Distinct antigenic features of linear epitopes at the N-terminus and C-terminus of 65 kDa glutamic acid decarboxylase (GAD65): implications for autoantigen modification during pathogenesis

Abstract

Autoantibodies to 65 kDa glutamic acid decarboxylase (GAD65) are produced in many patients with autoimmune polyendocrine syndrome type II (APS-II) or stiff-man syndrome (SMS) and are heterogeneous in their epitope specificities, recognizing both conformational and linear determinants. Major linear epitopes of GAD, which are recognized by autoantibodies in a minority of these patients, occur in the N-terminal and C-terminal regions. We have investigated antibody recognition of the N- and C-termini of GAD65 in relation to their structural features as an approach to understanding what modifications to the native GAD structure may occur that facilitate the generation of antibodies specific to linear epitopes in these regions during the autoimmune pathogenesis. A monoclonal antibody specific to the N-terminus of GAD65 bound both native and denatured GAD in ELISA, whereas monoclonal and polyclonal antibodies specific to the C-terminus of GAD bound only denatured GAD. These antibodies were epitope mapped using random peptide phage-display libraries and the epitopes related to a previously proposed structural model of GAD65. This has led us to propose that the alpha-helical secondary structure of the C-terminus of GAD65 must be denatured to generate linear epitopes. In contrast, the N-terminus is both surface exposed and linear in the native structure, but may be masked by membrane interactions, which must be broken to facilitate recognition by B cells.

Fig. 1

Examples of serum antibodies binding to N- and C-terminal peptides of GAD, expressed as O.D. of binding to the relevant peptide minus the O.D. of binding to an irrelevant peptide (δO.D.). (a) and (b) show reactivity with the N-terminal peptide, and (c) shows reactivity with the C-terminal peptide. (DM- and DM+ indicate non-diabetic and diabetic APS-II patients, respectively.)

Fig. 2

Sequences of peptides selected by N-MoAb from an M13 pIII linear 12-mer phage peptide library (a), or a T7 linear 9-mer phage peptide library (b). The number assigned to each clone is indicated on the left. Amino acid homologies (including conservative substitutions) between peptides are indicated by vertical lines; amino acid homologies with the relevant section of GAD65 sequence (shown above the peptide sequences) are indicated in bold. *Indicates the position of a stop codon in the DNA insert encoding the peptide.

Fig. 3

Sequences of peptides selected by C-MoAb from an M13 pIII linear 12-mer phage peptide library. The number assigned to each clone is indicated on the left. Amino acid homologies (including conservative substitutions) between peptides are indicated by vertical lines; amino acid homologueies with the relevant section of GAD65 sequence (shown above the peptide sequences) are indicated in bold.

Fig. 4

Sequences of peptides selected by C-pc antibody from an M13 pIII linear 12-mer phage peptide library (a), or a T7 constrained 9-mer phage peptide library (b). The number assigned to each clone is indicated on the left. Amino acid homologueies (including conservative substitutions) between peptides are indicated by vertical lines; amino acid homologies with the relevant section of GAD65 sequence (shown above the peptide sequences) are indicated in bold. *Indicates the position of a stop codon in the DNA insert encoding the peptide.

Fig. 5

(a) Helix wheel projection of residues 568–582 of GAD65, predicted by Schwartz et al. [19]. The shading of the residues indicates the involvement of each amino acid in the epitopes of C-MoAb and/or C-pc antibody as predicted by the selection of phage-displayed peptides by these antibodies (see Figs 3 and 4), as follows: no shading, no involvement; light shading, C-MoAb epitope only; medium shading, C-pc antibody epitope only; dark shading, both C-MoAb and C-pc antibody epitopes. Percentages indicate residue accessibility within the three-dimensional model of this region of GAD65 (see Fig. 5b) as predicted by the Swiss-PdbViewer version 3·7b2. (b) Three-dimensional model of the C-terminal region of GAD65 [19] shaded to indicate accessible amino acids recognized by C-MoAb and C-pc antibody.

Fig. 5

(a) Helix wheel projection of residues 568–582 of GAD65, predicted by Schwartz et al. [19]. The shading of the residues indicates the involvement of each amino acid in the epitopes of C-MoAb and/or C-pc antibody as predicted by the selection of phage-displayed peptides by these antibodies (see Figs 3 and 4), as follows: no shading, no involvement; light shading, C-MoAb epitope only; medium shading, C-pc antibody epitope only; dark shading, both C-MoAb and C-pc antibody epitopes. Percentages indicate residue accessibility within the three-dimensional model of this region of GAD65 (see Fig. 5b) as predicted by the Swiss-PdbViewer version 3·7b2. (b) Three-dimensional model of the C-terminal region of GAD65 [19] shaded to indicate accessible amino acids recognized by C-MoAb and C-pc antibody.

Fig. 6

Helix wheel projection of residues 4–11 of GAD65. The shading of the residues indicates the involvement of each amino acid in the epitope of N-MoAb as predicted by the selection of phage-displayed peptides by N-MoAb, as follows: no shading, no involvement; light shading, predicted by selected M13 pIII linear 12-mer peptides; medium shading, predicted by selected T7 linear 9-mer peptides; dark shading, predicted by both selected M13 linear 12-mer peptides and T7 linear 9-mer peptides.