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

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 [35S]-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 adult-onset 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 structure-defined 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. Fil: Faccinetti, Natalia Ines. Consejo Nacional de Investigaciones Cientiâ­ficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; ArgentinaFil: Guerra, Luciano Lucas. Consejo Nacional de Investigaciones Cientiâ­ficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; ArgentinaFil: Penas Steinhardt, Alberto. Consejo Nacional de Investigaciones Cientiâ­ficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; ArgentinaFil: Iacono, Ruben Francisco. Consejo Nacional de Investigaciones Cientiâ­ficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; ArgentinaFil: Frechtel, Gustavo D.. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo; ArgentinaFil: Trifone, Liliana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutierrez"; ArgentinaFil: Poskus, Edgardo. Consejo Nacional de Investigaciones Cientiâ­ficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; ArgentinaFil: Trabucchi, Aldana. Consejo Nacional de Investigaciones Cientiâ­ficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; ArgentinaFil: Valdez, Silvina Noemi. Consejo Nacional de Investigaciones Cientiâ­ficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; Argentin