title: Identification of delta/notch-like epidermal growth factor-related receptor as the Tr antigen in paraneoplastic cerebellar degeneration.
abstract: OBJECTIVE
  Anti-Tr is among the better described autoantibodies in paraneoplastic cerebellar degeneration (PCD) combined with Hodgkin lymphoma (HL); however, the Tr antigen remains unidentified.
  
  
  METHODS
  We used immunoprecipitation of total rat brain extract followed by mass spectrometry to identify the antigen recognized by anti-Tr-positive sera. By Western blotting and cell-based assays, we tested a total of 12 anti-Tr-positive and 246 control sera and determined the region of the epitope recognized by the anti-Tr antibodies. Deletion and mutant constructs were generated to further map the antigenic region.
  
  
  RESULTS
  Mass spectrometry analysis of immunopurified rat brain extract using 4 different anti-Tr-positive sera led to the identification of Delta/Notch-like epidermal growth factor-related receptor (DNER) as the Tr antigen. All but 1 of 246 control samples were negative in the HeLa cell-based screening assay, whereas 12 of the 12 anti-Tr-positive sera stained hemagglutinin-tagged DNER-expressing cells. Only 1 control subject with HL but no ataxia was found to be both DNER and Tr positive. Using deletion constructs, we pinpointed the main epitope to the extracellular domain. Knockdown of endogenous DNER in hippocampal and N-glycosylation mutations abolished the anti-Tr staining, indicating that glycosylation of DNER is required for it to be recognized by anti-Tr antibodies.
  
  
  CONCLUSIONS
  DNER is the antigen detected by anti-Tr-positive sera. Presence of anti-Tr antibodies in patients with PCD and HL or HL only can now be screened quickly and reliably by using a cell-based screening assay.
authors.affiliation: Cell Biology, Faculty of Science, Utrecht University, Utrecht, the Netherlands.
authors.forename: Esther
authors.initials: E
authors.lastname: de Graaff
authors.forename: Peter
authors.initials: P
authors.lastname: Maat
authors.forename: Esther
authors.initials: E
authors.lastname: Hulsenboom
authors.forename: Robert
authors.initials: R
authors.lastname: van den Berg
authors.forename: Martin
authors.initials: M
authors.lastname: van den Bent
authors.forename: Jeroen
authors.initials: J
authors.lastname: Demmers
authors.forename: Pieternella J
authors.initials: PJ
authors.lastname: Lugtenburg
authors.forename: Casper C
authors.initials: CC
authors.lastname: Hoogenraad
authors.forename: Peter
authors.initials: P
authors.lastname: Sillevis Smitt
mesh_terms: D000328:Adult; D000368:Aged; D000818:Animals; D001323:Autoantibodies / Q000097:blood*; D002461:Cell Line, Transformed; D002648:Child; D005260:Female; D006031:Glycosylation; D006624:Hippocampus / Q000473:pathology; D006689:Hodgkin Disease / Q000276:immunology; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D008875:Middle Aged; D009419:Nerve Tissue Proteins / Q000276:immunology* / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D020362:Paraneoplastic Cerebellar Degeneration / Q000276:immunology* / Q000378:metabolism*; D051381:Rats; D011956:Receptors, Cell Surface / Q000276:immunology / Q000378:metabolism*; D020080:Tandem Repeat Sequences / Q000276:immunology*; D055815:Young Adult; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D001323:Autoantibodies; C462501:DNER protein, human; D009419:Nerve Tissue Proteins; D011956:Receptors, Cell Surface; D001616:beta-Galactosidase