Analysis of GAD65 autoantibodies in Stiff-Person syndrome patients.
Autoantibodies to the 65-kDa isoform of glutamate decarboxylase GAD65 (GAD65Ab) are strong candidates for a pathological role in Stiff-Person syndrome (SPS). We have analyzed the binding specificity of the GAD65Ab in serum and cerebrospinal fluid (CSF) of 12 patients with SPS by competitive displacement studies with GAD65-specific rFab-derived from a number of human and mouse mAbs specific for different determinants on the Ag. We demonstrate considerable differences in the epitope specificity when comparing paired serum and CSF samples, suggesting local stimulation of B cells in the CSF compartment of these patients. Moreover, these autoantibodies strongly inhibit the enzymatic activity of GAD65, thus blocking the formation of the neurotransmitter gamma-aminobutyric acid. The capacity of the sera to inhibit the enzymatic activity of GAD65 correlated with their binding to a conformational C-terminal Ab epitope. Investigation of the inhibitory mechanism revealed that the inhibition could not be overcome by high concentrations of glutamate or the cofactor pyridoxal phosphate, suggesting a noncompetitive inhibitory mechanism. Finally, we identified a linear epitope on amino acids residues 4-22 of GAD65 that was recognized solely by autoantibodies from patients with SPS but not by serum from type 1 diabetes patients. A mAb (N-GAD65 mAb) recognizing this N-terminal epitope was successfully humanized to enhance its potential therapeutic value by reducing its overall immunogenicity.
Raju
Raghavanpillai
R
Neuromuscular Diseases Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.
Foote
Jefferson
J
Banga
J Paul
JP
Hall
Tyler R
TR
Padoa
Carolyn J
CJ
Dalakas
Marinos C
MC
Ortqvist
Eva
E
Hampe
Christiane S
CS
D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D001323:Autoantibodies / Q000097:blood / Q000134:cerebrospinal fluid / Q000276:immunology*; D001667:Binding, Competitive; D018604:Epitope Mapping; D018985:Epitopes, B-Lymphocyte / Q000276:immunology; D005260:Female; D005968:Glutamate Decarboxylase / Q000276:immunology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007527:Isoenzymes / Q000276:immunology*; D008297:Male; D051379:Mice; D008875:Middle Aged; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D016750:Stiff-Person Syndrome / Q000276:immunology*
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D018985:Epitopes, B-Lymphocyte; D007140:Immunoglobulin Fab Fragments; D007527:Isoenzymes; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2