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


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
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.
García-Herrero
C M
CM
Department of Biochemistry and Molecular Biology III, Faculty of Medicine, Complutense University of Madrid, Madrid, Spain.
Galán
M
M
Vincent
O
O
Flández
B
B
Gargallo
M
M
Delgado-Alvarez
E
E
Blázquez
E
E
Navas
M A
MA
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000293:Adolescent; D000328:Adult; D019943:Amino Acid Substitution; D001483:Base Sequence; D015992:Body Mass Index; D002648:Child; D017931:DNA Primers; D003924:Diabetes Mellitus, Type 2 / Q000201:enzymology / Q000235:genetics*; D005260:Female; D005941:Glucokinase / Q000235:genetics* / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006706:Homeostasis; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation
D048868:Adaptor Proteins, Signal Transducing; D017931:DNA Primers; C485100:GCKR protein, human; D005982:Glutathione Transferase; D005941:Glucokinase