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Objective: Identify the subcellular location and potential binding partners of two cerebellar degeneration-related proteins, CDR2L and CDR2, associated with anti-Yo-mediated paraneoplastic cerebellar degeneration. Methods: Cancer cells, rat Purkinje neuron cultures, and human cerebellar sections were exposed to cerebrospinal fluid and serum from patients with paraneoplastic cerebellar degeneration with Yo antibodies and with several antibodies against CDR2L and CDR2. We used mass spectrometry-based proteomics, super-resolution microscopy, proximity ligation assay, and co-immunoprecipitation to verify the antibodies and to identify potential binding partners. Results: We confirmed the CDR2L specificity of Yo antibodies by mass spectrometry-based proteomics and found that CDR2L localized to the cytoplasm and CDR2 to the nucleus. CDR2L co-localized with the 40S ribosomal protein S6, while CDR2 co-localized with the nuclear speckle proteins SON, eukaryotic initiation factor 4A-III, and serine/ arginine-rich splicing factor 2. Interpretation: We showed that Yo antibodies specifically bind to CDR2L in Purkinje neurons of PCD patients where they potentially interfere with the function of the ribosomal machinery resulting in disrupted mRNA translation and/or protein synthesis. Our findings demonstrating that CDR2L interacts with ribosomal proteins and CDR2 with nuclear speckle proteins is an important step toward understanding PCD pathogenesis.
ObjectiveInvestigate the value of including cerebellar degeneration-related protein 2-like (CDR2L) as a marker in commercial diagnostic tests for anti-Yo–associated paraneoplastic cerebellar degeneration (PCD).MethodsWe included sera and CSF samples from 24 patients with suspected PCD (6 of whom had PCD with underlying gynecologic or breast cancer), who were positive for Yo antibodies using the commercially available, paraneoplastic neurologic syndromes (PNS) 14 Line Assay from Ravo Diagnostika. The samples were further evaluated using the EUROLINE PNS 12 Ag Line Assay and a cell-based assay (CBA) from Euroimmun. For confirmation of positive lineblot results, we used indirect immunofluorescence of rat cerebellar sections. We also tested all samples in 2 assays developed in-house: a CBA for CDR2L and a Western blot analysis using recombinant cerebellar degeneration-related protein 2 (CDR2) and CDR2L proteins.ResultsIn PNS 14 and PNS 12 Ag Line Assays, anti-CDR2 reactivity was observed for 24 (100%) and 20 (83%) of the 24 samples, respectively. Thirteen of 24 subjects (54%) were also positive using the Euroimmun CBA. Rat cerebellar immunofluorescence was the best confirmatory test. In our in-house CBA for CDR2L and Western blot for CDR2 and CDR2L, only the 6 patients with confirmed PCD reacted with CDR2L.ConclusionsCommercially available tests for Yo antibody detection have low specificity for PCD because these assays use CDR2 as antigen. By adding a test for CDR2L, which is the major Yo antigen, the accuracy of PCD diagnosis greatly improved.Classification of EvidenceThis study provides Class III evidence that a CBA for CDR2L accurately identifies patients with PCD.
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