The core cysteines, (C909) of islet antigen-2 and (C945) of islet antigen-2β, are crucial to autoantibody binding in type 1 diabetes

Abstract

Cysteines are thought integral to conformational epitopes of islet antigen-2 (IA-2) autoantibodies (IA-2A), possibly through disulfide bond formation. We therefore investigated which cysteines are critical to IA-2A binding in patients with newly diagnosed type 1 diabetes. All 10 cysteines in the intracellular domain of IA-2 were modified to serine by site-directed mutagenesis, and the effects of these changes on autoantibody binding in comparison with wild-type control were investigated by radiobinding assay. Mutation of the protein tyrosine phosphatase (PTP) core cysteine (C909) in IA-2 caused large reductions in autoantibody binding. In contrast, little or no reduction in binding was seen following substitution of the other cysteines. Modification of the core cysteine (C945) in IA-2β also greatly reduced autoantibody binding. Lysine substitution of glutamate-836 in IA-2 or glutamate-872 in IA-2β resulted in modest reductions in binding and identified a second epitope region. Binding to IA-2 PTP and IA-2β PTP was almost abolished by mutation of both the core cysteine and these glutamates. The core cysteine is key to the major PTP conformational epitope, but disulfide bonding contributes little to IA-2A epitope integrity. In most patients, at disease onset, >90% of antibodies binding to the PTP domain of IA-2 recognize just two epitope regions.

FIG. 1.

Alignment of IA-2ic and IA-2β PTP sequences in pSP64 and pGEM, respectively. The IA-2 PTP sequence in pGEM is the same as IA-2ic, except it begins at amino acid 687 marked by the inverted triangle. Nonconserved amino acids are shaded. The highly conserved PTP core sequence is boxed (907–917) and differs by one amino acid between IA-2 and IA-2β. All 10 cysteines (boxed) in IA-2ic and cysteine at position at 945 in IA-2β were substituted with serine. Cysteine at position 909 and the corresponding cysteine in IA-2β (position 945) were changed to alanine. Glutamate at position 836 in IA-2 and 872 in IA-2β (circled) was changed to a lysine. Tryptophan at position 799 was changed to alanine in IA-2 PTP. The WPAE motif is also marked (boxed 875–878). The numbers above the sequence refer to the amino acid positions in IA-2ic. Sequences were obtained from GenBank accession numbers L18983 (IA-2) and Y08569 (IA-2β).

FIG. 2.

Binding (cpm) shown by 70 different IA-2–positive BOX sera of labels made using IA-2ic (A), IA-2 PTP (B), and IA-2β PTP (C) plasmid with mutations at amino acid position 909 (945 in IA-2β) to serine (open triangles) and alanine (filled squares). The dashed line represents equivalent binding with or without the amino acid mutation.

FIG. 3.

A box and whisker plot showing percentage binding by 70 IA-2A–positive patient sera of IA-2ic labels containing the 10 different cysteine-to-serine mutations in comparison with binding of WT label. Mutation of C909 resulted in 68% reduction in binding, but mutation of the other nine cysteines caused little reduction or slight increases in binding by the sera. The median percentage binding of WT is represented by the line in the box. The box contains the interquartile range. The region within the whiskers represents 80% of the sera, whereas outliers are sera between 1.5 (open circle) and 3 (asterisk) interquartile ranges from the end of the box.

FIG. 4.

Binding (cpm) shown by 70 different IA-2A–positive BOX sera to labeled proteins made using IA-2ic (A), IA-2 PTP (B), and IA-2β PTP (C) (open triangles), the IA-2 PTP E836K (E872K in IA2-β) (open squares), and the IA-2 and IA2-β PTP double mutants E836K/C909S and E872K/C945S (filled circles). The dashed line represents equivalent binding with or without the amino acid mutation.

FIG. 5.

Binding (cpm) shown by 58 different IA-2A–positive BOX sera to labeled proteins made using IA-2 PTP WT and IA-2 PTP plasmid with mutation of tryptophan at position 799 to alanine (open triangles). The dashed line represents equivalent binding with or without the amino acid mutation.

FIG. 6.

A: General overview ribbon diagram of the overlaid structures of IA-2 PTP (cyan) residues 687–976 and IA-2β (magenta) residues 724–1012 showing the side chains of amino acids important for antibody binding. The position of C909 in IA-2 and C945 in IA-2β is indicated to show its proximity to the WPAE epitope. Residues 831–836 and 867–872 indicate the position of epitopes affected by the glutamate-to-lysine mutations. Residues 952–958 and 988–994 form a ridge separating two clefts, one of which holds the core cysteine. The positions of tryptophan 799 and glutamine 862 are also shown. B shows the positions of the cysteine residues in IA-2 PTP (cyan) and IA-2β (magenta). C shows the effects of mutating C909 in WT (green) to alanine (magenta), serine (cyan) and oxidizing the cysteine by adding sulphonate (blue). All minimized models were superimposed in InsightII using C-α positions for the whole proteins. RMSDs were calculated comparing all non–hydrogen atom positions in the loops of the mutated C909 models with WT.