IDEHU   05542
INSTITUTO DE ESTUDIOS DE LA INMUNIDAD HUMORAL PROF. RICARDO A. MARGNI
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Prokariotic expression of major autoantigens from Diabetes Mellitus fused to thioredoxin: IA-2 and ZnT8
Autor/es:
GUERRA, L. L.; FACCINETTI, N. I.; ROVITTO, B.D.; IACONO, R. F.; POSKUS, E.; TRABUCCHI, A.; VALDEZ, S.N.
Lugar:
Medellín
Reunión:
Congreso; 11° Congress of the Latin American Association of Immunology & 10° Colombian Congress of Allergy, Asthma and Immunology; 2015
Institución organizadora:
Latin American Association of Immunology
Resumen:
Most patients with Type 1 Diabetes Mellitus (T1DM) present autoantibodies to several pancreatic beta-cell proteins, including IA-2, glutamic acid decarboxylase (GAD), insulin/proinsulin and zinc transporter 8 (ZnT8). The measurement of autoantibodies directed against IA-2 (IA-2A), GAD (GADA), insulin/proinsulin (IAA/PAA) and ZnT8 (ZnT8A) is a powerful analytical tool for early diagnosis and classification of several forms of DM. They are usually assessed by Radioligand Binding Assay (RBA), still considered the reference method. However, this methodology requires radioactive tracers employing [125I] or [35S]-methionine, making it environmentally inappropriate, expensive and limited to authorized laboratories.The neuroendocrine antigen IA-2 is one of the major autoantigen in T1DM. It is a 106 kDa transmembrane protein expressed in neural, pituitary and pancreatic beta-cells. IA-2 is an enzymatically inactive member of the tyrosine phosphatase family, involved in regulating insulin secretion. IA-2 native conformation is essential for IA-2A detection because they are mostly directed against discontinuous epitopes, which are mainly confined to the intracytoplasmic moiety (residues 604?979). Assessment of IA-2A presence contributes to predicting the likelihood of developing T1DM. Approximately, 65% of recent-onset T1DM patients are IA-2A positive. On the other hand, Zinc transporter 8 (ZnT8) is a multipass transmembrane protein which has been recently identified as a novel autoantigen in patients with autoimmune DM. It is a 369 amino acid protein, encoded by the SLC30A8 gene located in the chromosome 8q24.11. ZnT8 transports zinc ions from the beta-cell cytoplasm into insulin-secretory vesicles where they are essential for the proper storage and secretion of insulin. The expression of ZnT8 is remarkably restricted, being almost exclusively confined to pancreatic beta-cells. ZnT8A have been detected in more than 60% of patients with T1DM and in more than 10% of adult-onset diabetic patients. ZnT8-reactive sera mostly recognize the C-terminal domain of the molecule (amino acids 268-369), where a variant residue at amino acid 325 is located. This non-synonymous single nucleotide polymorphism changes Arginine (Arg) to Tryptophan (Trp). Previous studies have demonstrated that ZnT8A positive patients display specific immunoreactivities against the hybrid dimeric construction ZnT8-Arg-Trp325.The aim of this study was to express and recover properly folded recombinant IA-2 and ZnT8 from Escherichia coli, applicable to the development of non-radiometric immunoassays for autoantibodies detection.The codon-optimized sequences coding for the intracellular domain of IA-2 (IA-2ic) and the C-terminal region of ZnT8 were each cloned into pTrxFus vector in order to obtain them fused to thioredoxin (TrxIA-2ic and TrxZnT8, respectively). GI724 and GI698 E. coli strains were transformed either with pTrxIA-2ic or pTrxZnT8, and cultured at 30°C. Protein expression was induced with Trp 100 µg/mL at 37°C for GI724 or 20°C for GI698.After expression induction at different culture times, cells lysates from both strains were obtained. TrxIA-2ic and TrxZnT8 were purified by affinity chromatography from the intracellular soluble fraction (ISF). The expression and purification steps were analyzed by SDS-PAGE and Western Blot (WB) using a polyclonal serum to thioredoxin (Trx). These techniques revealed bands compatible with TrxIA-2ic and TrxZnT8 expected theoretical molecular weights (~55.4 and ~37.5 kDa, respectively) from both bacteria strains.The highest TrxIA-2ic expression was achieved in E. coli GI724, yielding 9 mg from ISF/L culture, whereas TrxZnT8 purification from ISF was achieved from strain GI698, yielding 2 mg TrxZnT8/L culture. The purified fusions proteins were subjected to proteolytic digestion with trypsin and chymotrypsin. The peptide profile obtained after digestion was analyzed by Orbitrap mass spectrometry, achieving 63.25% and 73.61% coverage for TrxIA-2ic and TrxZnT8 sequences, respectively.The immunochemical ability of TrxIA-2ic and TrxZnT8 to compete with [35S]IA-2 or [35S]ZnT8 (synthesized with rabbit reticulocyte lysate system) were assessed qualitatively by incubating 30 IA-2A or ZnT8A positive patients sera in the presence of 0.2 μM TrxIA-2ic or 0.7 μM TrxZnT8. Results were expressed as standard deviation scores(SDs). All sera became virtually negative under antigen excess (mean SDs changed from 17.07 to 0.34 for TrxIA-2ic, and 44.88 to 0.78 for TrxZnT8).Radiometric quantitative competition assays with 4 IA-2A and 5 ZnT8A positive patients sera were performed by adding TrxIA-2ic (31.0 pM to 0.6 µM) or TrxZnT8 (37.0 pM to 0.7 µM), using [35S]IA-2 or [35S]ZnT8. All dose-response curves showed similar protein concentration that caused 50% inhibition (53.3 to 1.1 nM for TrxIA-2ic and 41.0 to 1.4 nM for TrxZnT8).Recombinant IA-2 and ZnT8 were successfully expressed and purified from E. coli as fusion proteins with Trx. They were both purified from ISF by affinity chromatography. We identify them and demonstrate their proper immunochemical behaviour by displacement of radiochemical tracer-binding to autoantibodies.The availability of immunochemically competent IA-2 and ZnT8 would encourage researchers to improve current and developed novel, low-cost immunoassays for massive screening of individuals at risk of T1DM.