RESUMO
ICA512/PTPRN is a receptor tyrosine-like phosphatase implicated in the biogenesis and turnover of the insulin secretory granules (SGs) in pancreatic islet beta cells. Previously we found biophysical evidence that its luminal RESP18 homology domain (RESP18HD) forms a biomolecular condensate and interacts with insulin in vitro at close-to-neutral pH, that is, in conditions resembling those present in the early secretory pathway. Here we provide further evidence for the relevance of these findings by showing that at pH 6.8 RESP18HD interacts also with proinsulin-the physiological insulin precursor found in the early secretory pathway and the major luminal cargo of ß-cell nascent SGs. Our light scattering analyses indicate that RESP18HD and proinsulin, but also insulin, populate nanocondensates ranging in size from 15 to 300 nm and 10e2 to 10e6 molecules. Co-condensation of RESP18HD with proinsulin/insulin transforms the initial nanocondensates into microcondensates (size >1 µm). The intrinsic tendency of proinsulin to self-condensate implies that, in the ER, a chaperoning mechanism must arrest its spontaneous intermolecular condensation to allow for proper intramolecular folding. These data further suggest that proinsulin is an early driver of insulin SG biogenesis, in a process in which its co-condensation with RESP18HD participates in their phase separation from other secretory proteins in transit through the same compartments but destined to other routes. Through the cytosolic tail of ICA512, proinsulin co-condensation with RESP18HD may further orchestrate the recruitment of cytosolic factors involved in membrane budding and fission of transport vesicles and nascent SGs.
Assuntos
Insulina , Proinsulina , Insulina/química , Proinsulina/análise , Proinsulina/química , Proinsulina/metabolismo , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/análise , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/metabolismo , Vesículas Secretórias/química , Vesículas Secretórias/metabolismoRESUMO
Type 1 diabetes islet cell autoantigen 512 (ICA512/IA-2) is a tyrosine phosphatase-like intrinsic membrane protein involved in the biogenesis and turnover of insulin secretory granules (SGs) in pancreatic islet ß-cells. Whereas its membrane-proximal and cytoplasmic domains have been functionally and structurally characterized, the role of the ICA512 N-terminal segment named "regulated endocrine-specific protein 18 homology domain" (RESP18HD), which encompasses residues 35-131, remains largely unknown. Here, we show that ICA512 RESP18HD residues 91-131 encode for an intrinsically disordered region (IDR), which in vitro acts as a condensing factor for the reversible aggregation of insulin and other ß-cell proteins in a pH and Zn2+-regulated fashion. At variance with what has been shown for other granule cargoes with aggregating properties, the condensing activity of ICA512 RESP18HD is displayed at a pH close to neutral, i.e. in the pH range found in the early secretory pathway, whereas it is resolved at acidic pH and Zn2+ concentrations resembling those present in mature SGs. Moreover, we show that ICA512 RESP18HD residues 35-90, preceding the IDR, inhibit insulin fibrillation in vitro Finally, we found that glucose-stimulated secretion of RESP18HD upon exocytosis of SGs from insulinoma INS-1 cells is associated with cleavage of its IDR, conceivably to prevent its aggregation upon exposure to neutral pH in the extracellular milieu. Taken together, these findings point to ICA512 RESP18HD being a condensing factor for protein sorting and granulogenesis early in the secretory pathway and for prevention of amyloidogenesis.
Assuntos
Amiloide/metabolismo , Insulina/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/metabolismo , Amiloide/genética , Animais , Linhagem Celular Tumoral , Humanos , Concentração de Íons de Hidrogênio , Insulina/genética , Proteínas Intrinsicamente Desordenadas/genética , Proteínas do Tecido Nervoso/genética , Ratos , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/genética , Zinco/metabolismoRESUMO
There is a scarcity of data of zinc transporter-8 autoantibody (ZnT8A) on mixed populations such as Brazilian. Therefore, we evaluated the relevance of ZnT8A for type 1 diabetes (T1D) diagnosis and the role of ZnT8 coding gene (SLC30A8) in T1D predisposition. Patients with T1D (n = 629; diabetes duration = 11 (6-16) years) and 651 controls were genotyped for SLC30A8 rs16889462 and rs2466295 variants (BeadXpress platform). ZnT8 triple antibody was measured by ELISA; glutamic acid decarboxylase (GAD65A) and protein tyrosine phosphatase (IA-2A) autoantibodies by radioimmunoassay. RESULTS: Znt8A was detected in 68.7% of recent-onset T1D patients and 48.9% of the entire patient cohort, similar to GAD65A (68.3% and 47.2%) and IA-2A (64.8% and 42.4%) positivities respectively. ZnT8A was the only antibody in 8.4% of patients. Znt8A and IA2A frequencies and titers were independent of gender and ethnicity, whereas GAD65A titers were greater in females. The diabetes duration-dependent decline in ZnT8A frequency was similar to GAD65A and IA-2A. The SLC30A8 rs2466293 AG + GG genotypes were associated with T1D risk in non-European descents (56.2% × 42.9%; p = 0.018), and the GG genotype with higher ZnT8A titers in recent-onset T1D: 834.5 IU/mL (711.3-2190.0) × 281 IU/mL (10.7-726.8); p = 0.027. Conclusion ZnT8A detection increases T1D diagnosis rate even in mixed populations. SLC30A8 rs2466293 was associated with T1D predisposition in non-European descents.
Assuntos
Autoanticorpos/metabolismo , Diabetes Mellitus Tipo 1/diagnóstico , Polimorfismo de Nucleotídeo Único , Transportador 8 de Zinco/genética , Transportador 8 de Zinco/imunologia , Adolescente , Adulto , Brasil/etnologia , Estudos de Coortes , Diabetes Mellitus Tipo 1/etnologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Glutamato Descarboxilase/imunologia , Humanos , Masculino , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/imunologia , População Branca/genética , Adulto JovemRESUMO
BACKGROUND: The insulinoma associated protein tyrosine phosphatase 2 (IA-2) is one of the immunodominant autoantigens involved in the autoimmune attack to the beta-cell in Type 1 Diabetes Mellitus. In this work we have developed a complete and original process for the production and recovery of the properly folded intracellular domain of IA-2 fused to thioredoxin (TrxIA-2ic) in Escherichia coli GI698 and GI724 strains. We have also carried out the biochemical and immunochemical characterization of TrxIA-2icand design variants of non-radiometric immunoassays for the efficient detection of IA-2 autoantibodies (IA-2A). RESULTS: The main findings can be summarized in the following statements: i) TrxIA-2ic expression after 3 h of induction on GI724 strain yielded ≈ 10 mg of highly pure TrxIA-2ic/L of culture medium by a single step purification by affinity chromatography, ii) the molecular weight of TrxIA-2ic (55,358 Da) could be estimated by SDS-PAGE, size exclusion chromatography and mass spectrometry, iii) TrxIA-2ic was properly identified by western blot and mass spectrometric analysis of proteolytic digestions (63.25 % total coverage), iv) excellent immunochemical behavior of properly folded full TrxIA-2ic was legitimized by inhibition or displacement of [35S]IA-2 binding from IA-2A present in Argentinian Type 1 Diabetic patients, v) great stability over time was found under proper storage conditions and vi) low cost and environmentally harmless ELISA methods for IA-2A assessment were developed, with colorimetric or chemiluminescent detection. CONCLUSIONS: E. coli GI724 strain emerged as a handy source of recombinant IA-2ic, achieving high levels of expression as a thioredoxin fusion protein, adequately validated and applicable to the development of innovative and cost-effective immunoassays for IA-2A detection in most laboratories.
Assuntos
Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/imunologia , Escherichia coli/metabolismo , Engenharia de Proteínas/métodos , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/imunologia , Tiorredoxinas/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Diabetes Mellitus Tipo 1/sangue , Escherichia coli/genética , Feminino , Humanos , Testes Imunológicos/métodos , Masculino , Pessoa de Meia-Idade , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Tiorredoxinas/genética , Adulto JovemRESUMO
OBJECTIVE: While it is known that there is progression to diabetes in <10 years in 70% of children with two or more islet autoantibodies, predictors of the progression to diabetes are only partially defined. RESEARCH DESIGN AND METHODS: The Environmental Determinants of Diabetes in the Young (TEDDY) study has observed 8,503 children who were at increased genetic risk for autoimmune diabetes. Insulin autoantibodies (IAAs), GAD65 autoantibodies (GADAs), and insulinoma-associated protein 2 autoantibodies (IA-2As) were measured every 3 months until 4 years of age and every 6 months thereafter; if results were positive, the autoantibodies were measured every 3 months. RESULTS: Life table analysis revealed that the cumulative incidence of diabetes by 5 years since the appearance of the first autoantibody differed significantly by the number of positive autoantibodies (47%, 36%, and 11%, respectively, in those with three autoantibodies, two autoantibodies, and one autoantibody, P < 0.001). In time-varying survival models adjusted for first-degree relative status, number of autoantibodies, age at first persistent confirmed autoantibodies, and HLA genotypes, higher mean IAA and IA-2A levels were associated with an increased risk of type 1 diabetes in children who were persistently autoantibody positive (IAAs: hazard ratio [HR] 8.1 [95% CI 4.6-14.2]; IA-2A: HR 7.4 [95% CI 4.3-12.6]; P < 0.0001]). The mean GADA level did not significantly affect the risk of diabetes. CONCLUSIONS: In the TEDDY study, children who have progressed to diabetes usually expressed two or more autoantibodies. Higher IAA and IA-2A levels, but not GADA levels, increased the risk of diabetes in those children who were persistently autoantibody positive.
Assuntos
Autoanticorpos/metabolismo , Diabetes Mellitus Tipo 1/imunologia , Anticorpos Anti-Insulina/metabolismo , Ilhotas Pancreáticas/imunologia , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/imunologia , Criança , Pré-Escolar , Progressão da Doença , Feminino , Genótipo , Glutamato Descarboxilase/imunologia , Antígenos HLA-DQ/imunologia , Antígenos HLA-DR/imunologia , Humanos , Lactente , Masculino , Fatores de RiscoRESUMO
Phogrin/IA-2ß and ICA512/IA-2 are two paralogs receptor-type protein-tyrosine phosphatases (RPTP) that localize in secretory granules of various neuroendocrine cells. In pancreatic islet ß-cells, they participate in the regulation of insulin secretion, ensuring proper granulogenesis, and ß-cell proliferation. The role of their cytoplasmic tail has been partially unveiled, while that of their luminal region remains unclear. To advance the understanding of its structure-function relationship, the X-ray structure of the mature ectodomain of phogrin (ME phogrin) at pH 7.4 and 4.6 has been solved at 1.95- and 2.01-Å resolution, respectively. Similarly to the ME of ICA512, ME phogrin adopts a ferredoxin-like fold: a sheet of four antiparallel ß-strands packed against two α-helices. Sequence conservation among vertebrates, plants and insects suggests that the structural similarity extends to all the receptor family. Crystallized ME phogrin is monomeric, in agreement with solution studies but in striking contrast with the behavior of homodimeric ME ICA512. The structural details that may cause the quaternary structure differences are analyzed. The results provide a basis for building models of the overall orientation and oligomerization state of the receptor in biological membranes.
Assuntos
Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/química , Sequência de Aminoácidos , Sítios de Ligação/genética , Cristalografia por Raios X , Concentração de Íons de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Dobramento de Proteína , Multimerização Proteica , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/metabolismo , Homologia de Sequência de Aminoácidos , Soluções , Relação Estrutura-AtividadeRESUMO
The receptor-type protein-tyrosine phosphatase (RPTP) phogrin is localized at the membrane of secretory granules of pancreatic islet ß-cells and, similarly to the closely related ICA512, plays a role in the regulation of insulin secretion, in ensuring proper granulogenesis and stability, and in the regulation of ß-cell growth. The mature membraneproximal ectodomain of phogrin (MPE phogrin) was produced as a recombinant protein and characterized. CD, fluorescence, controlled proteolysis, size-exclusion chromatography, and multi-angle light scattering showed that it is a properlyfolded monomeric domain. Equilibrium experiments, in the presence of guanidinium chloride and thermal unfolding, suggest a two-state mechanism with a ΔG of 2.3-3.3 kcal/mol, respectively. The study establishes common features and differences of MPE phogrin and the homologous ectodomain of ICA512. A homology model of phogrin was built based in the x-ray structure of MPE ICA512. The model is a starting point for modeling the entire receptor and for testing the quaternary structure and interactions of this protein in vivo. A description of the membrane insertion mode and putative interacting surfaces of this large protein is fundamental for the understanding of its biological function.
Assuntos
Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/química , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/metabolismo , Animais , Dicroísmo Circular , Camundongos , Modelos Moleculares , Estrutura Terciária de Proteína , Desdobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TermodinâmicaRESUMO
Changes in the clinical presentation of diabetes mellitus in childhood and adolescence associated with obesity have resulted in an overlap of the two most common types of diabetes with a greater clinical heterogeneity. In order to characterize the type of diabetes at onset and assess the effect of obesity, 50 children with diabetes were studied. The patients were divided into two groups according to their nutritional status at diagnosis (over-weight/obese vs. normal weight). Insulin reserve was evaluated by measuring basal C-peptide and stimulated C-peptide in response to a mixed meal (MMTT) as well as HLA-DQB1 genotype, antibodies, and family history of risk factors for metabolic disease. Of all 50 patients, 38% was overweight/obese, 84% had a positive family history of metabolic syndrome, 82% had positive antibodies, and 100% were positive for the high-risk HLA-DQB1 genotype. No significant differences were found in fasting C-peptide or glycemic index/C-peptide levels between the two groups. In the overweight/obese group C-peptide response to MMTT showed higher levels at 60 and 120 minutes (p = 0.02 and 0.03) and the area under the curve for C-peptide was also higher (1.77 ng / ml vs. 5.5 ng/ ml, p = 0.0007) than in the normal-weight group. In conclusion, overweight/obese patients with type 1A diabetes had a greater pancreatic reserve, suggesting that nutritional status may accelerate disease onset.
Assuntos
Peptídeo C/sangue , Diabetes Mellitus Tipo 1/diagnóstico , Adolescente , Autoimunidade , Biomarcadores/sangue , Distribuição de Qui-Quadrado , Criança , Diabetes Mellitus Tipo 1/genética , Feminino , Genótipo , Glutamato Descarboxilase/sangue , Cadeias beta de HLA-DQ/sangue , Humanos , Anticorpos Anti-Insulina/sangue , Masculino , Síndrome Metabólica/complicações , Obesidade/complicações , Estudos Prospectivos , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/sangue , Fatores de RiscoRESUMO
PURPOSE: The objective of this study was to determine the frequencies of autoantibodies to heterogeneous islet-cell cytoplasmic antigens (ICA), glutamic acid decarboxylase(65) (GAD(65)A), insulinoma-associated antigen-2 (IA-2A) and insulin (IAA)-and human leukocyte antigen (HLA) class II markers (HLA-DR and -DQ) in first degree relatives of heterogeneous Brazilian patients with type I diabetes (T1DM). A major focus of this study was to determine the influence of age, gender, proband characteristics and ancestry on the prevalence of autoantibodies and HLA-DR and -DQ alleles on disease progression and genetic predisposition to T1DM among the first-degree relatives. METHODS: IAA, ICA, GAD(65)A, IA-2A and HLA- class II alleles were determined in 546 first-degree-relatives, 244 siblings, 55 offspring and 233 parents of 178 Brazilian patients with T1DM. RESULTS: Overall, 8.9% of the relatives were positive for one or more autoantibodies. IAA was the only antibody detected in parents. GAD(65) was the most prevalent antibody in offspring and siblings as compared to parents and it was the sole antibody detected in offspring. Five siblings were positive for the IA-2 antibody. A significant number (62.1%) of siblings had 1 or 2 high risk HLA haplotypes. During a 4-year follow-up study, 5 siblings (expressing HLA-DR3 or -DR4 alleles) and 1 offspring positive for GAD(65)A progressed to diabetes. CONCLUSIONS: The data indicated that the GAD(65) and IA-2 antibodies were the strongest predictors of T1DM in our study population. The high risk HLA haplotypes alone were not predictive of progression to overt diabetes.
Assuntos
Autoanticorpos/sangue , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Adolescente , Adulto , Fatores Etários , Idoso , Autoanticorpos/imunologia , Biomarcadores/sangue , Brasil , Criança , Pré-Escolar , Progressão da Doença , Família , Feminino , Predisposição Genética para Doença , Genótipo , Glutamato Descarboxilase/imunologia , Antígenos HLA-DQ/imunologia , Antígenos HLA-DR/imunologia , Humanos , Lactente , Insulina/imunologia , Masculino , Pessoa de Meia-Idade , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/imunologia , Fatores Sexuais , Adulto JovemAssuntos
Síndrome Antifosfolipídica/imunologia , Autoanticorpos/sangue , Glutamato Descarboxilase/imunologia , Insulina/imunologia , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/imunologia , Adulto , Síndrome Antifosfolipídica/sangue , Brasil , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RadioimunoensaioRESUMO
ICA512 (or IA-2) is a transmembrane protein-tyrosine phosphatase located in secretory granules of neuroendocrine cells. Initially, it was identified as one of the main antigens of autoimmune diabetes. Later, it was found that during insulin secretion, the cytoplasmic domain of ICA512 is cleaved and relocated to the nucleus, where it stimulates the transcription of the insulin gene. The role of the other parts of the receptor in insulin secretion is yet to be unveiled. The structures of the intracellular pseudocatalytic and mature extracellular domains are known, but the transmembrane domain and several intracellular and extracellular parts of the receptor are poorly characterized. Moreover the overall structure of the receptor remains to be established. We started to address this issue studying by X-ray crystallography the structure of the mature ectodomain of ICA512 (ME ICA512) and variants thereof. The variants and crystallization conditions were chosen with the purpose of exploring putative association interfaces, metal binding sites and all other structural details that might help, in subsequent works, to build a model of the entire receptor. Several structural features were clarified and three main different association modes of ME ICA512 were identified. The results provide essential pieces of information for the design of new experiments aimed to assess the structure in vivo.
Assuntos
Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/química , Sítios de Ligação , Cálcio/química , Núcleo Celular/metabolismo , Cristalização , Cristalografia por Raios X/métodos , DNA/metabolismo , Dimerização , Humanos , Concentração de Íons de Hidrogênio , Insulina/química , Modelos Moleculares , Conformação Molecular , Mapeamento de Interação de Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Solventes/química , Propriedades de SuperfícieRESUMO
IA-2 (insulinoma-associated protein 2) is a protein-tyrosine phosphatase receptor located in secretory granules of neuroendocrine cells. Initially, it attracted attention due to its involvement in the autoimmune response associated to diabetes. Later it was found that upon exocytosis, the cytoplasmic domain of IA-2 is cleaved and relocated to the nucleus, where it enhances the transcription of the insulin gene. A concerted functioning of the whole receptor is to be expected. However, very little is known about the structure and function of the transmembrane and extracellular domains of IA-2. To address this issue, we solved the x-ray structure of the mature ectodomain of IA-2 (meIA-2) to 1.30A resolution. The fold of meIA-2 is related to the SEA (sea urchin sperm protein, enterokinase, agrin)) domains of mucins, suggesting its participation in adhesive contacts to the extracellular matrix and providing clues on how this kind of molecule may associate and form homo- and heterodimers. Moreover, we discovered that meIA-2 is self-proteolyzed in vitro by reactive oxygen species, suggesting the possibility of a new shedding mechanism that might be significant in normal function or pathological processes. Knowledge of meIA-2 structure should facilitate the search of its possible ligands and molecular interactions.
Assuntos
Modelos Moleculares , Dobramento de Proteína , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/química , Autoimunidade/imunologia , Cristalografia por Raios X , Diabetes Mellitus/genética , Diabetes Mellitus/imunologia , Exocitose/genética , Exocitose/imunologia , Matriz Extracelular , Humanos , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/imunologia , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Homologia Estrutural de Proteína , Relação Estrutura-AtividadeRESUMO
The receptor protein tyrosine phosphatase superfamily (RPTP) includes proteins with a single transmembrane, one or more intracellular phosphatase, and a variety of extracellular domains. The 106-kDa insulinoma-associated protein (IA-2, ICA512) receptor is unique among RPTP members because: (a) it has a single, phosphatase-like intracellular domain identified as one of the most prominent self antigens in autoimmune diabetes; (b) its extracellular region bears no sequence similarity to known domains; (c) it is present in the membrane of secretory granules in neurons and pancreatic beta-cells where it suffers a complex processing; and (d) it has very poorly understood biological properties. In this work, we describe the expression, purification, and physicochemical characterization of residues 449-576 of IA-2 (IA-2ec(449-576)). Judging from CD, fluorescence, hydrodynamic, and thermal unfolding analyses, this fragment forms an autonomously folding unit with tight packing and well-defined secondary and tertiary structure. CD analysis suggests that about 25% of IA-2ec(449-576) residues are alpha-helical, whereas about the same amount are in beta-sheet structure. The availability of soluble and folded IA-2ec(449-576) is a step forward toward the characterization of a part of IA-2 at atomic detail, which may provide new insight in the biology of diabetes, the neurotransmission process, and the dynamic of secretory granules.
Assuntos
Autoantígenos/biossíntese , Autoantígenos/química , Proteínas de Membrana/biossíntese , Proteínas de Membrana/química , Proteínas Tirosina Fosfatases/biossíntese , Proteínas Tirosina Fosfatases/química , Proteínas Recombinantes/biossíntese , Sequência de Aminoácidos , Autoantígenos/genética , Escherichia coli/metabolismo , Humanos , Proteínas de Membrana/genética , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
The intracellular domain of insulinoma-associated protein (IA-2), IA-2ic, is a prominent antigen in autoimmune diabetes, and autoantibodies to it are early markers of the disease. The high-yield expression of properly folded IA-2ic is needed for basic research and crucial for low-cost immunoassays aimed at the detection of these autoantibodies in diagnostic and preventive medicine. In previous work, the expression of IA-2ic fused to glutathione S-transferase or to a biotinylatable peptide was reported; however, these methods had very poor yield. Here we show that, utilizing a codon-optimized gene, up to 80 mg of pure and properly folded autoantigen per litre of Escherichia coli culture may be obtained. Furthermore, the addition of a C-terminal His-tag greatly facilitates IA-2ic purification without compromising either its immunoreactivity or its expression yield. To take advantage of the recombinant antigen, an enzyme immunoassay format was developed which proved to be highly specific and sensitive.