Conformation-dependent GAD65 autoantibodies in diabetes.
OBJECTIVE
Conformation-dependent autoantibodies directed against GAD65 are markers of Type 1 diabetes. In this study we aimed to determine whether the substitution of GAD65 with GAD67 amino acids would affect the binding of conformation-dependent GAD65 autoantibodies.


METHODS
We used PCR-based site-directed mutagenesis to generate a series of mutated GAD65 cDNA constructs in which specific GAD65 coding sequences for regions of the protein critical for autoantibody binding were replaced with GAD67 coding sequences.


RESULTS
The introduction of a point mutation at position 517, substituting glutamic acid with proline, markedly reduced the binding of disease-associated GAD65 antibodies. The binding of GAD65 antibodies to the E517P mutant was reduced in the sera of all newly diagnosed Type 1 diabetes patients ( n=85) by a mean of 72% ( p<0.0001) compared with binding to wild-type GAD65. Patients with latent autoimmune diabetes in adults ( n=24) showed a similar reduction in binding (79% reduction, p<0.0001). First-degree relatives who subsequently progressed to Type 1 diabetes ( n=12) showed a reduction in binding of 80% compared with a reduction of only 65% among relatives who had not progressed to disease ( n=38; p=0.025). In healthy GAD65Ab-positive individuals who did not progress to diabetes during a 9-year follow-up period ( n=51), binding to GAD65-E517P was reduced by only 28% compared with binding to wild-type GAD65.


CONCLUSIONS
Differences in autoantibody binding to wild-type GAD65 versus GAD65-E517P may provide predictive information about Type 1 diabetes risk beyond that provided by the presence or absence of GAD65 autoantibodies. Lack of binding to mutant GAD65-E517P defines GAD65-positive individuals who are at higher risk of developing diabetes.
Luo
D
D
Department of Medicine, University of Washington, R. H. Williams Laboratory, 1959 N. E. Pacific Street, Box 357710, Seattle, WA 98195-7710, USA.
Gilliam
L K
LK
Greenbaum
C
C
Bekris
L
L
Hampe
C S
CS
Daniels
T
T
Richter
W
W
Marcovina
S M
SM
Rolandsson
O
O
Landin-Olsson
M
M
Kockum
I
I
Lernmark
A
A
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001323:Autoantibodies / Q000097:blood / Q000737:chemistry*; D001483:Base Sequence; D001665:Binding Sites; D003922:Diabetes Mellitus, Type 1 / Q000097:blood / Q000276:immunology*; D018450:Disease Progression; D005190:Family; D005968:Glutamate Decarboxylase / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011817:Rabbits; D012016:Reference Values
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D007527:Isoenzymes; D010446:Peptide Fragments; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2