葡萄糖激酶基因E339K突变的功能学研究

Objective To explore the molecular mechanisms of glucokinase (GCK) E339K mutation resulting in maturity-onset diabetes of the young-2 (MODY2).Methods Fasting plasma glucose (FPG), oral glucose tolerance test (OGTT) overload 2 h glucose (2hPG), glycosylated hemoglobin Alc (HbAlc) and fasting insulin (FIns) level were measured, respectively.Mutant glutathione S-transferase (GST)-GCK-cDNA was constructed with site-directed mutagenesis.Wild type and mutant GCK protein expressed in E.Coli were purified with affinity chromatography.Enzymatic kinetics and thermal stability were tested with enzyme-coupled analysis.Results Compared with non-mutants, mutants had higher FPG (6.92 ± 0.95) mmol/L vs (4.70 ± 0.35) mmol/L, P＜0.001], 2hPG (9.00 ± 1.49 ) mmol/L vs (5.51 ± 0.86) mmol/L,P＜0.001],HbAlc(6.46 ± 0.69)% vs(4.83 ± 0.30)%,P＜0.01],and lower FIns level (6.15 ± 1.97 ) mIU/L vs ( 10.79 ± 4.93 ) mIU/L, P ＜ 0.01], HOMA-β (34.16 ±3.62 vs 172.53 ± 76.58, P ＜ 0.001 ).This mutation induced lower protein yield ( 12.7 ±1.72) mg/L vs ( 16.2 ± 2.65 ) mg/L, P ＜ 0.01], lower appetency for glucose S0.5: ( 13.96 ± 1.89)mmol/L vs (5.92±0.99)mmol/L, P＜0.001] and ATP Km:(3.27 ±1.14) mmol/L vs (0.30±0.09)mmol/L, P＜0.001], lower catalytic ability Kcat:(1.62 ±0.35)/s vs (25.18 ±2.10)/s, P＜0.001].It also showed protein thermal instability.Conclusion Glucokinase gene E339K mutation promotes the development of MODY2 by affecting protein yield and protein stability as well as the enzymatic kinetics of GCK.

The functional analysis of glucokinase gene E339K mutation