1996 Fiscal Year Final Research Report Summary
Analysis of the autoantigen gene associated with paraneoplastic neurologic syndromes
| Project/Area Number |
07670736
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Neurology
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| Research Institution | Kanazawa Medical University |
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Principal Investigator |
SAKAI Koichiro Kanazawa Medical University Department of Neurology Assistant Professor, 医学部, 助教授 (70225754)
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| Project Period (FY) |
1995 – 1996
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| Keywords | Paraneoplastic syndromes / autoantigen / neuronal protein / leucine zipper / RNA-binding protein |
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| Research Abstract |
The PCD17 protein is a paraneoplastic cerebellar degeneration (PCD)-associated neural antigen wichi is recognized by anti-Purkinje cell antibodies in serum or cerebrospinal fluid of PCD patients with gynecologic or breast cancer. (i) We isolated the truncatedhuman pcd17 clone, pcd17.2, by screening a human cerebellar cDNA library by using a probe corresponding to the leucine zipper domain of the pcd17, which is a paraneoplastic cerebellar degeneration-associated gene, and performing 3' rapid amplification of the cDNA ends. The 1,410 nucleotides of the 3' end of the pcd 17.2 gene were identical to those of the 3' end of pcd17.1 (the original pcd17), but the 329 nucleotides of the 5' end were different. The deduced protein PCD17.2 encoded on pcd17.2 is truncated at the carboxyl-terminus with PCD17.1 protein. Immunoblot analysis demonstrated that the bacterially expressed protein from the pcd17.2 cDNA was recognized by the anti-Purkinje cell autoantibodies. These two truncated proteins ar
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e transcribed and translated from the truncated homologues on the genome. These results evidence the presence of two isoforms of the leucine zipper Purkinje cytoplasmic autoantigens. (ii) Homodimerization of the pcd17.1 and pcd17.2 genes was not demonstrated by the two-hybrid system and the GST-fused protein co-precipitation assay. (iii) Transactivation of the pcd 17 which is fused downward the GAL4 DNA binding domain in the yeast cells which contain GAL1 promoter fused with a beta galactosidase reporter gene was demonstrated by beta galactosidase assay. (iv) The three RNA recognition motives (RRMs) of the paraneoplastic limbic encephalomyelitis-associated antigen PLE21 was analyzed for its antigenicity for the anti neuronal nuclear antibodies. All of the three RRMs were recognized by the antibodies. (v) The RNA binding activities of the PLE 21 protein was demonstrated in the AU-rich element of the 3' untranslated region of the c-fos and c-myc mRNA.The first and the second RRMs were recognized by all of the antibodies examined, but the third RRMs are recognized by some of the antibodies and not by others. (vi) The PCD17 and PLE21 protein and pcd17 and ple21 mRNA were localized in the rat cerebellum and hippocampus by immunohistochemistry and by in situ hybridization histochemistry using a cRNA probe. Both the pcd17 mRNA and the ple21 mRNA in the cerebellum and the ple21 mRNA in the hippocampus were expressed in agreement with the distributions of the PCD17-and PLE21-immunoreactivities. However, intense hybridization signals of the pcd17 were detected in most of cells in the hippocampus in which notable PCD17-immunoreactivity was undetected. These observations indicate that the PCD17 protein is selectively expressed by some neuronal population, and suggest that some negative translational or posttranscriptional mechanisms of the PCD17 mRNA may exist in neurons of the hippocampus. Less
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