title: Site-directed mutagenesis of K396R of the 65 kDa glutamic acid decarboxylase active site obliterates enzyme activity but not antibody binding.
abstract: 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.
authors.affiliation: Department of Medicine, University of Washington, Seattle 98195, USA.  champe@u.washington.edu
authors.forename: C S
authors.initials: CS
authors.lastname: Hampe
authors.forename: L P
authors.initials: LP
authors.lastname: Hammerle
authors.forename: A
authors.initials: A
authors.lastname: Falorni
authors.forename: J
authors.initials: J
authors.lastname: Robertson
authors.forename: A
authors.initials: A
authors.lastname: Lernmark
mesh_terms: 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
chemical_list: 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