Functional Study And Analysis Of A Novel Glucokinase Gene I404N Mutation

Objective: To investigate the clinical and molecular genetic characteristics of Maturity-onset diabetes of the young type 2(MODY2) caused by glucokinase(GCK) gene mutation and the molecular mechanism of the novel GCK gene I404 N mutation.Methods:1.One patient and his family who met the clinical criteria for MODY2 molecular genetic testing were performed exon polymerase chain reaction(PCR)amplification and sequencing.2.Validation and analysis two alleles of the GCK locus in 100 healthy individuals.3.Through gene synthesis,vector expression construction(with glutathione S transferase-GST tag),Escherichia Coli(E.coli)cell expression,and protein purification,wild-type and mutant GST-GCK recombinant proteins with good purity were obtained.4.Enzyme functional studies were performed by enzyme coupling analysis.4.1 Enzyme Kinetic Analysis In the presence of 4 mM ATP,a different concentration of glucose dilutions between 0.4 and 150 mM or in the presence of 100 mM glucose,a different concentration of ATP dilutions between 0.02 and 4 mM,and the absorbance values of the reaction were continuously measured by spectrophotometry.The enzyme kinetic parameters were calculated using SigmaPlot 13.0.4.2 Thermal stability analysis A certain amount of wild-type and mutant GCK proteins were incubated respectively in a water bath at 25,30,37,45,50,55 and 60 ? for 30 minutes or at 55 ? for 5,10,15,20,25,30 minutes.The absorbance value of the reaction were continuously measured by spectrophotometry.4.3 Optimal pH analysis A certain amount of wild type and mutant GCK proteins were treated respectively in Tris-HCL buffer at pH 6.8,7.0,7.4,7.5,8.0,8.8 for 30 minutes.The absorbance value of the reaction were continuously measured by spectrophotometry.5.Statistical analysis The measurement data were characterized by mean ± standard deviation,and the t test or non-parametric Mann-Whitney test was used for comparison between the two groups.The data was analyzed using statistical software IBM SPSS Statistics 21.Bilateral P < 0.05 was considered statistically significant.Results:1.Gene sequencing revealed the proband and his father had a GCK gene I404 N mutation,which has not been reported.2.Two alleles of the GCK locus from 100 healthy individuals were validated and analyzed,and the mutation was not detected as a control.3.Compared with wild-type GCK protein,the production of mutant GCK protein decreased.4.Enzyme kinetic analysis showed that compared with wild-type GCK protein,the mutant GCK protein changed,the S0.5 value increased by 2.9 times,the glucose inflection point increased by 4.9 times,the ATP-Km value increased by 7.9 times,and the Kcat decrease to 6.1% of the wild type,the relative activity index decreased to 0.2% of the wild type.5.Thermal stability analysis showed that the mutant GCK protein showed a significant decrease in enzyme activity as the incubation temperature increased or the incubation time increased compared to the wild-type GCK protein.6.The optimal pH analysis showed that compared with the wild-type GCK protein,the mutant GCK protein showed significant changes in enzyme activity under the influence of pH and the enzyme activity was the lowest under normal conditions(pH 7.4-7.5).Conclusion:1.We first discovered the GCK gene I404 N mutation family,and the mutation was co-segregated with hyperglycemia.2.The GCK gene I404 N mutation resulted in the decrease of the affinity of the enzyme to glucose and ATP,the decrease of the thermal stability and the change of the optimal PH value,which lead to a disorder of glucose metabolism in the proband.