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.
Eur J Endocrinol. 2016 Feb;174(2):157-65. doi: 10.1530/EJE-15-0681. Epub 2015 Nov 13.
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.
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.
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.
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.
为了深入了解锌转运体 8（ZnT8）表位的结构，我们研究了位置 325 氨基酸在抗原中的作用及其二聚体构象对 ZnT8 自身抗体（ZnT8A）识别的影响。
为此，我们设计了几种 ZnT8 末端结构域变体：单体携带 Arg325 或 Trp325，同源二聚体 ZnT8-Arg-Arg325 和 ZnT8-Trp-Trp325，以及异源二聚体 ZnT8-Arg-Trp325。两组阿根廷糖尿病患者通过放射性配体结合分析（RBA）使用 [（35）S]-ZnT8 变体进行分析：i）100 名新诊断的胰岛素依赖型 1 型糖尿病患者和 ii）282 名进展缓慢至胰岛素需求的非肥胖成年起病型糖尿病患者。此外，还评估了美国糖尿病协会（ADA）提供的 50 名 1 型糖尿病患者和 100 名正常对照血清，以计算每种抗原变体的 ZnT8A 检测的敏感性和特异性。还通过 RBA 测试了其他常规β细胞自身抗体。
在 100 名阿根廷 1 型糖尿病患者中，有 65 名患者为 ZnT8A+。其中，8 名患者识别所有重组形式的 ZnT8，大多数患者（56 名）对异源二聚体反应。此外，在 282 名非肥胖成年起病型糖尿病患者中，有 46 名患者为 ZnT8A+，而 29 名患者仅对二聚体有反应。此外，在 1 型糖尿病患者中，ZnT8A 的特异性反应为 9.0%，在非肥胖成年起病型糖尿病患者中为 10.3%。
RBA 中获得的异源二聚体变体信号值显著更高。当 ZnT8A 与其他β细胞自身抗体联合使用时，在两组中均发现了体液自身免疫的检测增加。包含同型二聚体免疫反应性肽揭示了可能类似于体内自身抗原实际状态的四级结构定义表位的存在。最后，1 型和非肥胖成年起病型糖尿病患者表现出的 ZnT8A 不同特征表明，这两种疾病的自身免疫过程的性质不同。
