Identification of GRASP-1 as a novel 97 kDa autoantigen localized to endosomes.
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.
Stinton
Laura M
LM
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, 3330 Hospital Dr N.W., Calgary, AB, Canada T2N 4N1.
Selak
Sanja
S
Fritzler
Marvin J
MJ
D000293:Adolescent; D000595:Amino Acid Sequence; D000918:Antibody Specificity / Q000276:immunology; D001324:Autoantigens / Q000276:immunology* / Q000378:metabolism; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000276:immunology*; D045744:Cell Line, Tumor; D011992:Endosomes / Q000276:immunology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D050957:HSP110 Heat-Shock Proteins; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000276:immunology; D006801:Humans; D006851:Hydrochloric Acid / Q000737:chemistry; D047468:Immunoprecipitation; D007239:Infections / Q000097:blood / Q000276:immunology / Q000473:pathology; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000276:immunology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D011928:Raynaud Disease / Q000097:blood / Q000276:immunology / Q000473:pathology; D012008:Recurrence; D017386:Sequence Homology, Amino Acid; D013684:Telangiectasis / Q000097:blood / Q000276:immunology / Q000473:pathology; D033921:Vesicular Transport Proteins
D001324:Autoantigens; D002352:Carrier Proteins; D000939:Epitopes; C485827:GRIPAP1 protein, human; D050957:HSP110 Heat-Shock Proteins; C121993:HSPA4 protein, human; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D033921:Vesicular Transport Proteins; C093846:early endosome antigen 1; D006851:Hydrochloric Acid