Natalia I Faccinetti, Luciano L Guerra, Alberto Penas Steinhardt, Ruben F Iacono, Gustavo D Frechtel1, Liliana Trifone1, Edgardo Poskus, Aldana Trabucchi, Silvina N Valdez. 1. Chair of ImmunologySchool of Pharmacy and Biochemistry, University of Buenos Aires (UBA), and 'Prof. Ricardo A. Margni' Humoral Immunity Studies Institute (IDEHU), National Research Council (CONICET)-UBA, Ciudad Autónoma de Buenos Aires, ArgentinaGenetic DivisionClinical Hospital, UBA, Genetic, Immunology, Metabolism Institute (INIGEM), CONICET-UBA, Ciudad Autónoma de Buenos Aires, ArgentinaDiabetes and Nutrition ServiceNational Pediatric Hospital 'Dr. Ricardo Gutierrez', Ciudad Autónoma de Buenos Aires, Argentina.
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 β-cell autoantibodies were also tested by RBA. RESULTS: Of the 100 Argentinian type 1 diabetic patients, 65 were ZnT8A+. 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 ZnT8A+, 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 β-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.
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 diabeticpatients were subjected to analysis using [(35)S]-ZnT8 variants by radioligand binding assay (RBA): i) 100 new-onset, insulin-dependent, type 1 diabeticpatients and ii) 282 slowly progressing to insulin requirement, non-obese adult-onset diabeticpatients. In addition, 50 type 1 diabeticpatients 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 β-cell autoantibodies were also tested by RBA. RESULTS: Of the 100 Argentinian type 1 diabeticpatients, 65 were ZnT8A+. 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 diabeticpatients 46 were ZnT8A+, 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 diabeticpatients. 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 β-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 diabeticpatients suggest the different nature of autoimmune processes underlying both pathologies.
Authors: Natalia I Faccinetti; Luciano L Guerra; Adriana V Sabljic; Silvina S Bombicino; Bruno D Rovitto; Ruben F Iacono; Edgardo Poskus; Aldana Trabucchi; Silvina N Valdez Journal: Microb Cell Fact Date: 2017-11-13 Impact factor: 5.328