Glucokinase (GCK) mutations and their characterization in MODY2 children of southern Italy

Abstract

Type 2 Maturity Onset Diabetes of the Young (MODY2) is a monogenic autosomal disease characterized by a primary defect in insulin secretion and hyperglycemia. It results from GCK gene mutations that impair enzyme activity. Between 2006 and 2010, we investigated GCK mutations in 66 diabetic children from southern Italy with suspected MODY2. Denaturing High Performance Liquid Chromatography (DHPLC) and sequence analysis revealed 19 GCK mutations in 28 children, six of which were novel: p.Glu40Asp, p.Val154Leu, p.Arg447Glyfs, p.Lys458_Cys461del, p.Glu395_Arg397del and c.580-2A>T. We evaluated the effect of these 19 mutations using bioinformatic tools such as Polymorphism Phenotyping (Polyphen), Sorting Intolerant From Tolerant (SIFT) and in silico modelling. We also conducted a functional study to evaluate the pathogenic significance of seven mutations that are among the most severe mutations found in our population, and have never been characterized: p.Glu70Asp, p.His137Asp, p.Phe150Tyr, p.Val154Leu, p.Gly162Asp, p.Arg303Trp and p.Arg392Ser. These seven mutations, by altering one or more kinetic parameters, reduced enzyme catalytic activity by >40%. All mutations except p.Glu70Asp displayed thermal-instability, indeed >50% of enzyme activity was lost at 50°C/30 min. Thus, these seven mutations play a pathogenic role in MODY2 insurgence. In conclusion, this report revealed six novel GCK mutations and sheds some light on the structure-function relationship of human GCK mutations and MODY2.

Figure 1. View of the mutations p.Phe150Tyr and p.Val154Leu in the whole structures and in their local environment. A

: The structures of GCK in the super-open inactive form (PDB code code: 1v4t) and in the closed active form (PDB code code: 1v4s) are shown on the left and on the right, respectively. In the closed form (right) the sugar is shown as a yellow stick. In both panels, the large domain is in the same orientation and is circled. It is clear that the small domain undergoes a large conformational variation from the super-open to the closed form. Specifically the region embodying the residues Phe150 (blue sphere) and Val154 (red sphere) dramatically changes its orientation. B: Close-up view of the wild-type closed structure showing the hydrophobic core rich in aromatic/hydrophobic amino acids. Leu79, Phe123, Phe148, Phe150, Val154, Leu164, Trp167 and Phe171 form an intricate network of stabilizing hydrophobic interactions. C: Structure of GCK closed structure containing the both the Tyr150 and Leu154 mutated residues. Introduction of the oxydryl group (red) of Tyr150 within the hydrophobic core disrupts the interactions present in the wt-enzyme. The replacement of Val154 by leucine produces only small changes in the closed form.

Figure 2. Distribution of the selected GCK mutations.

The structure of GCK in the active closed form is shown (PDB code: 1v4s); the small and large domains are drawn in cyan and red, respectively; glucose appears as a yellow stick. Mutation sites are shown as green or blue spheres.

Figure 3. Effect of temperature on the stability of GST-GCK mutants

. Stock enzyme solutions were diluted to 250 µg/ml in storage buffer containing 30% glycerol, 50 mM glucose, 10 mM glutathione, 5 mM DTT, 200 mM KCl and 50 mMTris/HCl, pH 8.0. Panel A: The enzyme solutions were incubated for 30 min at temperatures ranging from 30 to 55°C and then assayed at 30°C as described in the Methods section. Panel B: The enzyme solutions were incubated for periods of time from 5 to 60 min at 50°C. Results are means and SEM of three independent enzyme preparations for each mutant except for GST-GCK (Phe150Tyr) which corresponds to two independent enzyme preparations. (*) p≤0.03, (†) p<0.008.