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

Tongtong Dai; Yun Yang; Juanjuan Zhang; Xiaoyu Ma; Lifen Chen; Caiping Zhang; Sheng Lv; Lin Li; Renqiao Tang; Ni Zhen; Wenli Lu; Chuanyin Li; Ronggui Hu; Yuan Xiao; Zhiya Dong

doi:10.3389/fgene.2023.1120153

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

Front Genet. 2023 Apr 4:14:1120153. doi: 10.3389/fgene.2023.1120153. eCollection 2023.

Authors

Tongtong Dai¹, Yun Yang², Juanjuan Zhang¹, Xiaoyu Ma¹, Lifen Chen¹, Caiping Zhang¹, Sheng Lv¹, Lin Li¹, Renqiao Tang¹, Ni Zhen¹, Wenli Lu¹, Chuanyin Li³, Ronggui Hu⁴, Yuan Xiao¹, Zhiya Dong¹

Affiliations

¹ Department of Pediatrics, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
² School of Medicine, Guizhou University, Guiyang, China.
³ Cancer Center, Shanghai Tenth People's Hospital, School of Medicine, Tongji University, Shanghai, China.
⁴ State Key Laboratory of Molecular Biology, Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China.

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.