Abstract
Variant-specific zinc transporter 8 autoantibodies (ZnT8A) against either arginine (R) or tryptophan (W) at amino acid (aa) position 325 of the zinc transporter 8 (ZnT8) has been identified in type 1 diabetes (T1D) patients. Reciprocal cross-over tests revealed differences in half-maximal binding to indicate variable affinity of patient ZnT8 autoantibodies. Insufficient recombinant ZnT8 variant proteins have precluded detailed analyses of ZnT8 autoantibody affinity. The aims in the present study were to (i) generate recombinant ZnT8R- and ZnT8W-aa275-369 proteins; (ii) test the ZnT8R- and ZnT8W-aa275-369 proteins in reciprocal competitive radiobinding assays (RBA) against ZnT8R- and ZnT8W-aa268-369 labelled with 35S-methionine; and (iii) determine the specificity and affinity of sera specific for either ZnT8 arginine (R) or ZnT8 tryptophan (W) autoantibodies in newly diagnosed T1D patients. The results demonstrate, first, that it was possible to produce recombinant human MBP–ZnT8-aa275-369 protein purified to homogeneity for RBA reciprocal competition experiments. Secondly, high-titre ZnT8WA sera diluted to half maximal binding showed significant specificity for respective variants of either ZnT8R or ZnT8W. Thirdly, ZnT8WA-positive sera showed high affinity for ZnT8W compared to ZnT8RA for ZnT8R. These data demonstrate that T1D patients may have single amino acid-specific autoantibodies directed against either ZnT8R or ZnT8W and that the autoantibody affinity to the respective variant may be different. Further studies are needed to assess the mechanisms by which variant-specific ZnT8A of variable affinity develop and their possible role in the pathogenic process leading to the clinical onset of T1D.
Highlights
In type 1 diabetes (T1D), which is an organ-specific autoimmune disease, cells within the immune system attack the pancreatic islet beta cells [1,2]
The results showed that the long ZnT8R and ZnT8W (268–369) proteins, but not the short zinc transporter 8 (ZnT8) peptides, displaced radiolabelled ZnT8 (268–369) in binding to ZnT8 autoantibodies (ZnT8A)-specific T1D sera [28]
The selection of high-titre sera for the subsequent displacement experiments showed no differences in the end-point titres for ZnT8RA compared to ZnT8WA (P = 0·083)
Summary
In type 1 diabetes (T1D), which is an organ-specific autoimmune disease, cells within the immune system attack the pancreatic islet beta cells [1,2]. While the autoantibodies recognize discernible epitopes on insulin [12,13,14], GAD65 [15] and IA-2 [16,17], the B cell response against the ZnT8 protein is unique, as patients generate three variants of ZnT8 autoantibodies (ZnT8A). These autoantibody variants are directed against epitope(s) that include arginine (R), tryptophan (W) or glutamine (Q) at amino acid (aa) position 325 and may be displayed either alone or in combination in T1D patients [18,19]. The ZnT8 autoantigenicity is exceptional, as subjects at risk or newly diagnosed T1D patients may differ in risk dependent upon whether unique ZnT8RA, ZnT8WA or ZnT8Q are present [20,21]
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