Site-directed mutagenesis of K396R of the 65 kDa glutamic acid decarboxylase active site obliterates enzyme activity but not antibody binding.
The role of K396 in the enzymatic catalysis and the antigenicity of the 65 kDa isoform of glutamate decarboxylase (GAD65) was analyzed using the K396R GAD65 mutant. GAD65 is a major autoantigen in Type 1 diabetes and autoantibodies directed to GAD65 are widely used markers for this disease. We found that (1) recombinant human GAD65 is fully enzymatically active; (2) the K396R mutation abolished GAD65 activity; and (3) the K396R mutant retained full antigenicity to GAD65 autoantibodies in serum from Type 1 diabetes patients, but not to polyclonal antibodies raised to the catalytic domain.
Hampe
C S
CS
Department of Medicine, University of Washington, Seattle 98195, USA.  champe@u.washington.edu
Hammerle
L P
LP
Falorni
A
A
Robertson
J
J
Lernmark
A
A
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D001323:Autoantibodies / Q000276:immunology*; D001324:Autoantigens / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain / Q000276:immunology; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology*; D005968:Glutamate Decarboxylase / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007106:Immune Sera / Q000276:immunology; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D010843:Pichia / Q000235:genetics; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D019163:Reducing Agents / Q000378:metabolism; D014170:Transformation, Genetic
D001323:Autoantibodies; D001324:Autoantigens; D007106:Immune Sera; D007527:Isoenzymes; D011994:Recombinant Proteins; D019163:Reducing Agents; D011732:Pyridoxal Phosphate; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2