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01.02.2007 | Article

Functional analysis of human glucokinase gene mutations causing MODY2: exploring the regulatory mechanisms of glucokinase activity

verfasst von: C. M. García-Herrero, M. Galán, O. Vincent, B. Flández, M. Gargallo, E. Delgado-Alvarez, E. Blázquez, M. A. Navas

Erschienen in: Diabetologia | Ausgabe 2/2007

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Abstract

Aims/hypothesis

Glucokinase (GCK) acts as a glucose sensor in the pancreatic beta cell and regulates insulin secretion. In the gene encoding GCK the heterozygous mutations that result in enzyme inactivation cause MODY2. Functional studies of naturally occurring GCK mutations associated with hyperglycaemia provide further insight into the biochemical basis of glucose sensor regulation.

Materials and methods

Identification of GCK mutations in selected MODY patients was performed by single-strand conformation polymorphism and direct sequencing. The kinetic parameters and thermal stability of recombinant mutant human GCK were determined, and in pull-down assays the effect of these mutations on the association of GCK with glucokinase (hexokinase 4) regulator (GCKR, also known as glucokinase regulatory protein [GKRP]) and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB1, also known as PFK2) was tested.

Results

We identified three novel GCK mutations: the insertion of an asparagine residue at position 161 (inserN161) and two missense mutations (M235V and R308W). We also identified a fourth mutation (R397L) reported in a previous work. Functional characterisation of these mutations revealed that insertion of asparagine residue N161 fully inactivates GCK, whereas the M235V and R308W mutations only partially impair enzymatic activity. In contrast, GCK kinetics was almost unaffected by the R397L mutation. Although none of these mutations affected the interaction of GCK with PFKFB1, we found that the R308W mutation caused protein instability and increased the strength of interaction with GCKR.

Conclusions/interpretation

Our results show that different MODY2 mutations impair GCK function through different mechanisms such as enzymatic activity, protein stability and increased interaction with GCKR, helping further elucidate the regulation of GCK activity.

Fajans SS, Bell GI, Polonsky KS (2001) Molecular mechanisms and clinical pathophysiology of maturity-onset diabetes of the young. N Engl J Med 345:971–980PubMedCrossRef

Prisco F, Iafusco D, Franzese A, Sulli N, Barbetti F (2000) MODY 2 presenting as neonatal hyperglycaemia: a need to reshape the definition of “neonatal diabetes”? Diabetologia 43:1331–1332PubMedCrossRef

Pearson ER, Velho G, Clark P et al (2001) Beta-cell genes and diabetes: quantitative and qualitative differences in the pathophysiology of hepatic nuclear factor-1alpha and glucokinase mutations. Diabetes 50:S101–S107PubMedCrossRef

Matschinsky FM (1990) Glucokinase as glucose sensor and metabolic signal generator in pancreatic beta-cells and hepatocytes. Diabetes 39:647–652PubMedCrossRef

Zelent D, Najafi H, Odili S et al (2005) Glucokinase and glucose homeostasis: proven concepts and new ideas. Biochem Soc Trans 33:306–310PubMedCrossRef

Matschinsky FM (2005) Glucokinase, glucose homeostasis, and diabetes mellitus. Curr Diab Rep 5:171–176PubMedCrossRef

Van Schaftingen E, Detheux M, Veiga da Cunha M (1994) Short-term control of glucokinase activity: role of a regulatory protein. FASEB J 8:414–419PubMed

Shiota C, Coffey J, Grimsby J, Grippo JF, Magnuson MA (1999) Nuclear import of hepatic glucokinase depends upon glucokinase regulatory protein, whereas export is due to a nuclear export signal sequence in glucokinase. J Biol Chem 274:37125–37130PubMedCrossRef

Alvarez E, Roncero I, Chowen JA, Vazquez P, Blazquez E (2002) Evidence that glucokinase regulatory protein is expressed and interacts with glucokinase in rat brain. J Neurochem 80:45–53PubMedCrossRef

10.

Baltrusch S, Lenzen S, Okar DA, Lange AJ, Tiedge M (2001) Characterization of glucokinase-binding protein epitopes by a phage-displayed peptide library. Identification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as a novel interaction partner. J Biol Chem 276:43915–43923CrossRef

11.

Massa L, Baltrusch S, Okar DA, Lange AJ, Lenzen S, Tiedge M (2004) Interaction of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) with glucokinase activates glucose phosphorylation and glucose metabolism in insulin-producing cells. Diabetes 53:1020–1029PubMedCrossRef

12.

Munoz-Alonso MJ, Guillemain G, Kassis N, Girard J, Burnol AF, Leturque A (2000) A novel cytosolic dual specificity phosphatase, interacting with glucokinase, increases glucose phosphorylation rate. J Biol Chem 275:32406–32412PubMedCrossRef

13.

Rizzo MA, Piston DW (2003) Regulation of {beta} cell glucokinase by S-nitrosylation and association with nitric oxide synthase. J Cell Biol 161:243–248PubMedCrossRef

14.

Danial NN, Gramm CF, Scorrano L et al (2003) BAD and glucokinase reside in a mitochondrial complex that integrates glycolysis and apoptosis. Nature 424:952–956PubMedCrossRef

15.

Shiraishi A, Yamada Y, Tsuura Y et al (2001) A novel glucokinase regulator in pancreatic beta cells precursor of propionyl-CoA carboxylase beta subunit interacts with glucokinase and augments its activity. J Biol Chem 276:2325–2328PubMedCrossRef

16.

Davis EA, Cuesta-Munoz A, Raoul M et al (1999) Mutants of glucokinase cause hypoglycaemia- and hyperglycaemia syndromes and their analysis illuminates fundamental quantitative concepts of glucose homeostasis. Diabetologia 42:1175–1186PubMedCrossRef

17.

Kesavan P, Wang L, Davis E et al (1997) Structural instability of mutant beta-cell glucokinase: implications for the molecular pathogenesis of maturity-onset diabetes of the young (type-2). Biochem J 322:57–63PubMed

18.

Gloyn AL, Odili S, Zelent D et al (2005) Insights into the structure and regulation of glucokinase from a novel mutation (V62M), which causes maturity-onset diabetes of the young. J Biol Chem 280:14105–14113PubMedCrossRef

19.

Galan M, Vincent O, Roncero I et al (2006) Effects of novel maturity-onset diabetes of the young (MODY)-associated mutations on glucokinase activity and protein stability. Biochem J 393:389–396PubMedCrossRef

20.

Porter JR, Shaw NJ, Barrett TG, Hattersley AT, Ellard S, Gloyn AL (2005) Permanent neonatal diabetes in an Asian infant. J Pediatr 146:131–133PubMedCrossRef

21.

Miller SA, Dykes DD, Polesky HF (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16:1215PubMedCrossRef

22.

Stoffel M, Froguel P, Takeda J et al (1992) Human glucokinase gene: isolation, characterization, and identification of two missense mutations linked to early-onset non-insulin-dependent (type 2) diabetes mellitus. Proc Natl Acad Sci USA 89:7698–7702PubMedCrossRef

23.

Navas MA, Vaisse C, Boger S, Heimesaat M, Kollee LA, Stoffel M (2000) The human HNF-3 genes: cloning, partial sequence and mutation screening in patients with impaired glucose homeostasis. Hum Hered 50:370–381PubMedCrossRef

24.

Liang Y, Kesavan P, Wang LQ et al (1995) Variable effects of maturity-onset-diabetes-of-youth (MODY)-associated glucokinase mutations on substrate interactions and stability of the enzyme. Biochem J 309:167–173PubMed

25.

Perez JX, Roig T, Manzano A et al (2000) Overexpression of fructose 2,6-bisphosphatase decreases glycolysis and delays cell cycle progression. Am J Physiol Cell Physiol 279:C1359–C1365PubMed

26.

Rose MD, Winston F, Hieter P (1990) Methods in yeast genetics: a laboratory course manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

27.

Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbour Laboratory, Cold Spring Harbor, NY

28.

Veiga-da-Cunha M, Xu LZ, Lee YH, Marotta D, Pilkis SJ, Van Schaftingen E (1996) Effect of mutations on the sensitivity of human beta-cell glucokinase to liver regulatory protein. Diabetologia 39:1173–1179PubMedCrossRef

29.

Gloyn AL, Odili S, Buetter C, Njolstad PR, Shiota C, Magnuson MA, Matschinsky FM (2004) Glucokinase and the regulation of blood sugar. A mathematical model predicts the threshold for glucose stimulated insulin release for GCK gene mutations that cause hyper- and hypoglycemia. In: Matschinsky FM, Magnuson MA (eds) Glucokinase and glycemic disease: from basics to novel therapeutics. Front. Diabetes. Vol 16, Karger, Basel, Switzerland, pp 92–109CrossRef

30.

Kamata K, Mitsuya M, Nishimura T, Eiki J, Nagata Y (2004) Structural basis for allosteric regulation of the monomeric allosteric enzyme human glucokinase. Structure 12:429–438PubMedCrossRef

31.

Sagen JV, Odili S, Bjorkhaug L et al (2006) From clinicogenetic studies of maturity-onset diabetes of the young to unraveling complex mechanisms of glucokinase regulation. Diabetes 55:1713–1722PubMedCrossRef

32.

Payne VA, Arden C, Wu C, Lange AJ, Agius L (2005) Dual role of phosphofructokinase-2/fructose bisphosphatase-2 in regulating the compartmentation and expression of glucokinase in hepatocytes. Diabetes 54:1949–1957PubMedCrossRef

33.

Gidh-Jain M, Takeda J, Xu LZ et al (1993) Glucokinase mutations associated with non-insulin-dependent (type 2) diabetes mellitus have decreased enzymatic activity: implications for structure/function relationships. Proc Natl Acad Sci USA 90:1932–1936PubMedCrossRef

34.

Veiga-da-Cunha M, Courtois S, Michel A, Gosselain E, Van Schaftingen E (1996) Amino acid conservation in animal glucokinases. J Biol Chem 271:6292–6297PubMedCrossRef

35.

Baltrusch S, Francini F, Lenzen S, Tiedge M (2005) Interaction of glucokinase with the liver regulatory protein is conferred by leucine-asparagine motifs of the enzyme. Diabetes 54:2829–2837PubMedCrossRef

36.

Sayle RA, Milner-White EJ (1995) RASMOL: biomolecular graphics for all. Trends Biochem Sci 20:374–376PubMedCrossRef

Titel: Functional analysis of human glucokinase gene mutations causing MODY2: exploring the regulatory mechanisms of glucokinase activity
verfasst von: C. M. García-Herrero
M. Galán
O. Vincent
B. Flández
M. Gargallo
E. Delgado-Alvarez
E. Blázquez
M. A. Navas
Publikationsdatum: 01.02.2007
Verlag: Springer-Verlag
Erschienen in: Diabetologia / Ausgabe 2/2007
Print ISSN: 0012-186X
Elektronische ISSN: 1432-0428
DOI: https://doi.org/10.1007/s00125-006-0542-7

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Functional analysis of human glucokinase gene mutations causing MODY2: exploring the regulatory mechanisms of glucokinase activity