Functional Characterization of MODY2 Mutations Highlights the Importance of the Fine-Tuning of Glucokinase and Its Role in Glucose Sensing

Glucokinase (GK) acts as a glucose sensor in the pancreatic beta-cell and regulates insulin secretion. Heterozygous mutations in the human GK-encoding GCK gene that reduce the activity index increase the glucose-stimulated insulin secretion threshold and cause familial, mild fasting hyperglycaemia, also known as Maturity Onset Diabetes of the Young type 2 (MODY2). Here we describe the biochemical characterization of five missense GK mutations: p.Ile130Thr, p.Asp205His, p.Gly223Ser, p.His416Arg and p.Ala449Thr. The enzymatic analysis of the corresponding bacterially expressed GST-GK mutant proteins show that all of them impair the kinetic characteristics of the enzyme. In keeping with their position within the protein, mutations p.Ile130Thr, p.Asp205His, p.Gly223Ser, and p.His416Arg strongly decrease the activity index of GK, affecting to one or more kinetic parameters. In contrast, the p.Ala449Thr mutation, which is located in the allosteric activator site, does not affect significantly the activity index of GK, but dramatically modifies the main kinetic parameters responsible for the function of this enzyme as a glucose sensor. The reduced Kcat of the mutant (3.21±0.28 s−1 vs 47.86±2.78 s−1) is balanced by an increased glucose affinity (S0.5 = 1.33±0.08 mM vs 7.86±0.09 mM) and loss of cooperativity for this substrate. We further studied the mechanism by which this mutation impaired GK kinetics by measuring the differential effects of several competitive inhibitors and one allosteric activator on the mutant protein. Our results suggest that this mutation alters the equilibrium between the conformational states of glucokinase and highlights the importance of the fine-tuning of GK and its role in glucose sensing

Functional characterization of MODY2 mutations in the nuclear export signal of glucokinase

Glucokinase (GCK) plays a key role in glucose homeostasis. Heterozygous inactivating mutations in the GCK gene cause the familial, mild fasting hyperglycaemia named MODY2. Besides its particular kinetic characteristics, glucokinase is regulated by subcellular compartmentation in hepatocytes. Glucokinase regulatory protein (GKRP) binds to GCK, leading to enzyme inhibition and import into the nucleus at fasting. When glucose concentration increases, GCK-GKRP dissociates and GCK is exported to the cytosol due to a nuclear export signal (NES). With the aim to characterize the GCK-NES, we have functionally analysed nine MODY2 mutations located within the NES sequence. Recombinant GCK mutants showed reduced catalytic activity and, in most cases, protein instability. Most of the mutants interact normally with GKRP, although mutations L306R and L309P impair GCK nuclear import in cotransfected cells. We demonstrated that GCK-NES function depends on exportin 1. We further showed that none of the mutations fully inactivate the NES, with the exception of mutation L304P, which likely destabilizes its α-helicoidal structure. Finally, we found that residue Glu300 negatively modulates the NES activity, whereas other residues have the opposite effect, thus suggesting that some of the NES spacer residues contribute to the low affinity of the NES for exportin 1, which is required for its proper functioning. In conclusion, our results have provided functional and structural insights regarding the GCK-NES and contributed to a better knowledge of the molecular mechanisms involved in the nucleo-cytoplasmic shuttling of glucokinase. Impairment of this regulatory mechanism by some MODY2 mutations might contribute to the hyperglycaemia in the patients.Instituto de Salud Carlos IIIDepto. de Bioquímica y Biología MolecularFac. de MedicinaTRUEpu

Effect of mutation p.Ala449Thr on the GK interaction with GKRP.

<p>A) Inhibition of glucokinase activity by human GKRP. Enzyme activity was measured at 5 mM glucose as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030518#s2" target="_blank">Material and Methods</a>, in the presence of 10 µM S6P (left panel) or 0.2 mM F1P (right panel). Results are means ± SEM for three independent enzyme purifications assayed in triplicate. B) Two-hybrid interaction of GBD-GKRP with GAD-GK or GAD-GK(p.Ala449Thr) mutant. Yeast strain Y187 was used, and fusion proteins were expressed from pGBKT7 and pACTII derivatives. Values are means ± SEM from ß-galactosidase activity of six independent transformants. In control experiments, GBD-GKRP did not interact with GAD and GAD-GK did not interact with GBD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030518#pone.0030518-Galan1" target="_blank">[22]</a>.</p

Clinical characteristics of probands and <i>GCK</i> mutations.

<p>BMI: body mass index; FPG: fasting plasma glucose; OGTT: plasma glucose at 120 min after a standard oral glucose tolerance test (1.75 g per kg, máx. 75 g). Nucleotide numbering uses +1 as the A of the ATG initiation codon, based on the GenBank sequence # NM_000162. F, father; M, mother; S, sister; pGF, paternal grandfather; mGF, maternal grandfather; mGM, maternal grandmother. NA, not analysed. Underlined are those affected family members where mutation was checked by genotyping. Novel mutations are shown in bold.</p

Inhibition of GK activity by mannoheptulose (MH), N-acetylglucosamine (NAG) and palmitoyl-CoA.

<p>The GST-GK enzyme was assayed at 5 mM glucose with the indicated concentration of inhibitors as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030518#s2" target="_blank">Material and Methods</a>. Means and SEM of three independent enzyme preparations are shown.</p

Effect of temperature on the stability of the GST-GK(p.Ala449Thr) protein.

<p>Stock enzyme solutions were diluted to 250 µg/ml in buffer containing 30% glycerol, 50 mM glucose, 10 mM glutathione, 5 mM dithiothreitol, 200 mM KCl and 50 mM Tris/HCl, pH 8.0. A) The enzyme solutions were incubated for 30 min at different temperatures ranging from 30 to 55°C and then assayed at 30°C as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030518#s2" target="_blank">Material and Methods</a>. B) The enzyme solutions were incubated for different periods of time from 5 to 60 min at 50°C. Means and SEM of three independent enzyme preparations are shown.</p

Kinetic constants of human recombinant wild type and MODY2 mutant beta-cell GST-GK fusion proteins.

<p>Data represent means ± SEM of <i>n</i> separate experiments from at least two independent enzyme purifications. The Hill coefficient (nH) and the relative activity index (I_a) are unit less. (*) <i>p</i><0.0007; (**) <i>p</i> = 0.012.</p

Effect of GK synthetic activator LY2121260 on wild type and mutant GST-GK fusion proteins.

<p>GK activity was measured in standard conditions, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030518#s2" target="_blank">Material and Methods</a>, in the absence and presence of 10 µM GK activator. Since LY2121260 was dissolved in a buffer containing DMSO, all assays contained a final concentration of 0.8% DMSO. Data represent means ± SEM of at least four separate experiments from two independent enzyme purifications. Statistical significance has been estimated comparing values in the presence of LY2121260 versus their corresponding values in the presence of buffer. (**) <i>p</i><0.005; (*) <i>p</i> = 0.023.</p

Localization of mutations in the structural model for beta-cell glucokinase.

<p>A) Localization of mutated residues in the closed conformation of GK. A to E) An enlargement of the region of interest is shown in each panel. F) Representation of mutation p.Ala449Thr in the super-open conformation of GK. Wild type residues are in black, whereas the mutated residues are magenta. Surrounding disturbing structures are in blue. Glucose is represented in green whereas activator compound A is in yellow. The closed (1V4S) and the super-open (1V4T) conformations of GK <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030518#pone.0030518-Kamata1" target="_blank">[18]</a> are represented using the Pymol Molecular Graphics System (Schrödinger).</p

Effect of MODY2 mutations p.Gly223Ser and p.Ala449Thr on the affinity of GK for N-acetylglucosamine (NAG), mannoheptulose (MH) and palmitoyl-CoA.

<p>Concentrations of inhibitors causing 50% inhibition (I₅₀) were derived from Dixon plots as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030518#s2" target="_blank">Materials and Methods</a>. Data represent means ± SEM of at least four separate experiments from at least two independent enzyme purifications. (**) <i>p</i><0.006. (*) <i>p</i> = 0.03.</p