In islet-specific glucose-6-phosphatase-related protein, the beta cell antigenic sequence that is targeted in diabetes is not responsible for the loss of phosphohydrolase activity.
OBJECTIVE
There are three members of the glucose-6-phosphatase (G6Pase) family: (1) the liver/kidney/intestine G6Pase-alpha (encoded by G6PC), which is a key enzyme in glucose homeostasis; (2) the ubiquitous G6Pase-beta (encoded by G6PC3); and (3) the islet-specific G6Pase-related protein (IGRP, encoded by /G6PC2). While G6Pase-alpha and G6Pase-beta are functional glucose-6-phosphate hydrolases, IGRP possesses almost no hydrolase activity. This was unexpected since G6Pase-alpha is more closely related to IGRP than G6Pase-beta. Recently, amino acids 206-214 in IGRP were identified as a beta cell antigen targeted by a prevalent population of pathogenic CD8+ T cells in autoimmune diabetes, suggesting that this peptide confers functional specificity to IGRP. We therefore investigated the molecular events that inactivate IGRP activity and the effects of the beta cell antigen sequence on the stability and enzymatic activity of G6Pase-alpha.


METHODS
Studies were performed using site-directed mutagenesis and transient expression assays. Protein stability was evaluated by Western blotting, proteasome inhibitor studies and in vitro transcription-translation.


RESULTS
We showed that the residues responsible for G6Pase activity are more extensive than previously recognised. Introducing the IGRP antigenic motif into G6Pase-alpha does not completely destroy activity, although it does destabilise the protein. The low hydrolytic activity in IGRP is due to the combination of multiple independent mutations.


CONCLUSIONS
The loss of catalytic activity in IGRP arises from the sum of many sequence differences. G6Pase-alpha mutants containing the beta cell antigen sequence are preferentially degraded in cells, which prevents targeting by pathogenic CD8+ T cells. It is possible that IGRP levels in beta cells could dictate susceptibilities to diabetes.
Shieh
J-J
JJ
Section on Cellular Differentiation, Heritable Disorders Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-1830, USA.
Pan
C-J
CJ
Mansfield
B C
BC
Chou
J Y
JY
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers; D004285:Dogs; D005952:Glucose-6-Phosphatase / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D006801:Humans; D007515:Islets of Langerhans / Q000201:enzymology / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D010744:Phosphoric Monoester Hydrolases; D005952:Glucose-6-Phosphatase; C492194:G6PC2 protein, human