Mapping of novel autoreactive epitopes of the diabetes-associated autoantigen IA-2.
IA-2, a member of the tyrosine phosphatase family, has been identified as a dominant autoantigen in type 1 diabetes. To define humoral IA-2 epitopes, we generated a panel of IA-2 deletion mutants and chimeric proteins using the highly homologous tyrosine phosphatase-like protein IA-2beta. Analysis of autoantibody reactivity in 111 IA-2 antibody positive sera from patients with type 1 diabetes revealed that humoral epitopes cluster to several domains of the intracytoplasmic part of IA-2 [IA-2ic, amino acid (aa) 604-979]. Immunodominant epitopes were found in the first N-terminal 73 amino acids (56% positive), in the middle domain residing between residues 699-874 (45% positive) and the C-terminus depending on the presence of aa 931-979 (at least 37% positive). Competition experiments with overlapping peptides revealed that autoantibody binding towards the N-terminus was dependent on residues 621-628. In the C-terminal domain, two novel conformation-dependent epitopes were identified. The first epitope requires the presence of the C-terminal part of IA-2 (aa 933-979) and an IA-2-specific region between residues 771-932. Reactivity against the second epitope was dependent on intact C-terminal domains as well as residues in the middle (aa 887-932) and N-terminal regions (aa 604-771) which are conserved in IA-2 and IA-2beta. We here defined novel autoantigenic determinants in the N-terminus of IA-2 and characterized conformational epitopes residing in the C-terminal region or spanning from C-terminal residues to the N-terminal domain of IA-2ic. The identification of dominant target regions of diabetes-specific autoantibodies may help to elucidate the molecular mechanisms involved in the autoimmunity towards IA-2.
Seissler
J
J
German Diabetes Reseach Institute at the University of Dusseldorf. sei@dfi.uni-duesseldorf.de.
Schott
M
M
Morgenthaler
N G
NG
Scherbaum
W A
WA
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001323:Autoantibodies; D001324:Autoantigens / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D001483:Base Sequence; D018387:Codon, Initiator / Q000235:genetics; D003922:Diabetes Mellitus, Type 1 / Q000201:enzymology / Q000235:genetics* / Q000276:immunology*; D018604:Epitope Mapping; D016056:Immunodominant Epitopes; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
D001323:Autoantibodies; D001324:Autoantigens; D018387:Codon, Initiator; C096322:ICA512 autoantibody; D016056:Immunodominant Epitopes; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; C516863:Ptprn protein, mouse; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8