Cloning of a leucine-zipper protein recognized by the sera of patients with antibody-associated paraneoplastic cerebellar degeneration.

Abstract

Antibody-associated paraneoplastic cerebellar degeneration (the Yo syndrome) is an uncommon disorder in which an immune response is specifically directed against tumor tissue and the cerebellum. Screening of a A expression library has resulted in the isolation of cDNA clones that encode the major antigen recognized by serum from these patients. The fusion protein produced by the cDNA clones provides the basis of a simple diagnostic assay for this neurological syndrome. The occurrence of leucine-zipper and zinc-finger motifs in the predicted open reading frame suggests that this protein plays a role in the regulation of gene expression.Paraneoplastic cerebellar degeneration is a disorder of the cerebellum found in association with neoplasms of lung, ovary, breast, or Hodgkin disease (1). Neuropathological analysis of affected brains has revealed extensive loss of Purkinje cells, variable loss of granule and basket neurons, and proliferation of Bergman glia (2). The relationship between the primary tumor and the resultant cerebellar dysfunction is not clearly understood. The presence of infiltrating lymphocytes in some of the affected brains has suggested an immune mechanism (3).A clinically definable subset of patients with paraneoplastic cerebellar degeneration harbor a characteristic antibody that has been called anti-Yo (4). Sera from these patients react with antigens expressed in the Purkinje cells of the normal cerebellum and in the tumor tissue of affected individuals (5). There is also evidence of increased antibody synthesis in the affected brains (6). These observations suggest a model for the neurological dysfunction in which an immune response primarily directed against a tumor antigen is misdirected against similar antigens peculiar to the cerebellum. On Western blot analysis of Purkinje cells and tumor tissue, the anti-Yo sera react with at least two antigens, a major species of 62 kDa (CDR-62) and a minor species of 34 kDa (CDR-34) (where CDR is cerebellar degeneration related) (7). The gene encoding the minor antigen (CDR-34) has been isolated and characterized (8,9). We now report the isolation of cDNAs that encode the major Yo paraneoplastic antigen. Sequence analysis § revealed CDR-62 to be a member of a family of the leucine-zipper DNA binding proteins. The availability of the recombinant protein has provided a simple diagnostic assay for the presence of anti-Yo antibodies.MATERIALS AND METHODS Sera from patients with antibody-associated paraneoplastic cerebellar degeneration was obtained from their physicians.A HeLa cell A ZAP expression library was obtained from Stratagene.Screening of A HeLa Expression Library. Recombinant phage were screened at a density of 2 x 104 plaque-forming units per 150-mm plate of Escherichia coli XLI-Blue. After incubation for 6 hr at 370C, the plates were overlaid with filters soaked in isopropyl 8-D-thiogalactopyranoside (10 mM) and incubated for a further 12 hr at 370C. The filters were then removed and incubated with anti-Yo sera (IgG, 2 tug/ml) for 2 ...

Enteric Nervous System: Neuropathic Gastrointestinal Motility

“…The UNC-OSU group evaluated the prevalence of anti-ENS autoimmune antibodies and the antigens recognized by the antibodies in patients diagnosed with idiopathic IBS. The approach was based on one used productively for evaluation of anti-enteric neuronal antibodies in patients with gastrointestinal disorders associated with a paraneoplastic syndrome [37][38][39][40][41][42].…”

Managing Paraneoplastic Neurological Disorders

“…Anti-Yo antibodies are associated with breast cancer and tumors of the ovaries, endometrium, and fallopian tubes [4,5,48]. These antibodies are directed against the cerebellar degeneration-related (CDR) proteins that are expressed by Purkinje cells and the associated tumors [48,49]. CDR-2-specific cytotoxic T cells have been identified in the serum from patients with PCD, suggesting a pathogenic role for the cellular immune response in this PNS [14].…”

The cytoplasmic Purkinje onconeural antigen cdr2 down-regulates c-Myc function: implications for neuronal and tumor cell survival

“…We tested several aminoterminal constructs for activation in a yeast two-hybrid system, and found that constructs containing the cdr2 HLZ domain without the acidic domain were suitable for screening. Because cdr2 is expressed in HeLa cells (Fathallah-Shaykh et al 1991), we performed a yeast twohybrid screen of a HeLa cell cDNA library using the cdr2 HLZ domain as bait, and identified c-Myc as a specifically interacting clone (Table 1). This clone encoded the carboxylterminus of c-Myc, a region that includes the HLZ c-Myc interaction domain.…”

“…The immune response in PCD is characterized by high titers of serum and cerebrospinal fluid antibodies that recognize a ∼55-kD antigen in the patient's tumors and in cerebellar Purkinje neurons. PCD antisera has been used to clone cDNAs encoding several related target antigens (Dropcho et al 1987;Sakai et al 1990;Fathallah-Shaykh et al 1991), only one of which, cdr2, is expressed in PCD tumor specimens (Corradi et al 1997). The cdr2 gene is widely transcribed, but the protein has only been found to be expressed in cerebellar Purkinje neurons, some brainstem neurons, and spermatogonia (Corradi et al 1997), all immune-privileged sites.…”