Identification of GRASP-1 as a novel 97 kDa autoantigen localized to endosomes |
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Authors: | Stinton Laura M Selak Sanja Fritzler Marvin J |
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Affiliation: | Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, 3330 Hospital Dr N.W., Calgary, AB, Canada T2N 4N1. |
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Abstract: | We have identified an autoantigen that is recognized by antibodies from an 18-year-old female with a history of recurrent infections who later in her clinical course developed Raynaud's phenomenon and telangiectasias. By indirect immunofluorescence (IIF), the index serum produced a unique cytoplasmic discrete speckled (CDS) staining pattern that partially colocalized with early endosome antigen 1 (EEA1) but not Golgi complex or other cytoplasmic organelles in HEp-2 cells. When HEp-2 cells were treated with 0.1 N HCl, the cytoplasmic speckled staining of the index serum was markedly decreased, suggesting that the reactive antigen was soluble. Western blot analysis showed a reactive approximately 97 kDa protein in a saline soluble protein preparation from HeLa cells. Mass spectrometric analysis of the excised 97 kDa band that was immunoprecipitated from HeLa cell extracts identified GRASP-1 as a possible target. The index serum and anti-GRASP-1 antibodies colocalized to structures in the cytoplasm of HEp-2 cells. Synthetic peptides representing the full-length GRASP-1 protein were used to identify reactive epitopes. Like many other cytoplasmic autoantigens, GRASP-1 has numerous coiled-coil domains throughout the protein with the exception of short segments at the amino and carboxyl terminus. |
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Keywords: | cANCA cytoplasmic anti-neutrophil cytoplasmic antibodies CLIP cytoplasmic linker protein EEA1 early endosome antigen 1 ELISA enzyme linked immunosorbent assay GRASP-1 glutamate receptor interacting protein-associated protein-1 GW/GWB glycine (G) and tryptophan (W) rich proteins localized in distinct cytoplasmic foci referred to as GW bodies Hrs hepatocyte growth factor receptor substrate: LBPA, lysobisphosphatidic acid MS mass spectroscopy Autoantibodies Autoimmunity Endosomes GRASP-1 Coiled-coils |
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