Biochemical, biophysical, and functional properties of ICA512/IA-2 RESP18 homology domain.

BACKGROUND: ICA512 (or IA-2/PTPRN) is a transmembrane protein-tyrosine phosphatase located in secretory granules of neuroendocrine cells. Previous studies implied its involvement in generation, cargo storage, traffic, exocytosis and recycling of insulin secretory granules, as well as in ß-cell proliferation. While several ICA512 domains have been characterized, the function and structure of a large portion of its N-terminal extracellular (or lumenal) region are unknown. Here, we report a biophysical, biochemical, and functional characterization of ICA512-RESP18HD, a domain comprising residues 35 to 131 and homologous to regulated endocrine-specific protein 18 (RESP18). METHODS: Pure recombinant ICA512-RESP18HD was characterized by CD and fluorescence. Its binding to insulin and proinsulin was characterized by ELISA, surface plasmon resonance, and fluorescence anisotropy. Thiol reactivity was measured kinetically. Targeting of ΔRESP18HD ICA512-GFP to the membrane of insulinoma cells was monitored by immunofluorescence. RESULTS: ICA512-RESP18HD possesses a strong tendency to aggregate and polymerize via intermolecular disulfide formation, particularly at pH>4.5. Its cysteine residues are highly susceptible to oxidation forming an intramolecular disulfide between cysteine 53 and 62 and intermolecular disulfides via cysteine 40 and cysteine 47. The regulated sorting of ICA512 to secretory granules in INS-1 cells was impaired by deletion of RESP18HD. ICA512-RESP18HD binds with high-affinity to insulin and proinsulin. CONCLUSIONS: RESP18HD is required for efficient sorting of ICA512 to secretory granules. GENERAL SIGNIFICANCE: RESP18HD is a key determinant for ICA512 granule targeting.

Prokaryotic expression and characterization of the heterodimeric construction of ZnT8 and its application for autoantibodies detection in diabetes mellitus.

BACKGROUND: In the present work we described the recombinant production and characterization of heterodimeric construction ZnT8-Arg-Trp325 fused to thioredoxin using a high-performance expression system such as Escherichia coli. In addition, we apply this novel recombinant antigen in a non-radiometric method, with high sensitivity, low operational complexity and lower costs. RESULTS: ZnT8 was expressed in E. coli as a fusion protein with thioredoxin (TrxZnT8). After 3 h for induction, recombinant protein was obtained from the intracellular soluble fraction and from inclusion bodies and purified by affinity chromatography. The expression and purification steps, analyzed by SDS-PAGE and western blot, revealed a band compatible with TrxZnT8 expected theoretical molecular weight (≈ 36.8 kDa). The immunochemical ability of TrxZnT8 to compete with [35S]ZnT8 (synthesized with rabbit reticulocyte lysate system) was assessed qualitatively by incubating ZnT8A positive patient sera in the presence of 0.2-0.3 µM TrxZnT8. Results were expressed as standard deviation scores (SDs). All sera became virtually negative under antigen excess (19.26-1.29 for TrxZnT8). Also, radiometric quantitative competition assays with ZnT8A positive patient sera were performed by adding TrxZnT8 (37.0 pM-2.2 µM), using [35S]ZnT8. All dose-response curves showed similar protein concentration that caused 50% inhibition (14.9-0.15 nM for TrxZnT8). On the other hand, preincubated bridge ELISA for ZnT8A detection was developed. This assay showed 51.7% of sensitivity and 97.1% of specificity. CONCLUSIONS: It was possible to obtain with high-yield purified heterodimeric construction of ZnT8 in E. coli and it was applied in cost-effective immunoassay for ZnT8A detection.

Novel prokaryotic expression of thioredoxin-fused insulinoma associated protein tyrosine phosphatase 2 (IA-2), its characterization and immunodiagnostic application.

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.

X-ray structure of the mature ectodomain of phogrin.

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.

Flow cytometric microsphere-based immunoassay as a novel non-radiometric method for the detection of glutamic acid decarboxylase autoantibodies in type 1 diabetes mellitus.

The first measurable sign of arising autoimmunity in type 1 diabetes mellitus is the detection of autoantibodies against beta-cell antigens, such as glutamic acid decarboxylase (GAD65). GAD65 autoantibodies (GADA) are usually measured by the Radioligand Binding Assay (RBA). The aim of this work was to develop protocols of flow cytometric microsphere-based immunoassays (FloCMIA) which involved glutamic acid decarboxylase fused to thioredoxin (TrxGAD65) adsorbed on polystyrene microspheres. Detection of bound GADA was accomplished by the use of anti-human IgG-Alexa Fluor 488 (protocol A), anti-human IgG-biotin and streptavidin-dichlorotriazinyl aminofluorescein (DTAF) (protocol B) or TrxGAD65-biotin and streptavidin-DTAF (protocol C). Serum samples obtained from 46 patients assayed for routine autoantibodies at Servicios Tecnológicos de Alto Nivel (STAN-CONICET) were analyzed by RBA, ELISA and three alternative FloCMIA designs. Protocol C exhibited the highest specificity (97.8%) and sensitivity (97.4%) and a wide dynamic range (1.00-134.40 SDs). Samples obtained from 40 new-onset diabetic patients were also analyzed to further evaluate the performance of protocol C. The latter protocol showed a sensitivity of 58.6% and a prevalence of 47.5%. Two patients resulted positive only by FloCMIA protocol C and its SDs were higher than those of RBA and ELISA, showing a significantly wide dynamic range. In conclusion, FloCMIA proved to be highly sensitive and specific, requiring a low sample volume; it is environmentally adequate, innovative and represents a cost-effective alternative to traditional GADA determination by RBA and/or ELISA, making it applicable to most medium-complexity laboratories.

Expression and characterization of human proinsulin fused to thioredoxin in Escherichia coli.

Native proinsulin (PI) belongs to the class of the difficult-to-express proteins in Escherichia coli. Problems mainly arise due to its high proteolytic decay and troubles to reproduce the native disulphide pattern. In the present study, human PI was produced in E. coli as a fusion thioredoxin protein (Trx-PI). Such chimeric protein was obtained from the intracellular soluble fraction, and it was purified in one step by affinity chromatography on immobilized phenylarsine oxide. Trx-PI was also recovered from inclusion bodies and purified by anion exchange chromatography. The product identity and integrity were verified by mass analysis (22,173.5 Da) and mapping with Staphylococcus aureus V8 protease. Native PI folding was evaluated by biochemical and also by immunochemical analysis using specific sera from PI antibody-positive diabetic patients that recognise conformational discontinue epitopes. Dose-response curves showed identity between standard PI and Trx-PI. Moreover, surface plasmon resonance technique verified the correct conformation of the recombinant protein. The biochemical and immunochemical assays demonstrated the integrity of the chimera and the epitopes involved in the interaction with antibodies. In conclusion, it was possible to obtain with high-yield purified human PI as a fusion protein in E. coli and useful for analytical purposes.

Association of common variants in JAK2 gene with reduced risk of metabolic syndrome and related disorders.

BACKGROUND: Disturbances in leptin and insulin signaling pathways are related to obesity and metabolic syndrome (MS) with increased risk of diabetes and cardiovascular disease. Janus kinase 2 (JAK2) is a tyrosine kinase involved in the activation of mechanisms that mediate leptin and insulin actions. We conducted a population cross-sectional study to explore the association between two common variants in JAK2 gene and MS related traits in 724 Argentinean healthy male subjects. METHODS: A total of 724 unrelated men aged 37.11 ± 10.91 yr were included in a cross-sectional study. Physical examination, anthropometric measurements and biochemical analysis were determined by a standardized protocol. rs7849191 and rs3780378 were genotyped. Analyses were done separately for each SNP and followed up by haplotype analysis. RESULTS: rs7849191 and rs3780378 were both associated with reduced risk of MS [p = 0.005; OR (95%CI) = 0.52 (0.33-0.80) and p = 0.006; OR (95% CI) = 0.59 (0.40-0.86) respectively, assuming a dominant model]. rs3780378 T allele was associated with triglyceridemia values under 150 mg/dl [p = 0.007; OR (95%CI) = 0.610 (0.429-0.868)] and TT carriers showed lower triglycerides (p = 0.017), triglycerides/HDL-C ratio (p = 0.022) and lipid accumulation product (p = 0.007) compared to allele C carriers. The two-SNPs-haplotype analysis was consistent with single locus analysis. CONCLUSIONS: It was found for the first time, significant associations of JAK2 common variants and related haplotypes with reduced risk of MS. These findings could be explained by the role of JAK2 in insulin and/or leptin signaling.

Novel Flow Cytometric Immunoassay for Detection of Proinsulin Autoantibodies in Diabetes Mellitus Employing a Recombinant Autoantigen Expressed in E. coli.

Introduction: Insulin and proinsulin autoantibodies (IAA/PAA) are usually the first markers to appear in patients with type 1 Diabetes Mellitus (T1DM) and their prevalence ranges from 10 to 60% in the child-adolescent population. The reference method for IAA/PAA detection is the Radioligand Binding Assay (RBA), a highly specific and sensitive technique, but expensive and polluting. The aim of this work was to develop a novel flow cytometric microsphere-based immunoassay (FloCMIA) for PAA detection, employing recombinant human proinsulin (PI), as an alternative method to RBA, less expensive and harmful to the environment. Materials and Methods: Human PI was expressed as Thioredoxin fusion protein (TrxPI) in E. coli and a fraction was biotinylated. A double paratope model was used in which samples were incubated with TrxPI-biotin and microspheres adsorbed with TrxPI. The immune complexes were revealed using Streptavidin-Phycoerythrin. The geometric mean of the signals was analyzed, and the results were expressed as Standard Deviation scores (SDs). Sera from 100 normal human control and from 111 type 1 diabetic patients were evaluated by FloCMIA. To correlate the novel assay with RBA, 51 diabetic patients were selected, spanning a wide range of PAA reactivity by RBA. Results: The study of ROC curves allowed choosing a cut-off value of 3.0 SDs and the AUC was 0.705, indicating that FloCMIA has fair ability to distinguish between samples from each group. A prevalence of 50% for PAA was obtained in the population of diabetic patients studied. The specificity was 96% and the analytical sensitivity (percentage of patients RBA positive, also positive by FloCMIA) was 69%. There was a substantial agreement between methods (kappa statistic=0.700). Conclusions: A novel immunoassay based on flow cytometry that uses easy-to produce recombinant PI was developed. This assay constitutes an innovative and cost-effective alternative to RBA for the determination of PAA in patients' sera. The method developed here, presents good performance and a wide dynamic range together with a small required sample volume. Furthermore, these results make it possible to develop multiplex immunoassays that allow the combined detection of autoantibodies present in T1DM and other related autoimmune diseases.

Frequency of pancreatic beta-cell autoimmunity markers in patients with autoimmune thyroid disease.

A total of 305 ambulatory patients recruited at the Division of Endocrinology, Hospital de Clínicas, University of Buenos Aires, with autoimmune thyroid disease (AITD) were studied to search for associations between autoimmune thyroid disease and presence of serum markers of autoimmune diabetes mellitus. Screening for markers of pancreatic beta-cell autoimmunity was performed by radioligand binding assays (RBA) as follows: autoantibodies to glutamic acid decarboxylase (GADA) and proinsulin (PAA) were determined in all sera, whereas autoantibodies to protein tyrosine phosphatase (IA-2A) and insulin (IAA) were additionally measured in 200 sera randomly selected from the total collection. In addition, every GADA positive serum among the remaining 105 sera was systematically tested for the presence of IA-2A and IAA. In the cohort of 305 AITD patients 22 (7.2%) were previously diagnosed as type 1, type 2 or insulin-requiring type 2 diabetics. Ten of these patients presented serum marker positivity specific for beta-cell autoantigens and 12 were marker negative. On the other hand, considering the majority of non-diabetic AITD patients (n = 283), beta3-cell marker positivity was detected in 17 individuals (6.0%). The prevalence of autoimmune diabetes markers was much higher in the studied population than in the general population utilized as a control group, and GADA was the most frequent marker.

Expression and physicochemical characterization of an extracellular segment of the receptor protein tyrosine phosphatase IA-2.

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.

Development of 2 alternative enzyme-linked immunosorbent assays for routine screening of glutamic acid decarboxylase autoantibodies.

BACKGROUND: Antibodies to GAD65 (GADA) are considered highly predictive humoral markers of the type 1 diabetes mellitus and also of the insulin requirement in adult-onset patients presumptively classified as type 2 diabetics or LADA. METHODS: We present 2 methods for GADA assessment. The first one (fluid phase, ELISA Protocol A) is carried out in a 2-step procedure in which serum GADA are first allowed to react with a fixed dose of GAD65-biotin in solution and the residual free antigen is later assayed by a conventional ELISA. In the second test (solid phase, ELISA Protocol B) GADA are measured in an ELISA that depends on the ability of divalent autoantibodies to form a bridge between immobilized TrxGAD65 and liquid-phase biotinylated TrxGAD65. RESULTS: All normal control samples scored negative in both variants of ELISA and RBA, hence specificity was 100% for all methods; the relative sensitivity of ELISA Protocol A respect of the RBA was 94% and that of ELISA Protocol B was 76%. CONCLUSIONS: Although ELISA Protocol A exhibited a better performance in terms of relative sensitivity than ELISA Protocol B, the simplicity of execution and the intended use of the assay must also be taken in consideration for the final choice.

Use of cross-reactivity immunoassay to orient insulin replacement in diabetic patients with high levels of insulin antibodies.

The prevalence and high levels of anti-insulin antibodies (IA) have frequently been associated with brittle diabetes, lipodystrophy in the areas where the insulin is injected and/or poor metabolic control. When this happens the usual criterion adopted is the empirical change of insulin type and/or formulation intending to diminish the IA level and then to decrease the undesirable side-effects. Here, we present a rational two step radiometric method consisting in: A) a first-line radioligand binding assay (RBA) to assess IA in sera of these patients and detecting those with high levels. B) applying a displacement assay (RIA) to determine the in vitro cross-reactivity parameters (affinity constants and selectivity ratios) that quantify the relative degree of interaction between antibodies and alternative insulin analogs. From these results we conclude that conventional criteria for selection of insulin analogs, in terms of pharmacokinetic and pharmacodinamic parameters, should be complemented with an appropriate test to assess affinity parameters when high IA title is demonstrated. •This manuscript introduces a rational method to determine the appropriated insulin replacement when high insulin antibodies levels are present.•This protocol provides instructions and details in mathematical tools and laboratory processes for the analysis of serum samples.•This method proved to be successful in a single case and requires confirmation using a large group of patients.

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 [(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 ß-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 ß-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.

Inmunoensayo multiplex para el diagnóstico simultáneo de diabetes mellitus autoinmune y enfermedad celíaca / Multiplex immunoassay for simultaneous diagnostic of autoimmune diabetes mellitus and celiac disease

Introducción: la diabetes mellitus autoinmune (DMA) y la enfermedad celíaca (EC) son enfermedades crónicas, poligénicas y multifactoriales vinculadas con la disfunción del sistema inmune. Dado que es frecuente que un mismo paciente presente ambas patologías, la detección simultánea de los marcadores de autoinmunidad de DMA y EC sería una estrategia racional para mejorar el diagnóstico. Objetivos: desarrollar un inmunoensayo basado en citometría de flujo (FloCMIA multiplex) para la detección simultánea y discriminativa de marcadores de DMA (GADA e IA-2A) y de EC (tTgA). Materiales y métodos: las muestras analizadas consistieron en sueros provenientes de 35 individuos controles normales y 21 pacientes con diabetes mellitus tipo 1 (DM1). Se empleó un modelo de "doble paratope" incubando los sueros con una mezcla de microesferas de diferente fluorescencia interna, cada una adsorbida con un autoantígeno: TrxGAD, TrxIA-2 o H6-tTg, y una mezcla de dichos autoantígenos biotinilados. Los inmunocomplejos se detectaron con estreptavidina-ficoeritrina y se adquirió en un citómetro de flujo. Resultados: FloCMIA multiplex detectó GADA en el 76,2% de los pacientes e IA-2A en el 52,38% (sensibilidad analítica: 88,24 y 56,25% respectivamente,y especificidad: 85,71%) y tTgA en el 42,86% (sensibilidad analítica: 50,0%, y especificidad: 80,0%). Estos resultados se contrastaron con el ensayo de unión de radioligando para GADA e IA-2A y se detectaron 80,95 y 76,19% de los sueros respectivamente (especificidad: 100%), y con un ELISA para tTgA se detectó un 38,1% (especificidad: 97,1%). Conclusiones: FloCMIA multiplex permitió detectar y discriminar GADA, IA-2A y/o tTgA, -en un único acto analítico- en sueros de pacientes con DMA y/o EC. El novedoso inmunoensayo desarrollado simplifica el screening de la población a gran escala

Introduction: autoimmune diabetes mellitus (ADM) and celiac disease (CD) are chronic, polygenic and multifactorial diseases associated with immune system dysfunction. As it is frequent that a patient presents both pathologies, the simultaneous detection of autoimmunity markers of ADM and CD would be a rational strategy to improve the diagnosis. Objectives: to develop an immunoassay based on Flow Cytometry (FloCMIA multiplex) for the simultaneous and discriminative detection of markers for ADM (GADA and IA-2A) and CD (tTgA). Materials and methods: thirty five serum samples of control individuals and 21 type 1 diabetes mellitus (T1DM) patients were assayed. A "double bridge" model was used for the assay, incubating the serum samples with a mixture of microspheres containing different amount of internal fluorescence, each one adsorbed with an autoantigen: TrxGAD, TrxIA-2 or H6-tTg, and a mixture of the same biotinilated autoantigens. The immunocomplexes were detected using streptavidinphycoerytrin and then acquired in a flow cytometer. Results: FloCMIA multiplex detected GADA in 76.2% of the patients; IA-2A in 52.38% (analytical sensitivity: 88.24% and 56.25% respectively, and specificity: 85.71%) and tTgA in 42.86% (analytical sensitivity: 50.0%, and specificity: 80.0%). These results were compared with the radioligand binding assay for GADA and IA-2A, detecting 80.95% and 76.19% of the serum samples respectively (100% specificity), and with an ELISA for tTgA detecting 38.1% (97.1% specificity). Conclusions: FloCMIA multiplex allowed detecting and discriminating GADA, IA-2A and/or tTgA, -in a single assay- in serum samples of ADM and/or CD. The novel developed immunoassay simplifies the screening of the large scale population

A radioligand-binding assay for detecting antibodies specific for proinsulin and insulin using 35S-proinsulin.

A new radioligand-binding assay (RBA) is described for the detection of insulin/proinsulin-specific antibodies using 35S-labeled proinsulin produced by a cell-free reticulocyte extract. Direct use of the crude expression product in the RBA was not feasible because the protein failed to fold properly (or had incorrectly paired disulphide bridges) and purification was hindered by interfering by-products. A refolding protocol and a chromatographic procedure were devised that readily allowed production of purified and immunochemically competent 35S-labeled proinsulin. The new RBA was compared with the reference test, in which the tracer was standard 125I-insulin. The analysis included sera from 41 diabetic patients and 25 healthy controls. Twenty-six (63.4%) and 29 (70.7%) patients scored positive by RBA using 35S-PI and 125I-insulin, respectively. The methods showed a satisfactory correlation with r(2)=0.77 and a slope not significantly different from unity (m=1.16+/-0.10; 95% confidence interval). Since the nuclide used in the assay is 35S, the procedure is compatible with standard assays for GADA and IA-2A, and thus may permit combined assays for the major early markers of autoimmune diabetes.

Biophysical characterization of the membrane-proximal ectodomain of the receptor-type protein-tyrosine phosphatase phogrin.

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.

Characterization of insulin antibodies by Surface Plasmon Resonance in two clinical cases: brittle diabetes and insulin autoimmune syndrome.

In this study, the characterization of insulin (auto)antibodies has been described, mainly in terms of concentration (q), affinity (Ka) and Ig (sub)isotypes by Surface Plasmon Resonance (SPR) in two particular clinical cases of individuals with severe episodes of impaired glycemia. Subject 1 suffers from brittle diabetes associated with circulating insulin antibodies (IA) due to insulin treatment. Subject 2 has insulin autoantibodies (IAA) associated with hypoglycemia in spite of not being diabetic and not having ever received exogenous insulin therapy. After conventional screening for IA/IAA by radioligand binding assay (RBA), we further characterized IA/IAA in sera of both patients in terms of concentration (q), affinity (Ka) and Ig (sub)isotypes by means of SPR technology. In both cases, q values were higher and Ka values were lower than those obtained in type 1 diabetic patients, suggesting that IA/IAA:insulin immunocomplexes could be responsible for the uncontrolled glycemia. Moreover, subject 1 had a predominat IgG1 response and subject 2 had an IgG3 response. In conclusion, SPR technology is useful for the complete characterization of IA/IAA which can be used in special cases where the simple positive/negative determination is not enough to achieve a detailed description of the disease fisiopathology.

Surface plasmon resonance reveals a different pattern of proinsulin autoantibodies concentration and affinity in diabetic patients.

Type 1 diabetes mellitus (DM) is characterized by autoimmune aggression against pancreatic beta cells resulting in absolute deficiency of insulin secretion. The first detectable sign of emerging autoimmunity during the preclinical asymptomatic period is the appearance of diabetes-related autoantibodies. In children at risk for type 1 DM, high-affinity Insulin autoantibodies reactive to proinsulin, are associated with diabetes risk. Autoantibodies are usually measured by radioligand binding assay (RBA) that provides quasi-quantitative values reflecting potency (product between concentration and affinity) of specific autoantibodies. Aiming to improve the characterization of the specific humoral immune response, we selected surface plasmon resonance (SPR) as an alternative method to measure proinsulin autoantibodies (PAA). This novel technology has allowed real time detection of antibodies interaction and kinetic analysis. Herein, we have employed SPR to characterize the PAA present in sera from 28 childhood-onset (mean age 8.31±4.20) and 23 adult-onset diabetic patients (≥65 years old, BMI<30) in terms of concentration and affinity. When evaluating comparatively samples from both groups, childhood-onset diabetic patients presented lower PAA concentrations and higher affinities (median 67.12×10(-9) M and 3.50×10(7) M(-1), respectively) than the adults (median 167.4×10(-9) M and 0.84×10(7) M(-1), respectively). These results are consistent with those from the reference method RBA (Standard Deviation score median 9.49 for childhood-onset group and 5.04 for adult-onset group) where the binding can be directly related to the intrinsic affinity of the antibody, suggesting that there is a different etiopathogenic pathway between both types of clinical presentation of the disease. This technology has shown to be a useful tool for the characterization of PAAs parameters as an alternative to radioimmunoassay, with high versatility and reproducibility associated to low occupational and environmental risk. However, this technology is not eligible for routine marker screening, but this is a powerful technique for a fine description of the thermodynamic parameters of antigen-antibody interaction.

Detection and characterization of ZnT8 autoantibodies could help to screen latent autoimmune diabetes in adult-onset patients with type 2 phenotype.

Autoantibodies to zinc transporter 8 (ZnT8A) constitute an additional marker of autoimmune diabetes, complementing those already used in diagnosis support. ZnT8A could also be found in latent autoimmune diabetes of adults (LADA). The aim of this study was to evaluate the prevalence of ZnT8A in adult-onset diabetic patients in Argentinian population. A total of 271 patients diagnosed for diabetes at mean age 53.4 ± 10.9, body mass index ≤ 30, without insulin treatment for the first year of disease, and initially classified as type 2 diabetic patients were tested for ZnT8A using cDNA plasmids encoding the C-terminal domains (aa 268-369) carrying 325Arg, 325Trp, and a dimeric cDNA construct carrying both 325Arg and 325Trp (ZnT8 Arg-Trp325). We also analyzed proinsulin autoantibodies (PAA), glutamic acid decarboxylase autoantibodies (GADA), and protein tyrosine phosphatase IA-2 autoantibodies (IA-2A). A subset of 101 patients was followed during 6 years in order to analyze insulin requirement. Out of the 271 patients, 22.1% presented at least one humoral marker, 2.6% were PAA+, 12.5% were GADA+, 3.3% were IA-2A+, and 10.7% were ZnT8A+. Among the latter, 7.0% were ZnT8A-Arg325, 51.7% were ZnT8A-Trp325, and 62.1% were ZnT8A-Arg-Trp325. Furthermore, the prevalence of autoantibodies in the group of patients treated with insulin (n = 18) was 55.6%. These results demonstrated that a significant proportion of autoimmune adult-onset diabetic patients presented ZnT8A as the only humoral marker. Between them, the higher prevalence was for ZnT8A-Trp325. We suggest that screening for LADA patients, best performed with a minimal set of marker determination, must include at least the screening of GADA and ZnT8A-Arg-Trp325.

Functional characterization of TLR4 +3725 G/C polymorphism and association with protection against overweight.

Subclinical low-grade systemic inflammation has been associated with obesity, insulin resistance and metabolic syndrome (MS). Recent studies have highlighted the role of gut microbiota in these disorders. The toll-like receptor 4 (TLR4) plays a key role in the innate immune response activation. We studied two polymorphisms (+3725G/C and 11350G/C) in the 3' untranslated region (3'UTR) of the TLR4 gene that may alter its expression and their association with metabolic disorders related to systemic inflammation. We cloned the 3'UTR into a luciferase reporter system and compared wild-type 3'UTR (WT) and +3725C variant (MUT) constructs luciferase activities. MUT construct reduced the reporter gene activity by 30% compared to WT (P = 0.0001). To evaluate the association between these polymorphisms with biochemical and clinical overweight related variables, we conducted a population cross-sectional study in 966 men of Argentine general population. Considering smoking as a confounding variable that causes systemic inflammation, we studied these possible effects in both, smokers and nonsmokers. The 11350G/C polymorphism was not detected in our sample whereas the CC genotype of +3725 polymorphism was associated with lean subjects (p = 0.011) and higher Adiponectin levels (p = 0.021). Subjects without any NCEP/ATP III MS component were associated with this genotype as well (p = 0.001). These results were strengthened in nonsmokers, in which CC genotype was associated with lean subjects (p = 0.003) and compared with G carriers showed significantly lower BMI (25.53 vs. 28.60 kg/m2; p = 0.023) and waist circumference (89.27 vs. 97.51 cm; p = 0.025). None of these associations were found in smokers. These results showed that +3725C variant has a functional effect down-regulating gene expression and it could be considered as a predictive factor against overweight, particularly in nonsmokers. Considering the role of TLR4 in inflammation, these findings would suggest that the presence of +3725C variant could predict a lower prevalence of chronic metabolic disorders.