2003 Fiscal Year Final Research Report Summary
Clinical significance of islet protein presented by type 1 diabetes-susceptible
| Project/Area Number |
14571117
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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 |
Metabolomics
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| Research Institution | Okinaka Memorial Institute for Medical Research |
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Principal Investigator |
NAKANISHI Koji Okinaka Memorial Institute for Medical Research, Staff, 研究員 (80211423)
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| Project Period (FY) |
2002 – 2003
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| Keywords | Type 1 diabetes / MHC molecule / Antigenic peptides / Islet |
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| Research Abstract |
Susceptibility to type 1 diabetes is conferred by major histocompatibility antigens (MHC) and it is caused by T -cell mediated autoimmunity to pancreatic islet. The aim of this study is to identify islet-derived autoantigen which is presented by type 1 diabetes-susceptible MHC molecules. B lymphoblastoid cell lines were prepared from a type 1 diabetic patient and cultured to 10^<10> cells. The debris of fetal islet cell line (1B2C6) were added to one culture system, and the other culture was performed in media only. Cell surface proteins were obtained with treatment of lysis buffer containing NP-40. HLA-DR and --DQ molecules were obtained by affinity chromatography. After treatment of trifluoloacetic acid, the peptides in MHC molecules were analyzed by reverse phase HPLC. Comparing HPLC patterns identified 3 peaks which were specifically emerged under the pulse of the debris of 1B2C6. By sequencing the amino acids, we identified a peptide and their derived protein as heparin sulfate/heparin interacting protein (HIP). We could obtain cDNA of HIP from 1B2C6 cell lines by RT-PCR and expressed HIP protein in E. coli. Autoantibodies to HIP was not detected in type 1 diabetic patients by Western blotting. However, immunostaining of pancreatic section using antisera to HIP revealed specific localization of HIP in the islet. We identified the peptide that naturally processed and presented by type 1 diabetes-susceptible HLA molecule, DR4.
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