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.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Sequence
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Amino Acid Substitution / genetics*
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Animals
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Autoantibodies / immunology*
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Autoantigens / chemistry
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Autoantigens / genetics
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Autoantigens / immunology
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Autoantigens / metabolism
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Binding Sites
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Catalysis
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Catalytic Domain / immunology
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Diabetes Mellitus, Type 1 / immunology*
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Glutamate Decarboxylase / chemistry
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Glutamate Decarboxylase / genetics
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Glutamate Decarboxylase / immunology*
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Glutamate Decarboxylase / metabolism*
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Humans
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Hydrogen-Ion Concentration
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Immune Sera / immunology
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Isoenzymes / chemistry
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Isoenzymes / genetics
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Isoenzymes / immunology*
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Isoenzymes / metabolism*
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Kinetics
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Molecular Sequence Data
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Molecular Weight
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Mutagenesis, Site-Directed / genetics*
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Mutation / genetics
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Pichia / genetics
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Pyridoxal Phosphate / metabolism
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Radioimmunoassay
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Recombinant Proteins / biosynthesis
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Recombinant Proteins / chemistry
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Recombinant Proteins / immunology
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Recombinant Proteins / metabolism
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Reducing Agents / metabolism
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Transformation, Genetic
Substances
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Autoantibodies
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Autoantigens
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Immune Sera
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Isoenzymes
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Recombinant Proteins
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Reducing Agents
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Pyridoxal Phosphate
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Glutamate Decarboxylase
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glutamate decarboxylase 2