Hiroki Hirai

Selective screening of secretory vesicle-associated proteins for autoantigens in type 1 diabetes: VAMP2 and NPY are new minor autoantigens.

The four major autoantigens (IA-2, IA-2 beta, GAD65 and insulin) of type 1 diabetes are all associated with dense core or synaptic vesicles. This raised the possibility that other secretory vesicle-associated proteins might be targets of the autoimmune response in type 1 diabetes. To test this hypothesis 56 proteins, two-thirds of which are associated with secretory vesicles, were prepared by in vitro transcription/translation and screened for autoantibodies by liquid phase radioimmunoprecipitation. Two secretory vesicle-associated proteins, VAMP2 and NPY, were identified as new minor autoantigens with 21% and 9%, respectively, of 200 type 1 diabetes sera reacting positively. These findings add support to the hypothesis that secretory vesicle-associated proteins are particularly important, but not the exclusive, targets of the autoimmune response in type 1 diabetes. Selective screening of the human proteome offers a useful approach for identifying new autoantigens in autoimmune diseases.

Deletion of Ia-2 and/or Ia-2β in mice decreases insulin secretion by reducing the number of dense core vesicles

Aims/hypothesisIslet antigen 2 (IA-2) and IA-2β are dense core vesicle (DCV) transmembrane proteins and major autoantigens in type 1 diabetes. The present experiments were initiated to test the hypothesis that the knockout of the genes encoding these proteins impairs the secretion of insulin by reducing the number of DCV.MethodsInsulin secretion, content and DCV number were evaluated in islets from single knockout (Ia-2 [also known as Ptprn] KO, Ia-2β [also known as Ptprn2] KO) and double knockout (DKO) mice by a variety of techniques including electron and two-photon microscopy, membrane capacitance, Ca2+ currents, DCV half-life, lysosome number and size and autophagy.ResultsIslets from single and DKO mice all showed a significant decrease in insulin content, insulin secretion and the number and half-life of DCV (p < 0.05 to 0.001). Exocytosis as evaluated by two-photon microscopy, membrane capacitance and Ca2+ currents supports these findings. Electron microscopy of islets from KO mice revealed a marked increase (p < 0.05 to 0.001) in the number and size of lysosomes and enzymatic studies showed an increase in cathepsin D activity (p < 0.01). LC3 protein, an indicator of autophagy, also was increased in islets of KO compared with wild-type mice (p < 0.05 to 0.01) suggesting that autophagy might be involved in the deletion of DCV.Conclusions/interpretationWe conclude that the decrease in insulin content and secretion, resulting from the deletion of Ia-2 and/or Ia-2β, is due to a decrease in the number of DCV.

A New Luminescence Assay for Autoantibodies to Mammalian Cell–Prepared Insulinoma-Associated Protein 2

OBJECTIVE—Insulinoma-associated protein 2 (IA-2) is a major autoantigen in type 1 diabetes, and IA-2 autoantibodies are routinely detected by a liquid-phase radioimmunoprecipitation assay. The present experiments were initiated to develop a new assay that does not require the use of radioisotopes or autoantigens prepared in bacteria or by in vitro transcription/translation. RESEARCH DESIGN AND METHODS—IA-2 luciferase fusion protein was expressed in mammalian cells and assayed for autoantibodies by liquid-phase luciferase immunoprecipitation. RESULTS—Our study showed that there was no significant difference between the luciferase immunoprecipitation and the radioimmunoprecipitation assays in sensitivity and specificity, and comparison of the two assays revealed a high correlation coefficient (R2 = 0.805). CONCLUSIONS—The luciferase system offers a robust, inexpensive, nonradioactive method for the detection of autoantibodies to mammalian cell–prepared IA-2 and could be of practical value at the clinical level.

Comparison of Radioimmunoprecipitation With Luciferase Immunoprecipitation for Autoantibodies to GAD65 and IA-2β

OBJECTIVE To compare the sensitivity and specificity of luciferase immunoprecipitation (LIPS) with radioimmunoprecipitation (RIP) for the measurement of autoantibodies to the type 1 diabetes autoantigens glutamic acid decarboxylase 65 (GAD65) and insulinoma-associated protein (IA)-2β. RESEARCH DESIGN AND METHODS Sera from 49 type 1 diabetic patients and 100 nondiabetic control subjects from Diabetes Antibody Standardization Program 2007 were used to screen for autoantibodies to GAD65. An additional 200 type 1 diabetic patients and 200 nondiabetic control subjects were used to validate the GAD65 results and screen for autoantibodies to IA-2β. RESULTS LIPS showed equal sensitivity and specificity to RIP for detecting autoantibodies to GAD65 and IA-2β. Receiver-operating characteristic analysis revealed that the detection of autoantibodies to GAD65 and IA-2β by LIPS and RIP were not statistically different. CONCLUSIONS The LIPS assay does not require the use of radioisotopes or in vitro transcription/translation and is a practical alternative at the clinical level for the RIP assay.

Deletion of the secretory vesicle proteins IA-2 and IA-2β disrupts circadian rhythms of cardiovascular and physical activity

Targeted deletion of IA‐2 and IA‐2β, major autoantigens in type 1 diabetes and transmembrane secretory vesicle proteins, results in impaired secretion of hormones and neurotransmitters. In the present study, we evaluated the effect of these deletions on daily rhythms in blood pressure, heart rate, core body temperature, and spontaneous physical and neuronal activity. We found that deletion of both IA‐2 and IA‐2β profoundly disrupts the usual diurnal variation of each of these parameters, whereas the deletion of either IA‐2 or IA‐2β alone did not produce a major change. In situ hybridization revealed that IA‐2 and IA‐2β transcripts are highly but nonrhythmically expressed in the suprachiasmatic nuclei, the site of the brains master circadian oscillator. Electrophysiological studies on tissue slices from the suprachiasmatic nuclei showed that disruption of both IA‐2 and IA‐2β results in significant alterations in neuronal firing. From these studies, we concluded that deletion of IA‐2 and IA‐2β, structural proteins of secretory vesicles and modulators of neuroendocrine secretion, has a profound effect on the circadian system.—Kim, S. M., Power, A., Brown, T. M., Constance, C. M., Coon, S. L., Nishimura, T., Hirai, H., Cai, T., Eisner, C., Weaver, D. R., Piggins, H. D., Klein, D. C., Schnermann, J., Notkins, A. L. Deletion of the secretory vesicle proteins IA‐2 and IA‐2β disrupts circadian rhythms of cardiovascular and physical activity. FASEBJ. 23, 3226‐3232 (2009). www.fasebj.org

RESP18, a homolog of the luminal domain IA-2, is found in dense core vesicles in pancreatic islet cells and is induced by high glucose

The regulated endocrine-specific protein, RESP18, first found in the rat pituitary, was thought to be regulated by dopaminergic drugs. Bioinformatics studies showed that RESP18 shares sequence homology with the luminal region of IA-2, a dense core vesicle (DCV) transmembrane protein involved in insulin secretion. The present study was initiated to examine the genomic structure and subcellular localization of RESP18 and the effect of glucose on its expression. Human RESP18 was isolated from a pancreas cDNA library and its subcellular localization was determined by immunoelectron microscopy. MIN6 cells and mouse islets were used to study the effect of glucose on RESP18 expression. Bioinformatics analysis revealed that RESP18 and IA-2 are tandemly arranged within a 45 kb region on human chromosome 2 and share common intron-exon boundaries. By confocal microscopy, RESP18 was found in alpha, beta and delta cells in the pancreatic islets. Electron microscopy revealed that RESP18 is present in the lumen of DCVs. The expression of RESP18 in beta cells is markedly increased following exposure to high glucose and also elevated in the islets of diabetic, but not non-diabetic, NOD mice. We conclude that RESP18 is a luminal protein of DCVs and its expression is regulated by exposure to glucose.

Dense-core vesicle proteins IA-2 and IA-2β affect renin synthesis and secretion through the β-adrenergic pathway

IA-2 and IA-2beta, major autoantigens in type 1 diabetes, are transmembrane proteins in dense-core vesicles, and their expression influences the secretion of hormones and neurotransmitters. The present experiments were performed to examine whether IA-2 and IA-2beta modulate the release of renin from dense-core vesicles of juxtaglomerular granular cells in the kidney. Plasma renin concentration (PRC; ng angiotensin I.ml(-1).h(-1)) was significantly reduced in mice with null mutations in IA-2, IA-2beta, or both IA-2 and IA-2beta compared with wild-type mice (876 +/- 113, 962 +/- 130, and 596 +/- 82 vs. 1,367 +/- 93; P < 0.01, P < 0.02, and P < 0.001). Renin mRNA levels were reduced to 26.4 +/- 5.1, 39 +/- 5.4, and 35.3 +/- 5.5% of wild-type in IA-2-/-, IA-2beta-/-, and IA-2/IA-2beta-/- mice. Plasma aldosterone levels were not significantly different among genotypes. The regulation of PRC by furosemide and salt intake, and of aldosterone by salt intake, was maintained in all genotypes. IA-2 and IA-2beta expression did not colocalize with renin but showed overlapping immunoreactivity with tyrosine hydroxylase. While propranolol reduced PRC in wild-type mice, it had no effect on PRC in IA-2/ IA-2beta-/- mice. Renal tyrosine hydroxylase mRNA and immunoreactivity were reduced in IA-2/IA-2beta-/- mice as was the urinary excretion of catecholamines. We conclude that IA-2 and IA-2beta are required to maintain normal levels of renin expression and renin release, most likely by permitting normal rates of catecholamine release from sympathetic nerve terminals.

Loss of the transcriptional repressor PAG-3/Gfi-1 results in enhanced neurosecretion that is dependent on the dense-core vesicle membrane protein IDA-1/IA-2.

It is generally accepted that neuroendocrine cells regulate dense core vesicle (DCV) biogenesis and cargo packaging in response to secretory demands, although the molecular mechanisms of this process are poorly understood. One factor that has previously been implicated in DCV regulation is IA-2, a catalytically inactive protein phosphatase present in DCV membranes. Our ability to directly visualize a functional, GFP-tagged version of an IA-2 homolog in live Caenorhabditis elegans animals has allowed us to capitalize on the genetics of the system to screen for mutations that disrupt DCV regulation. We found that loss of activity in the transcription factor PAG-3/Gfi-1, which functions as a repressor in many systems, results in a dramatic up-regulation of IDA-1/IA-2 and other DCV proteins. The up-regulation of DCV components was accompanied by an increase in presynaptic DCV numbers and resulted in phenotypes consistent with increased neuroendocrine secretion. Double mutant combinations revealed that these PAG-3 mutant phenotypes were dependent on wild type IDA-1 function. Our results support a model in which IDA-1/IA-2 is a critical element in DCV regulation and reveal a novel genetic link to PAG-3-mediated transcriptional regulation. To our knowledge, this is the first mutation identified that results in increased neurosecretion, a phenotype that has clinical implications for DCV-mediated secretory disorders.

Engineering and expression of the intracellular domain of insulinoma-associated tyrosine phosphatase (IA-2ic), a type 1 diabetes autoantigen, in plants.

We have produced the recombinant intracellular domain of human IA-2 (IA-2ic), a diabetes-associated autoantigen, in plants. This was achieved by transient expression using agroinfiltration of Nicotiana benthamiana plants. The resulting plant-derived IA-2ic had the expected size, reacted with polyclonal and monoclonal antibodies specific to human IA-2ic and competitively inhibited radiolabeled IA-2ic in an immunoprecipitation assay. The expression level of recombinant IA-2ic was estimated to be 0.5% of the total soluble protein (TSP). Transient expression in plants has the potential to produce a large amount of human IA-2ic protein at low cost in a short period of time.

The minimal promoter region of the dense-core vesicle protein IA-2: transcriptional regulation by CREB

AbstractAimsIA-2 is a transmembrane protein found in the dense-core vesicles (DCV) of neuroendocrine cells and one of the major autoantigens in type 1 diabetes. DCV are involved in the secretion of hormones (e.g., insulin) and neurotransmitters. Stimulation of pancreatic β cells with glucose upregulates the expression of IA-2 and an increase in IA-2 results in an increase in the number of DCV. Little is known, however, about the promoter region of IA-2 or the transcriptional factors that regulate the expression of this gene.MethodsIn the present study, we constructed eight deletion fragments from the upstream region of the IA-2 transcription start site and linked them to a luciferase reporter.ResultsBy this approach, we have identified a short bp region (−216 to +115) that has strong promoter activity. We also identified a transcription factor, cAMP responsive element-binding protein (CREB), which binds to two CREB-related binding sites located in this region. The binding of CREB to these sites enhanced IA-2 transcription by more than fivefold. We confirmed these findings by site-directed mutagenesis, chromatin immunoprecipitation assays and RNAi inhibition.Conclusion Based on these findings, we conclude that the PKA pathway is a critical, but not the exclusive signaling pathway involved in IA-2 gene expression.