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Mapping of conformational autoantibody epitopes in ZNT8.

ABSTRACT: Identification of the major humoral epitopes in zinc transporter 8 (ZnT8) will expand the range of biomarkers for human type 1 diabetes and may provide clues to the mechanisms governing disease progression. Our initial studies suggested that most ZnT8-reactive sera recognize conformational epitopes in the final 100aa region of the molecule. Subsequently we identified residue 325 as a major determinant in two epitopes linked to a genetic polymorphism with high minor allele frequency (rs13266634). The goal of the current study was to extend this analysis to identify non-polymorphic epitopes in ZnT8.Although the carboxy-terminal domains of human and mouse ZnT8 are ?80% identical, the mouse probe is not precipitated by the majority of human type 1 diabetes sera. Thus to identify key residues we systematically 'humanized' the mouse probe at each position that differs and evaluated the probes in radio-immunoassays.As previously reported, only the alteration of Q>R325 by itself showed any restoration of binding to human sera. However, when clusters of structurally adjacent variant residues were also changed an additional region of antigenicity was revealed that depended on residues R332, E333, K336, and K340. Using a panel of 112 sera from recent onset subjects tested with the hC325Q and m-R325R332E333K336K340 probes, 39.3% of the subjects were ZnT8(Q)A+ , of which 38.6% (17/44) also recognized the mouse probe.We conclude that the mR-REKK probe identifies a third major epitope in ZnT8 that may add to the diagnostic utility of measuring autoantibodies to this molecule.

SUBMITTER: Wenzlau JM

PROVIDER: S-EPMC3664522 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Mapping of conformational autoantibody epitopes in ZNT8.

Wenzlau Janet M JM Frisch Lisa M LM Hutton John C JC Davidson Howard W HW

Diabetes/metabolism research and reviews 20111101 8

<h4>Background</h4>Identification of the major humoral epitopes in zinc transporter 8 (ZnT8) will expand the range of biomarkers for human type 1 diabetes and may provide clues to the mechanisms governing disease progression. Our initial studies suggested that most ZnT8-reactive sera recognize conformational epitopes in the final 100aa region of the molecule. Subsequently we identified residue 325 as a major determinant in two epitopes linked to a genetic polymorphism with high minor allele freq ...[more]

PMID: 22069278

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Project description:Background and aimsAutoantibodies against the zinc transporter 8 (ZnT8A) are common in type 1 diabetes (T1D). ZnT8A analyses are complicated by the fact that there are three variants of the autoantigen at amino acid position 325 representing ZnT8-R (Arginine), ZnT8-W (Tryptophan) and ZnT8-Q (Glutamin). The aims of the study were: 1) to develop an autoantigen triple mix Radio-Binding Assay (RBA) for ZnT8A; 2) to identify the individual ZnT8-R,-W,-QA reactivity and 3) to validate the triple mix ZnT8A RBA in children with newly diagnosed T1D.MethodsSerum samples were obtained from 2664 (56% males, n=1436) patients in the Swedish nationwide Better Diabetes Diagnosis (BDD) study representing patients with T1D (97%, n=2582), T2D (1.7%, n=46), MODY (1.0%, n=28) and secondary diabetes (0.3%, n=8). cDNA coding for the C-terminal end of each variant was prepared by site-directed mutagenesis and subcloned into a high efficiency in vitro transcription translation vector. The ZnT8 variants were labeled with 35S-methionine and used in a standard RBA separating free from autoantibody-bound autoantigen with Protein A-Sepharose.ResultsZnT8-TripleA was detected in 1678 (65%) patients with T1D, 4 (9%) T2D, 3 (11%) MODY and in none (0%) of the patients with secondary diabetes. Among the T1D patients ZnT8-RA was detected in 1351 (52%) patients, ZnT8-WA in 1209 (47%) and ZnT8-QA in 790 (31%) demonstrating that 1661 (64%) had one or several ZnT8A. The ZnT8-TripleA assay showed a false positive rate of 1.9% (n=49). Only 1.2% (n=32) of the T1D patients were false negative for ZnT8-TripleA compared to 0/46 (0%) of the T2D patients. The precision (intra assay CV) and reproducibility (inter assay CV) of the ZnT8-TripleA assay did not differ from the RBA of the individual ZnT8 variants.ConclusionWe conclude that the ZnT8-TripleA assay had low false positive and false negative rates. The ZnT8-TripleA assay would therefore be highly suitable not only to analyze patient with newly diagnosed diabetes but also for screening the general population since this assay demonstrated high sensitivity and very high specificity.

Project description:BackgroundThe nature of epitopes on Bet v 1 recognized by natural IgG antibodies of birch pollen allergic patients and birch pollen-exposed but non-sensitized subjects has not been studied in detail.ObjectiveTo investigate IgE and IgG recognition of Bet v 1 and to study the effects of natural Bet v 1-specific IgG antibodies on IgE recognition of Bet v 1 and Bet v 1-induced basophil activation.MethodsSera from birch pollen allergic patients (BPA, n = 76), allergic patients without birch pollen allergy (NBPA, n = 40) and non-allergic individuals (NA, n = 48) were tested for IgE, IgG as well as IgG1 and IgG4 reactivity to folded recombinant Bet v 1, two unfolded recombinant Bet v 1 fragments comprising the N-terminal (F1) and C-terminal half of Bet v 1 (F2) and unfolded peptides spanning the corresponding sequences of Bet v 1 and the apple allergen Mal d 1 by ELISA or micro-array analysis. The ability of Bet v 1-specific serum antibodies from non-allergic subjects to inhibit allergic patients IgE or IgG binding to rBet v 1 or to unfolded Bet v 1-derivatives was assessed by competition ELISAs. Furthermore, the ability of serum antibodies from allergic and non-allergic subjects to modulate Bet v 1-induced basophil activation was investigated using rat basophilic leukaemia cells expressing the human FcεRI which had been loaded with IgE from BPA patients.ResultsIgE antibodies from BPA patients react almost exclusively with conformational epitopes whereas IgG, IgG1 and IgG4 antibodies from BPA, NBPA and NA subjects recognize mainly unfolded and sequential epitopes. IgG competition studies show that IgG specific for unfolded/sequential Bet v 1 epitopes is not inhibited by folded Bet v 1 and hence the latter seem to represent cryptic epitopes. IgG reactivity to Bet v 1 peptides did not correlate with IgG reactivity to the corresponding Mal d 1 peptides and therefore does not seem to be a result of primary sensitization to PR10 allergen-containing food. Natural Bet v 1-specific IgG antibodies inhibited IgE binding to Bet v 1 only poorly and could even enhance Bet v 1-specific basophil activation.ConclusionIgE and IgG antibodies from BPA patients and birch pollen-exposed non-sensitized subjects recognize different epitopes. These findings explain why natural allergen-specific IgG do not protect against allergic symptoms and suggest that allergen-specific IgE and IgG have different clonal origin.

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Mapping of conformational autoantibody epitopes in ZNT8.

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Mapping of conformational autoantibody epitopes in ZNT8.

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