Molecular characterization of epitope (s) reactive with antibodies to asialoglycoprotein receptor in patients with autoimmune hepatitis

以去唾液酸糖蛋白受体(AGPR)-H1的全长cDNA克隆为模板，通过聚合酶链式反应获得了AGPR-H1胞外区和胞内区的部分编码区。利用这些片段在大肠杆菌中表达了胞外区和胞内区的重组AGPR蛋白。将重组蛋白纯化后用于检测自身免疫性肝炎(AIH)患者血清中的AGPR抗体，利用AGPR蛋白的胞外结构域检测AIH患者血清中的AGPR抗体，发现AIH患者对抗AGPR抗体的敏感性与AIH相似，而在慢性丙型肝炎患者中反应较弱。AIH患者血清与胞外AGPR蛋白有较强的免疫沉淀作用，而不与S…发生免疫共沉淀，其GD值较高更明显的沉淀与胞内结构域的蛋白质。仅在血清中检测到弱阳性信号，具有较强的酶联免疫吸附试验和免疫沉淀反应，提示在琼脂糖凝胶电泳法中，AIH中与AGPR抗体反应的表位(S)可能被降解。ELISA法和免疫沉淀法的结果表明，与AIH抗体反应的主要表位(S)可能位于AGPR蛋白的胞外区。这一发现支持抗AGPR抗体可能在AIH中起致病作用。肝细胞损伤可能通过抗体依赖的细胞介导的细胞毒作用而产生。在重组AGPR抗原的淋巴细胞刺激试验中，几例AIH患者外周血单个核细胞的淋巴细胞呈明显的增殖反应。为了阐明AIH自身免疫应答的病理意义，有必要进一步鉴定AIH抗原上的B细胞和T细胞表位(S)。较少

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 … More 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