Characterization of zinc transporter 8 (ZnT8) antibodies in autoimmune diabetic patients from Argentinian population using monomeric, homodimeric, and heterodimeric ZnT8 antigen variants

Abstract

Objective: In order to gain further knowledge of the structure of zinc transporter 8 (ZnT8) epitopes, we studied the role of the amino acid at position 325 in the antigen and its dimeric conformation for autoantibodies to ZnT8 (ZnT8A) recognition. Methods: For this purpose, several ZnT8 C-terminal domain variants were designed: monomer carrying Arg325 or Trp325, homo-dimers ZnT8-Arg-Arg325 and ZnT8-Trp-Trp325, and hetero-dimer ZnT8-Arg-Trp325. Two groups of Argentinian diabetic patients were subjected to analysis using [ 35 S]-ZnT8 variants by radioligand binding assay (RBA): i) 100 new-onset, insulin-dependent, type 1 diabetic patients and ii) 282 slowly progressing to insulin requirement, non-obese adult-onset diabetic patients. In addition, 50 type 1 diabetic patients and 100 normal control sera provided by the American Diabetes Association (ADA) were evaluated in order to calculate the sensitivity and specificity of ZnT8A assays for each antigenic variant. Other routine b-cell autoantibodies were also tested by RBA. Results: Of the 100 Argentinian type 1 diabetic patients, 65 were ZnT8AC. Out of them, 8 patients recognized all recombinant forms of ZnT8 and most patients (56) reacted against the heterodimer. Additionally, out of 282 non-obese adultonset diabetic patients 46 were ZnT8AC, whereas 29 patients recognized only dimers. Besides, exclusive reactivity against ZnT8A was found in 9.0% for type 1 diabetes mellitus and 10.3% for non-obese adult-onset diabetic patients. Conclusions: Significantly higher signal values in RBA were obtained with the heterodimeric variant. An increased detection of humoral autoimmunity was found in both groups when ZnT8A was employed in combination with the other b-cell autoantibodies. The inclusion of homodimeric immunoreactive peptides revealed the existence of quaternary structuredefined epitopes probably resembling the actual state of the autoantigen in vivo. Finally, the differential profiles of ZnT8A exhibited by type 1 and non-obese adult-onset diabetic patients suggest the different nature of autoimmune processes underlying both pathologies.

Immunological and clinical characteristics of latent autoimmune diabetes in the elderly

“…The present study shows ZnT8A as the second most prevalent antibody, with a high 6.50% in this particular population of autoimmune diabetes over 65 years of age. Our group had previously determined ZnT8A positivity in another cohort of Argentine patients, which rendered 29 (10.3%) ZnT8‐positive cases out of 282 nonobese adult patients with diabetes . Furthermore, the NIRAD study analysed ZnT8A in 196 patients with adult onset autoimmune diabetes (ADA) previously known to be GADA or IA‐2A‐positive, with results showing 18.6% ZnT8A detection .…”

Prokaryotic expression and characterization of the heterodimeric construction of ZnT8 and its application for autoantibodies detection in diabetes mellitus

“…3 b). Sera from 29 type 1 diabetic patients that scored positive by the reference method, RBA (with [ 35 S]-ZnT8), were used [ 2 , 12 ]. Also, 20 control sera were included in order to set a cut-off value.…”

“…Zinc transporter 8 (ZnT8) is a multipass transmembrane protein which has been identified as a novel autoantigen in patients with diabetes mellitus (DM), such as in type 1 DM and in latent autoimmune diabetes of the adults (LADA) [ 1 , 2 ]. It is a 369 amino acid protein, encoded by the SLC30A8 gene located in the chromosome 8q24.11.…”

“…Wenzlau et al and other authors have reported that ZnT8-reactive sera mostly recognize the C-terminal domain of the molecule (amino acids 268–369) [ 1 , 5 ]. ZnT8A have been detected in more than 60% of patients with type 1 DM [ 1 , 2 , 6 – 11 ] and in more than 10% of adult-onset diabetic patients [ 2 , 8 , 12 ], increasing the sensitivity in the screening of autoimmunity. Therefore, ZnT8A constitutes an additional prevalent marker to the preexisting triad, enabling the appropriate classification of diabetic patients as autoimmune DM and improving the efficacy of treatment.…”

“…We have previously characterized ZnT8A from type 1 diabetic patients and non-obese adult-onset diabetic patients employing different ZnT8 antigenic variants, since the C-terminal domain of the ZnT8 include the variant residue at amino acid 325. In that work, we concluded that the heterodimeric construction ZnT8-Arg-Trp325 showed higher prevalence and signal levels and increased dynamic range in RBA for ZnT8A detection in both group of patients, therefore we decided to continue working with this antigenic construction [ 2 ]. Although there are commercial ELISA kits for ZnT8A detection, most of them do not mention their specificity and sensitivity parameters.…”