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 1.30 Å 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. Protein-tyrosine phosphatases (PTP),2 together with the corresponding kinases, regulate cell division, growth, differentiation, and metabolism (1). There are cytoplasmic PTP as well as transmembrane receptors PTP (RPTP). The latter also participate in cell-cell and cell-matrix contacts, possess an impressive diversity of adhesive and multimerization modules (2), and have been involved in human diseases such as cancer, autoimmunity, and degenerative processes (1).Two paralog RPTPs, IA-2 (insulinoma-associated protein 2, also termed PTP35 or ICA512) and IA-2 ␤ (PTPR2, also known as phogrin or IAR), were identified as major autoantigens in type-1 diabetes mellitus (3). They have a signal peptide, an ectodomain, a single-pass transmembrane region, and a single intracellular PTP domain.IA-2 and IA-2 ␤ are prominent in the secretory granules (SG) of brain, pituitary, pancreatic islet, and adrenal endocrine cells (4). Although the physiological ligands for these receptors are unknown and their function is poorly understood, they are involved in hormone and neuropeptide secretion. Indeed, single-and double-knock-out mice lacking IA-2 suffer from glucose intolerance, impaired insulin secretion, and abnormal secretion of pituitary hormones and female infertility (5, 6).Processing of pro IA-2 by furin-like hormone convertases produces mature IA-2 (7), which lacks the signal peptide and an adjacent fragment (residues 1-448). Mature IA-2 reaches the plasma membrane during exocytosis and comprises extracellular (449 -575), transmembrane (576 -600), and cytoplasmic domains (601-979). High glucose levels up-regulate IA-2 (8), and insulin exocytosis triggers a Ca 2ϩ -dependent and -calpain-mediated cleavage of the IA-2 cytopl...

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.

Autoantibodies to the islet-cell 65-kDa variant of glutamate decarboxylase (GAD65) are found in most insulin-dependent diabetes mellitus (IDDM) patients many years before the appearance of clinical symptoms of the disease. As IDDM-preventive therapies may be available in the future, an international effort is taking place to develop widely applicable anti-GAD immunochemical tests. These tests would help to detect individuals at risk before the full installation of the disease and to enroll them in prevention programs. Autoantibodies to GAD65 are mostly directed to conformational epitopes, and the enzyme is a complex molecule with a prosthetic group and 15 cysteine residues. Thus, the conformational integrity of GAD65 is essential for an appropriate anti-GAD assay. Isolation of large amounts of GAD65 from pancreas or other tissues is impractical, and no successful production of properly folded GAD65 has been reported in bacteria. Native recombinant GAD65 for immunochemical tests is usually obtained from eukaryotic expression systems. Since the large-scale production of a recombinant protein in an eukaryotic system is expensive and technically difficult, we investigated the expression of GAD65 in Escherichia coli as an alternative. A number of DNA constructs intended to export the enzyme to the periplasmic space or to improve its cytoplasmic solubility were designed and tested. Our results provide a solution to the two main problems associated with the expression of GAD65 in E. coli: misfolding, leading to the formation of inclusion bodies ; and the presence of alternative initiation sites for translation that causes the preferential production of truncated variants of GAD65. We describe here the production of properly folded, fully active, and immunochemically competent GAD65 as an N-terminal fusion protein with thioredoxin. An account of the reactivity of the produced protein with sera of six IDDM patients is also presented.