Monoclonal antibody 76F distinguishes IA-2 from IA-2β and overlaps an autoantibody epitope

Piquer, Sandra; Valera, Lionel; Lampasona, Vito; Jardin-Watelet, Bénédicte; Roche, Stéphanie; Granier, Claude; Roquet, Françoise; Christie, Michael R.; Giordano, Tiziana; Malosio, Maria Luisa; Bonifacio, Ezio; Laune, Daniel

doi:10.1016/j.jaut.2005.12.001

Cited by 16 publications

(10 citation statements)

References 28 publications

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“…The mouse monoclonal antibodies (mAb) were an IgG2a anti-rat CgB (CIRO) from our laboratory [28]; an anti-insulin (Sigma-Aldrich, Milan, Italy) and an anti-human CgB (67-C7-2), gift of W. B. Huttner (Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany); an anti-trans-Golgi network (TGN) marker (TGN38) and an anti-GM130 (Golgi complex marker; BD Biosciences, San Jose, CA, USA); an antilysosomal-associated membrane protein 1 (LAMP1) (LYC16; Calbiochem, Darmstadt, Germany); two antiprotein tyrosine phosphatase-like protein-2 (ICA512/IA-2), mICA512cyto, gift of M. Solimena (Technical University of Dresden, Germany) [29] and 76F from our laboratory [30]. INS-1E lysates and incubation media were immunoblotted to assess antibody specificity (Electronic supplementary material [ESM] Fig.…”

Section: Cells and Antibodies Ins-1ementioning

confidence: 99%

Beta cell chromogranin B is partially segregated in distinct granules and can be released separately from insulin in response to stimulation

et al. 2008

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Aims/hypothesis We investigated, in three beta cell lines (INS-1E, RIN-5AH, betaTC3) and in human and rodent primary beta cells, the storage and release of chromogranin B, a secretory protein expressed in beta cells and postulated to play an autocrine role. We asked whether chromogranin B is stored together with and discharged in constant ratio to insulin upon various stimuli. Methods The intracellular distribution of insulin and chromogranin B was revealed by immunofluorescence followed by three-dimensional image reconstruction and by immunoelectron microscopy; their stimulated discharge was measured by ELISA and immunoblot analysis of homogenates and incubation media. Results Insulin and chromogranin B, co-localised in the Golgi complex/trans-Golgi network, appeared largely segregated from each other in the secretory granule compartment. In INS-1E cells, the percentage of granules positive only for insulin or chromogranin B and of those positive for both was 66, 7 and 27%, respectively. In resting cells, both insulin and chromogranin B were concentrated in the granule cores; upon stimulation, chromogranin B (but not insulin) was largely redistributed to the core periphery and the surrounding halo. Strong stimulation with a secretagogue mixture induced parallel release of insulin and chromogranin B, whereas with 3-isobutyl-1-methylxantine and forskolin ± high glucose release of chromogranin B predominated. Weak, Ca 2+ -dependent stimulation with ionomycin or carbachol induced exclusive release of chromogranin B, suggesting a higher Ca 2+ sensitivity of the specific granules. Conclusions/interpretation The unexpected complexity of the beta cell granule population in terms

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Section: Cells and Antibodies Ins-1ementioning

confidence: 99%

Beta cell chromogranin B is partially segregated in distinct granules and can be released separately from insulin in response to stimulation

et al. 2008

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“…Then chimeric molecules of human ␣3(IV)NC1 and ␣1(IV)NC1 expressed in a mammalian cell line were used for epitope mapping (6 -9). Hellmark et al (8) identified nine critical amino acid residues in the amino-terminal part of the ␣3(IV)NC1 sequence (positions 17,18,19,21,24,27,28,31, and 57) and produced a recombinant construct named S2 that expresses these substitutions in the ␣1(IV) background (8). In other studies, two regions that harbor conformational anti-GBM epitopes had been defined at residues 17 to 31 and 127 to 141 of the ␣3(IV)NC1 domain, which were named as E A and E B , respectively (7).…”

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confidence: 99%

Antigen and Epitope Specificity of Anti–Glomerular Basement Membrane Antibodies in Patients with Goodpasture Disease with or without Anti-Neutrophil Cytoplasmic Antibodies

Yang¹,

Hellmark²,

Zhao³

et al. 2007

Journal of the American Society of Nephrology

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Goodpasture disease (GP) is defined by the presence of anti-glomerular basement membrane (anti-GBM) antibodies and rapidly progressive glomerulonephritis. Besides anti-GBM, many patients with GP produce anti-neutrophil cytoplasmic antibodies (ANCA). For elucidation of the pathophysiologic significance of ANCA in this setting, epitope and antigen specificity of the anti-GBM antibodies and antigen specificity of ANCA were studied. Bovine testis ␣(IV)NC1 (tNC1); recombinant human ␣1, ␣3, ␣4, and ␣5(IV)NC1 (r␣1 through r␣5); and three chimeric proteins that contain previously defined epitope regions designated E A , E B , and S2 were used to examine the anti-GBM antibodies by ELISA in 205 Chinese patients with GP with or without ANCA. In the 205 anti-GBM antibody-positive sera, 63 (30.7%) were also ANCA positive (61 myeloperoxidase-ANCA and six proteinase 3-ANCA, four being triple positive). All 205 sera recognized tNC1 and r␣3(IV)NC1. In the double-positive group, 54.0, 66.7, 71.4% of the sera could recognize r␣1, r␣4, and r␣5, respectively, compared with 49.3, 60.6, and 55.6% for patients with anti-GBM antibodies alone. The levels of the antibodies to r␣3, tNC1, and the ␣3/␣1 ratio were lower in the double-positive group than that in patients with anti-GBM antibody alone (P < 0.05). Most of the sera could recognize the epitope regions E A , E B , and S2, but the absorbance values to E A , E B , and S2 were lower in double-positive group (P < 0.05). Double-positive patients had a broader spectrum of anti-GBM antibodies and lower levels of antibodies against ␣3(IV)NC1 compared with that of patients with anti-GBM antibodies alone.

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“…Monoclonal antibodies recognizing epitopes in the JM (MAb-76F4B), PTP (MAb-A5), and PTPβ (MAb-A9–19 and MAb-beta593) regions were provided by Dr. E. Bonifacio (18). Antibody binding was assessed by radiobinding assay as described above, but using protein G-sepharose (GE Healthcare) for immunoprecipitation.…”

Section: Methodsmentioning

confidence: 99%

“…Two linear epitopes have been identified in the JM region [amino acids 611–620 and 621–630 (12,18)], but the conformational epitopes found in the PTP region are more diverse (6,19–22). PTP residues shown to be important for antibody binding include tryptophan 799, glutamate 836, asparagine 838, tyrosine 855, asparagine 858, and glutamine 862 (23–25).…”

mentioning

confidence: 99%

The Core Cysteines, (C909) of Islet Antigen-2 and (C945) of Islet Antigen-2β, Are Crucial to Autoantibody Binding in Type 1 Diabetes

et al. 2012

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Cysteines are thought integral to conformational epitopes of islet antigen-2 (IA-2) autoantibodies (IA-2A), possibly through disulfide bond formation. We therefore investigated which cysteines are critical to IA-2A binding in patients with newly diagnosed type 1 diabetes. All 10 cysteines in the intracellular domain of IA-2 were modified to serine by site-directed mutagenesis, and the effects of these changes on autoantibody binding in comparison with wild-type control were investigated by radiobinding assay. Mutation of the protein tyrosine phosphatase (PTP) core cysteine (C909) in IA-2 caused large reductions in autoantibody binding. In contrast, little or no reduction in binding was seen following substitution of the other cysteines. Modification of the core cysteine (C945) in IA-2β also greatly reduced autoantibody binding. Lysine substitution of glutamate-836 in IA-2 or glutamate-872 in IA-2β resulted in modest reductions in binding and identified a second epitope region. Binding to IA-2 PTP and IA-2β PTP was almost abolished by mutation of both the core cysteine and these glutamates. The core cysteine is key to the major PTP conformational epitope, but disulfide bonding contributes little to IA-2A epitope integrity. In most patients, at disease onset, >90% of antibodies binding to the PTP domain of IA-2 recognize just two epitope regions.

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Monoclonal antibody 76F distinguishes IA-2 from IA-2β and overlaps an autoantibody epitope

Cited by 16 publications

References 28 publications

Beta cell chromogranin B is partially segregated in distinct granules and can be released separately from insulin in response to stimulation

Beta cell chromogranin B is partially segregated in distinct granules and can be released separately from insulin in response to stimulation

Antigen and Epitope Specificity of Anti–Glomerular Basement Membrane Antibodies in Patients with Goodpasture Disease with or without Anti-Neutrophil Cytoplasmic Antibodies

The Core Cysteines, (C909) of Islet Antigen-2 and (C945) of Islet Antigen-2β, Are Crucial to Autoantibody Binding in Type 1 Diabetes

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