GCK exonic mutations induce abnormal biochemical activities and result in GCK-MODY

Abstract

Objective: Glucokinase-maturity-onset diabetes of the young (GCK-MODY; MODY2) is a rare genetic disorder caused by mutations in the glucokinase (GCK) gene. It is often under- or misdiagnosed in clinical practice, but correct diagnosis can be facilitated by genetic testing. In this study, we examined the genes of three patients diagnosed with GCK-MODY and tested their biochemical properties, such as protein stability and half-life, to explore the function of the mutant proteins and identify the pathogenic mechanism of GCK-MODY. Methods: Three patients with increased blood glucose levels were diagnosed with MODY2 according to the diagnostic guidelines of GCK-MODY proposed by the International Society for Pediatric and Adolescent Diabetes (ISPAD) in 2018. Next-generation sequencing (whole exome detection) was performed to detect gene mutations. The GCK gene and its mutations were introduced into the pCDNA3.0 and pGEX-4T-1 vectors. Following protein purification, enzyme activity assay, and protein immunoblotting, the enzyme activity of GCK was determined, along with the ubiquitination level of the mutant GCK protein. Results: Genetic testing revealed three mutations in the GCK gene of the three patients, including c.574C>T (p.R192W), c.758G>A (p.C253Y), and c.794G>A (p.G265D). The biochemical characteristics of the protein encoded by wild-type GCK and mutant GCK were different, compared to wild-type GCK, the enzyme activity encoded by the mutant GCK was reduced, suggesting thermal instability of the mutant GST-GCK. The protein stability and expression levels of the mutant GCK were reduced, and the enzyme activity of GCK was negatively correlated with the levels of fasting blood glucose and HbA1c. In addition, ubiquitination of the mutant GCK protein was higher than that of the wild-type, suggesting a higher degradation rate of mutant GCK than WT-GCK. Conclusion: GCK mutations lead to changes in the biochemical characteristics of its encoded proteins. The enzyme activities, protein expression, and protein stability of GCK may be reduced in patients with GCK gene mutations, which further causes glucose metabolism disorders and induces MODY2.

Keywords: GCK; GCK-MODY; biochemical activity; gene variants; glucokinase.

FIGURE 1

Pedigree of MODY2 family in this study and mutations carried by proband. (A). GCKc.758G > A (p. C253Y) carried by proband 1 confirmed by Sanger sequencing. The normal site was only the base G, whereas the same site in proband 1 was G and A. (B). GCK c.574C > T (p. R192W) carried by proband 2 was confirmed by Sanger sequencing. The normal site was only base C, whereas the same site in proband 2 was C and T. (C). GCK c.794G > A (p. G265D) carried by proband 3 was confirmed by Sanger sequencing. The normal site was only base G, whereas the same site in proband 3 was G and A. (D–F). Squares represent males and circles represent females. Filled black symbols indicate members carrying mutations and having clinical symptoms, and empty symbols indicate family members without mutations. The proband is marked with a black arrow. (G–H). Schematic view of human GCK protein and gene showing the location of the three variants involved in our study. GCK is a 465-residue, 52-kDa enzyme comprising two domains, hereafter referred to as the large domain (green) and the small domain (blue). The overall structure of the protein was comprised of a large and a small globular domain connected by a hinge made up of three flexible loops. The binding site for the activator was localized in the hinge region. The mutations identified in this study were located in two regions. GCK contains 10 exons, and the three mutations detected in this study were located in exons 5 and 7.

FIGURE 2

Mutation of the GCK gene reduces enzyme activity and protein expression. (A). Protein content was detected using the Coomassie Brilliant Blue method. The same volume (10 μL) of protein solution was added to each loading well, and the same band width indicated the same protein concentration. The GST protein (23 kDa) was used as a control. (B). The enzyme activity was detected after the protein was heated in a water bath at 37°C, 42°C, and 50°C for 30 min. The graph shows that the enzyme activity of the wild-type protein was much higher than that of the mutant protein at the same temperature. At 42°C, the wild-type protein still contained at least 50% enzyme activity, whereas the mutant protein lost its activity. (C). Detection of protein levels in WT GCK and its mutants by immunoblotting. Empty vector or FLAG-tagged wild-type GCK/GCK (c.758G>A)/GCK (c.794G>A)/GCK (c.574C>T) were transfected into HEK293T cells, and the protein levels of FLAG-tagged protein and GAPDH were detected by immunoblotting. The expression of the wild-type protein was higher than that of the mutant protein. We detected the gray scale of the bands by ImageJ and used ANOVA test to verify whether there were differences in protein expression. P‹0.05, significant different.

FIGURE 3

Analysis of the protein stability of wild type and mutant GCK in mammalian cells. (A). HEK293T cells transfected with plasmids containing GCK-WT or its mutants were treated with CHX (1 μg/μL) for 0, 2 and 4 h. Total cell lysates were analyzed for GCK protein levels using western blotting. (B). ImageJ was used to detect the gray levels of wild-type and mutant GCK proteins at 0 h and to homogenize them to better compare the degradation rates of proteins. The results were calculated from three independent experiments. Each bar represents the mean ± SD. (C-D). The ubiquitination level of FLAG-GCK with the C253Y/G265D/R192W mutation was higher than that of the WT FLAG-tagged protein. Ubiquitination of the wild-type GCK protein was less than that of the mutants. FLAG-tagged wild-type or mutant GCK were expressed in HEK293T cells and immunoprecipitated using anti-FLAG beads, followed by immunoblotting with anti-Ub to detect ubiquitination signals.