1998 Fiscal Year Final Research Report Summary
Molecular characterization of epitope (s) reactive with antibodies to asialoglycoprotein receptor in patients with autoimmune hepatitis
| Project/Area Number |
09670532
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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 |
Gastroenterology
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| Research Institution | Shinshu University |
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Principal Investigator |
IMAI Haruhiko Shinshu University Hospital, Instructor, 医学部・附属病院, 助手 (70291383)
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| Co-Investigator(Kenkyū-buntansha) |
KIYOSAWA Kendo Shinshu University School of Medicine, Professor, 医学部, 教授 (30020829)
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| Project Period (FY) |
1997 – 1998
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| Keywords | autoimmune hepatitis / asialoglycoprotein receptor / autoantibody / molecular biology |
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| Research Abstract |
The partial cDNA fragments encoding the extra- and intracellular domain of asialoglycoprotein receptor (AGPR)-Hl was obtained by polymerase chain reaction from a full-length AGPR-Hl cDNA clone. Using these cDNA fragments, recombinant AGPR proteins of extra- and intracellular domain were expressed in E.coli. Recombinant proteins were purified and subjected to an enzyme-linked immunosorbent assay (ELISA) to detect antibodies to AGPR in sera from autoimmune hepatitis (AIH), By using extracellular domain of AGPR protein in ELISA, a similar sensitivity to pick-up anti-AGPR antibodies was demonstrated in AIH and less reactivity was detected in AIH and chronic hepatitis C.In order to confirm an antigen-antibody system in AIH, an immunoprecipitation method was applied using biotinilated recombinant AGPR proteins as antigen and Protein G-agarose as carrier. Sera from AIH, which showed high GD values in ELISA, demonstrated a strong immunoprecipitation with the extracellular AGPR protein and no s
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ignificant precipitation with intracellular domain of the protein. Weakly positive signals were only detected with sera which gave a strong reactivity in ELISA and immunoprecipitation, suggesting that epitope(s) reactive with antibodies to AGPR in AIH might be degraded during SDS acrylamide gel electrophoresis. The results from ELISA and immunoprecipitation suggested that main epitope(s), which were reactive with antibodies in AIH, could be located on the extracellular domain of the AGPR protein. This finding support that anti-AGPR antibodies might play an pathogenic role in AIH.Hepatocytic injury could be generated via antibody-dependent cell-mediated cytotoxicity. Lymphocytes from peripheral blood mononuclear cells in several AIH patients showed a significant proliferation in a lymphocyte stimulation test with recombinant AGPR antigen. Further characterization of B and T cell epitope(s) on AGPR antigens is necessary to elucidate the pathological significance of autoimmune response to AGPR in AIH. Less
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