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Structural and functional study of the GlnB22-insulin mutant responsible for maturity-onset diabetes of the young.

ABSTRACT: The insulin gene mutation c.137G>A (R46Q), which changes an arginine at the B22 position of the mature hormone to glutamine, causes the monogenic diabetes variant maturity-onset diabetes of the young (MODY). In MODY patients, this mutation is heterozygous, and both mutant and wild-type (WT) human insulin are produced simultaneously. However, the patients often depend on administration of exogenous insulin. In this study, we chemically synthesized the MODY mutant [GlnB22]-insulin and characterized its biological and structural properties. The chemical synthesis of this insulin analogue revealed that its folding ability is severely impaired. In vitro and in vivo tests showed that its binding affinity and biological activity are reduced (both approximately 20% that of human insulin). Comparison of the solution structure of [GlnB22]-insulin with the solution structure of native human insulin revealed that the most significant structural effect of the mutation is distortion of the B20-B23 ?-turn, leading to liberation of the B chain C-terminus from the protein core. The distortion of the B20-B23 ?-turn is caused by the extended conformational freedom of the GlnB22 side chain, which is no longer anchored in a hydrogen bonding network like the native ArgB22. The partially disordered [GlnB22]-insulin structure appears to be one reason for the reduced binding potency of this mutant and may also be responsible for its low folding efficiency in vivo. The altered orientation and flexibility of the B20-B23 ?-turn may interfere with the formation of disulfide bonds in proinsulin bearing the R46Q (GlnB22) mutation. This may also have a negative effect on the WT proinsulin simultaneously biosynthesized in ?-cells and therefore play a major role in the development of MODY in patients producing [GlnB22]-insulin.

SUBMITTER: Krizkova K

PROVIDER: S-EPMC4244080 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Structural and functional study of the GlnB22-insulin mutant responsible for maturity-onset diabetes of the young.

Křížková Květoslava K Veverka Václav V Maletínská Lenka L Hexnerová Rozálie R Brzozowski Andrzej M AM Jiráček Jiří J Žáková Lenka L

PloS one 20141125 11

The insulin gene mutation c.137G>A (R46Q), which changes an arginine at the B22 position of the mature hormone to glutamine, causes the monogenic diabetes variant maturity-onset diabetes of the young (MODY). In MODY patients, this mutation is heterozygous, and both mutant and wild-type (WT) human insulin are produced simultaneously. However, the patients often depend on administration of exogenous insulin. In this study, we chemically synthesized the MODY mutant [GlnB22]-insulin and characterize ...[more]

PMID: 25423173

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Project description:It is important to identify patients with Maturity-onset diabetes of the young (MODY) as a molecular diagnosis determines both treatment and prognosis. Genetic testing is currently expensive and many patients are therefore not assessed and are misclassified as having either type 1 or type 2 diabetes. Biomarkers could facilitate the prioritisation of patients for genetic testing. We hypothesised that patients with different underlying genetic aetiologies for their diabetes could have distinct metabolic profiles which may uncover novel biomarkers. The aim of this study was to perform metabolic profiling in urine from patients with MODY due to mutations in the genes encoding glucokinase (GCK) or hepatocyte nuclear factor 1 alpha (HNF1A), type 2 diabetes (T2D) and normoglycaemic control subjects. Urinary metabolic profiling by Nuclear Magnetic Resonance (NMR) and ultra performance liquid chromatography hyphenated to Q-TOF mass spectrometry (UPLC-MS) was performed in a Discovery set of subjects with HNF1A-MODY (n = 14), GCK-MODY (n = 17), T2D (n = 14) and normoglycaemic controls (n = 34). Data were used to build a valid partial least squares discriminate analysis (PLS-DA) model where HNF1A-MODY subjects could be separated from the other diabetes subtypes. No single metabolite contributed significantly to the separation of the patient groups. However, betaine, valine, glycine and glucose were elevated in the urine of HNF1A-MODY subjects compared to the other subgroups. Direct measurements of urinary amino acids and betaine in an extended dataset did not support differences between patients groups. Elevated urinary glucose in HNF1A-MODY is consistent with the previously reported low renal threshold for glucose in this genetic subtype. In conclusion, we report the first metabolic profiling study in monogenic diabetes and show that, despite the distinct biochemical pathways affected, there are unlikely to be robust urinary biomarkers which distinguish monogenic subtypes from T2D. Our results have implications for studies investigating metabolic profiles in complex traits including T2D.

Project description:Heterozygous glucokinase (GCK) mutations cause a subtype of maturity-onset diabetes of the young (GCK-MODY). Over 600 GCK mutations have been reported of which ?65% are missense. In many cases co-segregation has not been established and despite the importance of functional studies in ascribing pathogenicity for missense variants these have only been performed for <10% of mutations. The aim of this study was to determine the minimum prevalence of GCK-MODY amongst diabetic subjects in Slovakia by sequencing GCK in 100 Slovakian probands with a phenotype consistent with GCK-MODY and to explore the pathogenicity of identified variants through family and functional studies. Twenty-two mutations were identified in 36 families (17 missense) of which 7 (I110N, V200A, N204D, G258R, F419S, c.580-2A>C, c.1113-1114delGC) were novel. Parental DNA was available for 22 probands (covering 14/22 mutations) and co-segregation established in all cases. Bioinformatic analysis predicted all missense mutations to be damaging. Nine (I110N, V200A, N204D, G223S, G258R, F419S, V244G, L315H, I436N) mutations were functionally evaluated. Basic kinetic analysis explained pathogenicity for 7 mutants which showed reduced glucokinase activity with relative activity indices (RAI) between 0.6 to <0.001 compared to wild-type GCK (1.0). For the remaining 2 mutants additional molecular mechanisms were investigated. Differences in glucokinase regulatory protein (GKRP) -mediated-inhibition of GCK were observed for both L315H & I436N when compared to wild type (IC(50) 14.6±0.1 mM & 20.3±1.6 mM vs.13.3±0.1 mM respectively [p<0.03]). Protein instability as assessed by thermal lability studies demonstrated that both L315H and I436N show marked thermal instability compared to wild-type GCK (RAI at 55°C 8.8±0.8% & 3.1±0.4% vs. 42.5±3.9% respectively [p<0.001]). The minimum prevalence of GCK-MODY amongst Slovakian patients with diabetes was 0.03%. In conclusion, we have identified 22 GCK mutations in 36 Slovakian probands and demonstrate that combining family, bioinformatic and functional studies can aid the interpretation of variants identified by molecular diagnostic screening.

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Structural and functional study of the GlnB22-insulin mutant responsible for maturity-onset diabetes of the young.

Publications

Structural and functional study of the GlnB22-insulin mutant responsible for maturity-onset diabetes of the young.

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