorphism.

Article Snippet: A human G6PC1 cDNA (Accession number BC130478 ; IMAGE clone number 40146509) was purchased from Transomic technologies.

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Nonsynonymous single-nucleotide polymorphisms in the G6PC2 gene affect protein expression, enzyme activity, and fasting blood glucose

doi: 10.1016/j.jbc.2021.101534

Figure Lengend Snippet: Analysis of the effect of human G6PC2 SNPs that affect amino acids that are not associated with GSD1a in the context of human G6PC1

Article Snippet: A human G6PC1 cDNA (Accession number BC130478 ; IMAGE clone number 40146509) was purchased from Transomic technologies.

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Nonsynonymous single-nucleotide polymorphisms in the G6PC2 gene affect protein expression, enzyme activity, and fasting blood glucose

doi: 10.1016/j.jbc.2021.101534

Figure Lengend Snippet: Analysis of the suppression of glucose-stimulated fusion gene expression by G6PC2 in 832/13 cells. 832/13 cells were transiently cotransfected with the −7248/+62 G6pc1 - luciferase fusion gene (2 μg), an expression vector encoding Renilla luciferase (0.5 μg) and expression vectors (1 μg) encoding either wild type (WT), catalytically dead (D; AA 174 Ala), or variant (AA8, AA114) G6PC2. Following transfection, cells were incubated for 18 to 20 h in serum-free medium in the presence of 2 or 11 mM glucose. Cells were then harvested and luciferase activity assayed as described in . Results show mean data −/+ SD (n = 3–6) and were calculated as the ratio of firefly: Renilla luciferase activity. Statistical comparisons to 11 mM WT were made using ANOVA with a Dunnett's Multiple Comparisons post-hoc test; ∗ p < 0.05. G6PC, glucose-6-phosphatase catalytic subunit.

Article Snippet: A human G6PC1 cDNA (Accession number BC130478 ; IMAGE clone number 40146509) was purchased from Transomic technologies.

Techniques: Gene Expression, Luciferase, Expressing, Plasmid Preparation, Variant Assay, Transfection, Incubation, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Nonsynonymous single-nucleotide polymorphisms in the G6PC2 gene affect protein expression, enzyme activity, and fasting blood glucose

doi: 10.1016/j.jbc.2021.101534

Figure Lengend Snippet: Analysis of the effect of human G6PC2 SNPs on G6PC1 protein expression. 832/13 cells were transiently transfected with pcDNA3.1D expression vectors encoding human G6PC1 with a C terminal V5 His Tag. Following transfection, cells were incubated for 18–20 h in serum-containing media. Cells were subsequently harvested and protein expression assayed by Western blotting as described in . G6PC1 expression was assessed using an anti-V5 antibody and equal protein loading was confirmed by measurement of actin expression. Representative blots are shown. G6PC, glucose-6-phosphatase catalytic subunit; SNP, single-nucleotide polymorphism.

Article Snippet: A human G6PC1 cDNA (Accession number BC130478 ; IMAGE clone number 40146509) was purchased from Transomic technologies.

Techniques: Expressing, Transfection, Incubation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Nonsynonymous single-nucleotide polymorphisms in the G6PC2 gene affect protein expression, enzyme activity, and fasting blood glucose

doi: 10.1016/j.jbc.2021.101534

Figure Lengend Snippet: Analysis of the effect of human G6PC2 SNPs that affect conserved amino acids associated with GSD1a in the context of human G6PC1, on G6PC1 protein expression. Human G6PC2 SNPs were identified that affect AAs conserved between mouse and human G6PC1 and mouse and human G6PC2 where a mutation is known to cause GSD1a in the context of human G6PC1 ( Table 1 ). The effect of these G6PC2 SNPs on G6PC1 protein expression was determined as described in Fig. 3 . Protein expression was quantified as described in . Results show mean data −/+ SD, n = 4–8. ∗ p < 0.05 vs. WT. G6PC, glucose-6-phosphatase catalytic subunit; SNP, single-nucleotide polymorphism.

Article Snippet: A human G6PC1 cDNA (Accession number BC130478 ; IMAGE clone number 40146509) was purchased from Transomic technologies.

Techniques: Expressing, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Nonsynonymous single-nucleotide polymorphisms in the G6PC2 gene affect protein expression, enzyme activity, and fasting blood glucose

doi: 10.1016/j.jbc.2021.101534

Figure Lengend Snippet: Analysis of the effect of human G6PC2 SNPs that affect amino acids that are not associated with GSD1a in the context of human G6PC1, on G6PC1 protein expression. Human G6PC2 SNPs were identified that affect AAs conserved between mouse and human G6PC1 and mouse and human G6PC2, that are not associated with GSD1a in the context of human G6PC1 ( Table 2 ). The effect of these G6PC2 SNPs on G6PC1 protein expression was determined as described in Fig. 3 . Protein expression was quantified as described in . Results show mean data −/+ SD, n = 4–10. ∗ p < 0.05 vs. WT. G6PC, glucose-6-phosphatase catalytic subunit; SNP, single-nucleotide polymorphism.

Article Snippet: A human G6PC1 cDNA (Accession number BC130478 ; IMAGE clone number 40146509) was purchased from Transomic technologies.

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Nonsynonymous single-nucleotide polymorphisms in the G6PC2 gene affect protein expression, enzyme activity, and fasting blood glucose

doi: 10.1016/j.jbc.2021.101534

Figure Lengend Snippet: Comparison of the effect of specific amino acid changes on the expression of mouse and human G6PC1. Human G6PC2 SNPs were identified that affect AAs conserved between mouse and human G6PC1 and mouse and human G6PC2 ( Tables 1 and ). The effect of these G6PC2 SNPs on mouse and human G6PC1 protein expression was determined as described in Fig. 3 . A representative blot is shown. G6PC, glucose-6-phosphatase catalytic subunit.

Article Snippet: A human G6PC1 cDNA (Accession number BC130478 ; IMAGE clone number 40146509) was purchased from Transomic technologies.

Techniques: Comparison, Expressing