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

Amino acids (AAs) changed by human G6PC2 SNPs that are conserved in human G6PC2, mouse G6pc2, human G6PC1 and mouse G6pc1 were identified using the UCSC Genome Browser (https://genome.ucsc.edu/) and HumSAVR (http://omictools.com/humsavar-tool) databases. The G6PC2 domain affected by each AA change was predicted by comparison with the proposed structure of G6PC1 [41]. 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 in situ enzyme assay (Figs 2 & 3). This assay measures the ability of G6pc1 to suppress glucose-stimulated fusion gene expression (Figs 2 & 3). 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 G6PC2 SNPs change AAs that are conserved in G6PC1, mutation of some of these AAs in G6PC1 causes GSD type 1a [40]. In each case the AA associated with GSD type 1a is shown in parentheses. In each case the G6PC2 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 (Fig 1). N.D., not determined; N.C., no change. **, these residues have been associated with variations in FBG in healthy individuals who do not have diabetes [50,52,53].