Autoreactive epitopes defined by diabets-associated human monoclonal-antibodies are localited in the middle and C-Terminal domains of the smaller form of glutamate-decarboxylase

The gamma-aminobutyrate-synthesizing enzyme glutamate decarboxylase (GAD; L-glutamate 1-carboxy-lyase, EC 4.1.1.15) is a major target of autoantibodies associated with both early and late stages of pancreatic beta-cell destruction and development of type 1 diabetes. We have used five monoclonal anti-islet-cell antibodies (MICAs 1,2,3,4, and 6) derived from a newly diagnosed diabetic patient to probe the autoimmune epitopes in the enzyme. All the MICAs specifically recognized the smaller GAD protein, GAD65, and did not recognize the nonallelic GAD67 protein. A series of N-terminal, C-terminal, and internal deletion mutants, as well as protein footprinting, were used to identify the target regions in GAD65. Immunoprecipitation revealed two major native epitope areas in the GAD65 molecule. The first, defined by MICAs 1 and 3, is destroyed by deleting 41 amino acids at the C terminus but is also dependent on intact amino acids 244-295. This epitope (or epitopes) may span both middle and C-terminal domains of the protein. The second conformational epitope region, defined by MICAs 4 and 6, is dependent on intact amino acids 245-295 but is not affected by deletion of 110 amino acids at the C terminus and is therefore confined to domain(s) in the middle of the molecule. MICA 2 recognizes a linear epitope close to the C terminus. Thus, the N-terminal domain of GAD65, which differs most significantly from GAD67, does not harbor the MICA epitopes. Rather subtle amino acid differences in the middle and C-terminal domains define the GAD65-specific autoimmune epitopes. Analysis of sera from 10 type 1 diabetic patients suggests that MICAs 1, 3, 4, and 6 represent a common epitope recognition in this disease, whereas the MICA 2 epitope is rare. Furthermore, autoantibodies in some sera are restricted to the MICA 1/3 epitope, suggesting that this epitope may represent a single dominant epitope in the early phases of beta-cell autoimmunity.

High-resolution autoreactive epitope mapping and structural modeling of the 65 kDa form of human glutamic acid decarboxylase

Steinunn Baekkeskov Hanahan

The smaller isoform of the GABA-synthesizing enzyme, glutamic acid decarboxylase 65 (GAD65), is unusually susceptible to becoming a target of autoimmunity affecting its major sites of expression, GABA-ergic neurons and pancreatic β-cells. In contrast, a highly homologous isoform, GAD67, is not an autoantigen. We used homolog-scanning mutagenesis to identify GAD65-specific amino acid residues which form autoreactive B-cell epitopes in this molecule. Detailed mapping of 13 conformational epitopes, recognized by human monoclonal antibodies derived from patients, together with two and three-dimensional structure prediction led to a model of the GAD65 dimer. GAD65 has structural similarities to ornithine decarboxylase in the pyridoxal-5′-phosphate-binding middle domain (residues 201-460) and to dialkylglycine decarboxylase in the C-terminal domain (residues 461-585). Six distinct conformational and one linear epitopes cluster on the hydrophilic face of three amphipathic α-helices in exons 14-16 in the C-terminal domain. Two of those epitopes also require amino acids in exon 4 in the N-terminal domain. Two distinct epitopes reside entirely in the N-terminal domain. In the middle domain, four distinct conformational epitopes cluster on a charged patch formed by amino acids from three α-helices away from the active site, and a fifth epitope resides at the back of the pyridoxal 5′-phosphate binding site and involves amino acid residues in exons 6 and 11-12. The epitopes localize to multiple hydrophilic patches, several of which also harbor DR∗0401-restricted T-cell epitopes, and cover most of the surface of the protein. The results reveal a remarkable spectrum of human autoreactivity to GAD65, targeting almost the entire surface, and suggest that native folded GAD65 is the immunogen for autoreactive B-cells.

Elsevier1999

Identification of the 64K autoantigen in insulin-dependent diabetes as the GABA-synthesizing enzyme glutamic acid decarboxylase

Steinunn Baekkeskov Hanahan

The pancreatic islet β-cell autoantigen of relative molecular mass 64,000 (64K), which is a major target of autoantibodies associated with the development of insulin-dependent diabetes mel-litus (IDDM) has been identified as glutamic acid decarboxylase, the biosynthesizing enzyme of the inhibitory neurotransmitter GABA (γ-aminobutyric acid). Pancreatic β cells and a subpopulation of central nervous system neurons express high levels of this enzyme. Autoantibodies against glutamic acid decarboxylase with a higher titre and increased epitope recognition compared with those usually associated with IDDM are found in stiff-man syndrome, a rare neurological disorder characterized by a high coincidence with IDDM.

1990

Isolation and Characterization of Human Monoclonal Autoantibodies to Glutamic Acid Decarboxylase

Steinunn Baekkeskov Hanahan

Production of human monoclonal autoantibodies to glutamic acid decarboxylase Mr 65,000 (GAD65), characterization of their isotype, binding affinity, V region sequences and competition with autoantibodies in patients' sera is described. Lymphocytes from a patient with Addison's disease who had GAD65 autoantibodies without diabetes were immortalised and fused to a mouse/human hybridoma. In addition, mouse monoclonal antibodies to GAD65 were produced using standard techniques. F(ab')2s from our monoclonals and the GAD6 mouse monoclonal were used in competition with intact monoclonals and sera from diabetic patients for binding to 125I-labelled GAD65 (amino acids 46-586). Reactivities of the human monoclonals with GAD 65,000/67,000 Mr chimeras were also studied. Variable region genes of human monoclonals were sequenced and analysed. The human monoclonals (n = 3) had affinity constants for GAD65 of 2.2 &#50 109, 5.8 &#50 109, 1.3 &#50 1010 mol/l&#109 1; affinities of the mouse monoclonals (n = 5) ranged from 1.1 &#50 108 to 5.4 &#50 1010 mol/l&#109 1. The binding of each of the human monoclonals was inhibited by GAD6 F(ab')2 and the binding of GAD6 antibody was inhibited by the human monoclonal F(ab')2s suggesting that the epitopes for these antibodies were overlapping. Studies with GAD65/GAD67 chimeras indicated that the human monoclonals reacted with C-terminal epitopes. The human monoclonals, GAD6 and 3/5 mouse monoclonals inhibited serum autoantibody binding to 125I-labelled GAD65. Overall, the human monoclonals were of high affinity, reacted with C-terminal epitopes and showed evidence of antigen driven maturation; they represented only a proportion of the repertoire of autoantibodies to GAD65 in the donor's serum and in the sera of patients with type-1 diabetes.

Taylor & Francis2002

High-resolution autoreactive epitope mapping and structural modeling of the 65 kDa form of human glutamic acid decarboxylase

Steinunn Baekkeskov Hanahan

Elsevier1999