Mapping of novel autoreactive epitopes of the diabetes-associated autoantigen IA-2

Abstract

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.

Fig. 1

IA-2 and IA-2β constructs used for the analysis of humoral IA-2 epitopes. Numbers represent amino acid position in IA-2 and IA-2β. A summary of reactivity patterns of 111 sera from patients with type 1 diabetes are indicated on the right margin.

Fig. 2

IA-2/IA-2β chimeric proteins used for the analysis of N- and C-terminal epitopes. Numbers represent amino acid position in IA-2 and IA-2ß. A summary of reactivity patterns of 105 sera from patients with type 1 diabetes which show no reactivity to IA-2β deletion mutants B1 and B2 is indicated on the right margin.

Fig. 3

Immunoprecipitation analyses of dominant humoral IA-2 and IA-2ß epitopes. Autoradiography of [³⁵S] methionine labelled reticulocyte lysates revealed that IA-2 and IA-2β deletion mutants were expressed as major bands after expression by in vitro transcription and translation. The figure demonstrates representative reactivity patterns against IA-2 deletion mutants (a) and IA-2β deletion mutants (b) obtained by immunoprecipitation using sera from patients with type 1 diabetes. Molecular weight markers (MWM) are given in the left margin.

Fig. 4

Analysis of immunoreactivity against IA-2/IA-2β chimeric proteins. Chimeras were expressed and labelled with [³⁵S] methionine by in vitro transcription and translation (upper panel). Antibody binding was analysed by immunoprecipitation with sera from patients with type 1 diabetes. Representative antibody patterns are illustrated against IA-2/IA-β chimeras (a) and IA-2β/IA-2 chimeric proteins (b). Molecular weight markers (MWM) are given on the left margin.

Fig. 5

Reactivity against the N-terminal epitope can be blocked by synthetic peptides. Sera were preincubated with 20 µg peptide 1–8 and then used for immunoprecipitation of [³⁵S] methionine labelled chimera CH3. Preincubation of sera with peptide 5 and 6 completely blocked the reactivity to chimera CH3. Peptides 1–4 and 7–8 do not inhibit binding of any sera. The lower panel demonstrates sequence homology between human IA-2 and mouse IA-2β harbouring the epitope for N-terminal IA-2 autoantibodies. Solid lines indicate identical amino acids (aa), points indicate aa with similar charge, polarity, or hydrophobicity. Numbers at peptides refer to the aa residues of human IA-2.