Glucokinase (GCK) acts as a glucose sensor and regulates β-cell insulin secretion. The heterozygous mutations in the gene encoding GCK cause a reduction of the enzyme activity, which results in a monogenic form of diabetes, maturity-onset diabetes of the young. In the present study, we identified and functionally characterized a novel missense mutation in the GCK gene, which results in a protein mutation Glu339 → Lys (E339K), from a Chinese family with hyperglycemia. The same GCK mutation that co-segregated with diabetes phenotype was identified in five members of this family but was not found in 200 healthy control individuals. We expressed and affinity-purified the GCK proteins from bacterial expression system that carries mutation (E339K) and fused to glutathione S-transferase. The expressed GCK protein was subjected to the measurement of its biochemical effects of the missense mutation on GCK activity. Our results showed that the mutation reduced the GCK protein yield. The enzymatic kinetics and the thermal stability analysis on the recombinant GCK proteins revealed that the mutation inactivates enzyme kinetics and severely impaired the GCK protein stability.
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Barrio R, Bellanné-Chantelot C, Moreno JC, Morel V, Calle H, Alonso M, Mustieles C (2002) Nine novel mutations in maturity-onset diabetes of the young (MODY) candidate genes in 22 Spanish families. J Clin Endocrinol Metab 87:2532–2539
Byrne MM, Sturis J, Clément K, Vionnet N, Pueyo ME, Stoffel M, Takeda J, Passa P, Cohen D, Bell GI (1994) Insulin secretory abnormalities in subjects with hyperglycaemia due to glucokinase mutations. J Clin Invest 93:1120–1130
Davis EA, Cuesta-Muñoz A, Raoul M, Buettger C, Sweet I, Moates M, Magnuson MA, Matschinsky FM (1999) Mutants of glucokinase cause hypoglycaemia- and hyperglycaemia syndromes and their analysis illuminates fundamental quantitative concepts of glucose homeostasis. Diabetologia 42:1175–1186
Ding S, Tribble ND, Kraft CA, Markwardt M, Gloyn AL, Rizzo MA (2010) Naturally occurring glucokinase mutations are associated with defects in posttranslational S-nitrosylation. Mol Endocrinol 24:171–177
Estalella I, Garcia-Gimeno MA, Marina A, Castaño L, Sanz P (2008) Biochemical characterization of novel glucokinase mutations isolated from Spanish maturity-onset diabetes of the young (MODY2) patients. J Hum Genet 53:460–466
Eto K, Sakura H, Shimokawa K, Kadowaki H, Hagura R, Akanuma Y, Yazaki Y, Kadowaki T (1993) Sequence variations of the glucokinase gene in Japanese subjects with NIDDM. Diabetes 42:1133–1137
Froguel P, Vaxillaire M, Sun F, Velho G, Zouali H, Butel MO, Lesage S, Vionnet N, Clément K, Fougerousse F, Tanizawa Y, Weissenbach J, Beckmann JS, Lathrop GM, Passa PH, Permutt MA, Cohen D (1992) Close linkage of glucokinase locus on chromosome 7p to early-onset non-insulin-dependent diabetes mellitus. Nature 356:162–164
Froguel P, Zouali H, Vionnet N, Velho G, Vaxillaire M, Sun F, Lesage S, Stoffel M, Takeda J, Passa P, Permutt MA, Beckmann JS, Bell GI, Cohen D (1993) Familial hyperglycaemia due to mutations in glucokinase—definition of a subtype of diabetes mellitus. N Engl J Med 328:697–702
Galán M, Vincent O, Roncero I, Azriel S, Boix-Pallares P, Delgado-Alvarez E, Diaz-Cadorniga F, Blazquez E, Navas MA (2006) Effects of novel maturity-onset diabetes of the young (MODY)-associated mutations on glucokinase activity and protein stability. Biochem J 393:389–396
García-Herrero CM, Galán M, Vincent O, Flández B, Gargallo M, Delgado-Alvarez E, Blázquez E, Navas MA (2007) Functional analysis of human glucokinase gene mutations causing MODY2: exploring the regulatory mechanisms of glucokinase activity. Diabetologia 50:325–333
Giuffrida FMA, Reis AF (2005) Genetic and clinical characteristics of maturity-onset diabetes of the young. Diabetes Obes Metab 7:318–326
Glaser B, Kesavan P, Heyman M, Davis E, Cuesta A, Buchs A, Stanley CA, Thornton PS, Permutt MA, Matschinsky FM, Herold KC (1998) Familial hyperinsulinism caused by an activating glucokinase mutation. N Engl J Med 338:226–230
Hattersley AT, Turner RC, Patel P, Permutt MA, Patel P, Tanizawa Y, Chiu KC, O’Rahilly S, Watkins PJ, Wainscoat JS (1992) Linkage of type 2 diabetes to the glucokinase gene. Lancet 339:1307–1310
Horikawa Y, Iwasaki N, Hara M, Furuta H, Hinokio Y, Cockburn BN, Lindner T, Yamagata K, Ogata M, Tomonaga O, Kuroki H, Kasahara T, Iwamoto Y, Bell GI (1997) Mutations in hepatocyte nuclear factor 1β gene (TCF2) associated with MODY. Nat Genet 17:384–385
Hwang JS, Shin CH, Yang SW, Jung SY, Huh N (2006) Genetic and clinical characteristics of Korean maturity-onset diabetes of the young (MODY) patients. Diabetes Res Clin Pract 74:75–81
Kesavan P, Wang L, Davis E, Cuesta A, Sweet I, Niswender K, Magnuson MA, Matschinsky FM (1997) Structural instability of mutant β-cell glucokinase: implications for the molecular pathogenesis of maturity-onset diabetes of the young (type-2). Biochem J 322:57–63
Liang Y, Kesavan P, Wang LQ, Niswender K, Tanizawa Y, Permutt MA, Magnuson MA, Matschinsky FM (1995) Variable effects of maturity-onset diabetes of the young (MODY)-associated glucokinase mutations on substrate interactions and stability of the enzyme. Biochem J 309:167–173
Lorini R, Klersy C, d’Annunzio G, Massa O, Minuto N, Iafusco D, Bellannè-Chantelot C, Frongia AP, Toni S, Meschi F, Cerutti F, Barbetti F, the Italian Society of Pediatric Endocrinology, Diabetiology (ISPED) Study Group (2009) Maturity-onset diabetes of the young in children with incidental hyperglycemia: a multicenter Italian study of 172 families. Diabetes Care 32:1864–1866
Mahalingam B, Cuesta-Munoz A, Davis EA, Matschinsky FM, Harrison RW, Weber IT (1999) Structural model of human glucokinase in complex with glucose and ATP: implications for the mutants that cause hypo- and hyperglycaemia. Diabetes 48:1698–1705
Malecki MT, Jhala US, Antonellis A, Fields L, Doria A, Orban T, Saad M, Warram JH, Montminy M, Krolewski AS (1999) Mutations in NEUROD1 are associated with the development of type 2 diabetes mellitus. Nat Genet 23:323–328
Matschinsky FM (1990) Glucokinase as glucose sensor and metabolic signal generator in pancreatic beta-cells and hepatocytes. Diabetes 39:647–652
Matschinsky FM (1996) Banting Lecture 1995: a lesson in metabolic regulation inspired by the glucokinase glucose sensor paradigm. Diabetes 45:223–241
Matschinsky FM (2005) Glucokinase, glucose homeostasis, and diabetes mellitus. Curr Diab Rep 5:171–176
Miller SP, Anand GR, Karschnia EJ, Bell GI, LaPorte DC, Lange AJ (1999) Characterization of glucokinase mutations associated with maturity-onset diabetes of the young type 2 (MODY-2): different glucokinase defects lead to a common phenotype. Diabetes 48:1645–1651
Njølstad PR, Søvik O, Cuesta-Muñoz A, Bjørkhaug L, Massa O, Barbetti F, Undlien DE, Shiota C, Magnuson MA, Molven A, Matschinsky FM, Bell GI (2001) Neonatal diabetes mellitus due to complete glucokinase deficiency. N Engl J Med 344:1588–1592
Osbak KK, Colclough K, Saint-Martin C, Beer NL, Bellanné-Chantelot C, Ellard S, Gloyn AL (2009) Update on mutations in glucokinase (GCK), which cause maturity-onset diabetes of the young, permanent neonatal diabetes, and hyperinsulinemic hypoglycemia. Hum Mut 30:1512–1526
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 54:1949–1957
Postic C, Shiota M, Niswender KD, Jetton TL, Chen Y, Moates JM, Shelton KD, Linder J, Cherrington AD, Magnuson MA (1999) Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre recombinase. J Biol Chem 274:305–315
Prisso 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 43:1331–1332
Ralph EC, Sun S (2009) Biochemical characterization of MODY2 glucokinase variants V62M and G72R reveals reduced enzymatic activities relative to wild type. Biochemistry 48:2514–2521
Sagen JV, Odili S, Bjørkhaug L, Zelent D, Buettger C, Kwagh J, Stanley C, Dahl-Jørgensen K, de Beaufort C, Bell GI, Han Y, Grimsby J, Taub R, Molven A, Søvik O, Njølstad PR, Matschinsky FM (2006) From clinicogenetic studies of maturity-onset diabetes of the young to unraveling complex mechanisms of glucokinase regulation. Diabetes 55:1713–1722
Schober E, Rami B, Grabert M, Thont A, Kapellen T, Reinehr T, Holl RW on behalf of the DPV-Wiss Initiative of the German Working Group for Paediatric Diabetology and the German Competence Network Diabetes Mellitus (2009) Phenotypical aspects of maturity-onset diabetes of the young (MODY diabetes) in comparison with type 2 diabetes mellitus (T2DM) in Children and adolescents: experience from a large multicentre database. Diabet Med 26:466–473
Staffers DA, Ferrer J, Clarke WL, Habener JF (1997) Early-onset type 2 diabetes mellitus (MODY4) linked to IPF1. Nat Genet 17:138–139
Stoffel M, Froguel P, Takeda J, Zouali H, Vionnet N, Nishi S, Weber IT, Harrison RW, Pilkis SJ, Lesage S (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 89:7698–7702
Stride A, Vaxillaire M, Tuomi T, Barbetti F, Njølstad PR, Hansen T, Costa A, Conget I, Pedersen O, Søvik O, Lorini R, Groop L, Froguel P, Hattersley AT (2002) The genetic abnormality in the beta cell determines the response to an oral glucose load. Diabetologia 45:427–435
Vaxillaire M, Froguel P (2008) Monogenic diabetes in the young, pharmacogenetics and relevance to multifactorial forms of type 2 diabetes. Endocr Rev 29:254–264
Vionnet N, Stoffel M, Takeda J, Yasuda K, Bell GI, Zouali H, Lesage S, Velho G, Iris F, Passa P, Froguel PH, Cohen D (1992) Nonsense mutation in the glucokinase gene causes early-onset non-insulin-dependent diabetes mellitus. Nature 356:721–722
Xu JY, Dan QH, Chan V, Wat NM, Tam S, Tiu SC, Lee KF, Siu SC, Tsang MW, Fung LM, Chan KW, Lam KS (2005) Genetic and clinical characteristics of maturity-onset diabetes of the young in Chinese patients. Eur J Hum Genet 13:422–427
Yamagata K, Furuta H, Oda N, Kaisaki PJ, Menzel S, Cox NJ, Fajans SS, Signorini S, Stoffel M, Bell GI (1996a) Mutations in the hepatocyte nuclear factor-4-alpha gene in maturity-onset diabetes of the young (MODY1). Nature 384:458–460
Yamagata K, Oda N, Kaisaki PJ, Menzel S, Furuta H, Vaxillaire M, Southam L, Cox RD, Lathrop GM, Boriraj VV, Chen X, Cox NJ, Oda Y, Yano H, Le Beau MM, Yamada S, Nishigori H, Takeda J, Fajans SS, Hattersley AT, Iwasaki N, Hansen T, Pedersen O, Polonsky KS, Bell GI (1996b) Mutations in the hepatocyte nuclear factor-1-alpha gene in maturity-onset diabetes of the young (MODY3). Nature 384:455–458
Yutani K, Ogasahara K, Aoki K, Kakuno T, Sugino Y (1984) Effect of amino acid residues on conformational stability in eight mutant proteins variously substituted at a unique position of the tryptophan synthase α-subunit. J Biol Chem 259:14076–14081
We thank the family in this study. Professors Franz M. Matschinsky, Pan Chen, and Charles A. Stanley are thanked for generously providing the wild-type GST-GCK-cDNA. This work was supported by Grants from the National Basic Research Program of China (2006CB503902), the Ministry of Health of the People’s Republic of China for Key Projects of Clinical Disciplines of Hospitals Affiliated to Ministry of Health (2007–2009), the Specialized Research Fund for the Doctoral Program of Higher Education of China (200805580070) to WJ, and Post-doctor Foundation of Ministry of Education (20060390754), Natural Science Foundation of Guangdong Province, China (8151008901000196), Medical Science Foundation of Guangdong Province, China (B2008045) to LH.
Conflict of interest
Y. Shen and M. Cai contributed equally to this work.
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Shen, Y., Cai, M., Liang, H. et al. Insight into the biochemical characteristics of a novel glucokinase gene mutation. Hum Genet 129, 231–238 (2011). https://doi.org/10.1007/s00439-010-0914-4
- Gestational Diabetes Mellitus
- Oral Glucose Tolerance Test
- Thermal Instability
- E339K Mutation
- Glutamic Acid Decarboxylase Antibody