Analysis of structure of proteasome-PCNA multiprotein conplex and its autoimmune response

蛋白酶体-PCNA多蛋白复合物结构分析及其自身免疫反应

• 批准号: 13670476
• 负责人: TAKASAKI Yoshinari
• 金额: $ 1.47万
• 依托单位: Juntendo University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670476/
• 关键词: proteasome; PA28γ; PCNA; autoimmunity; autoantibody; proteasome; DNA replication; autoantibody; autoantigen; autoimmunity

We have previously shown that a group of anti-PCNA monoclonal antibodies (mAbs) raised in our laboratory can react with PCNA bound to or interacting with other proteins associated with cell proliferation (PCNA complexes). In addition, we have found that proteasome is also interacting with PCNA. We purified the complexes by affinity chromatography using the mAbs, and also analyzed autoimmune response of proteins in the complex in lupus patients. We then found that 35% of SLE sera react with at least one component of the PCNA complex, which suggests that analysis of the targeted antigens may shed light on the mechanisms underlying autoimmune responses to proteins interacting with PCNA in SLE patients. To clarify this issue, we have identified elements of the PCNA complexes that are reactive to antibodies (Abs) in sera from SLE patients using 2-dimensional electrophoresis and ion-pair chromatography. Among the various elements of the complexes was a 37-kDa protein (PI 8.5) that specifically reacted with lupus sera, but not with sera from patients with other connective tissue diseases, and was later identified as glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Immunoblot analysis showed that lupus sera reactive with the 37-kDa protein specifically reacted with GAPDH, as did anti-GAPDH Abs purified from the sera. In addition, ELISAs revealed the presence of anti-GAPDH autoantibodies in 47% of lupus patients. Longitudinal analysis of the reactivity of lupus sera to PCNA complexes showed the autoimmune response to spread from GAPDH to other elements of PCNA complexes, and the presence of anti-GAPDH Abs was correlated with increased levels of serum PCNA. In addition, we have also found that lupus sera recognized proteasome activators as autoantigens, and there is a linkage of autoimmune response between PCNA and proteasome. Taken together, these findings suggest that "antige presentation" is playing a critical role to induce the autoimmune response to the PCNA complex.

我们之前已经证明，我们实验室生产的一组抗 PCNA 单克隆抗体 (mAb) 可以与与细胞增殖相关的其他蛋白质（PCNA 复合物）结合或相互作用的 PCNA 发生反应。此外，我们发现蛋白酶体也与PCNA相互作用。我们使用单克隆抗体通过亲和层析纯化了复合物，并分析了狼疮患者复合物中蛋白质的自身免疫反应。然后我们发现 35% 的 SLE 血清与 PCNA 复合物的至少一种成分发生反应，这表明对目标抗原的分析可能有助于揭示 SLE 患者对与 PCNA 相互作用的蛋白质产生自身免疫反应的机制。为了澄清这个问题，我们使用二维电泳和离子对色谱法鉴定了 PCNA 复合物中与 SLE 患者血清中的抗体 (Ab) 发生反应的元素。在复合物的各种元素中，有一种 37 kDa 的蛋白质 (PI 8.5) 与狼疮血清发生特异性反应，但与其他结缔组织疾病患者的血清不发生反应，后来被鉴定为甘油醛 3-磷酸脱氢酶 (GAPDH)。免疫印迹分析表明，与 37-kDa 蛋白反应的狼疮血清与 GAPDH 发生特异性反应，从血清中纯化的抗 GAPDH 抗体也是如此。此外，ELISA 显示 47% 的狼疮患者存在抗 GAPDH 自身抗体。狼疮血清对 PCNA 复合物反应性的纵向分析显示，自身免疫反应从 GAPDH 扩散到 PCNA 复合物的其他成分，并且抗 GAPDH 抗体的存在与血清 PCNA 水平升高相关。此外，我们还发现狼疮血清将蛋白酶体激活剂识别为自身抗原，并且PCNA和蛋白酶体之间存在自身免疫反应的联系。总而言之，这些发现表明“抗原呈递”在诱导 PCNA 复合物的自身免疫反应中发挥着关键作用。

项目成果

Takasaki Y, Kaneda K, Takeuchi K, Matsudaira R, Matsushita M, Yamada H, Nawata M, Ikeda K, Kaneda K, Hashimoto H: "Analysis of structure of proteasome-PCNA multiprotein complex and its autoimmune response in lupus patients."Ann N Y Acad Sci. 987. 316-318

Takasaki Y、Kaneda K、Takeuchi K、Matsudaira R、Matsushita M、Yamada H、Nawata M、Ikeda K、Kaneda K、Hashimoto H：“狼疮患者中蛋白酶体-PCNA 多蛋白复合物的结构及其自身免疫反应的分析。”Ann

Fritzler MJ, Wiik A, Tan EM, takasaki Y, et al.: "A critical evaluation of enzyme immunoassay kits for detection of antinuclear autoantibodies of defined specificity III."J.Rheumatol. 30. 2374-2381 (2003)

Fritzler MJ、Wiik A、Tan EM、takasaki Y 等人：“用于检测具有明确特异性的抗核自身抗体的酶免疫测定试剂盒的关键评估 III”。J.Rheumatol。

Suzuki K, Sawada T, Murakami A, Matsui T, Takasaki Y, et al.: "High diagnosis performance of ELISA detection of antibodies to citrullinated antigens in rheumatoid arthritis."Scad J Rheumatol. 32. 197-204 (2003)

Suzuki K、Sawada T、Murakami A、Matsui T、Takasaki Y 等人：“类风湿性关节炎中瓜氨酸抗原抗体 ELISA 检测的高诊断性能。”Scad J Rheumatol。

Matsudaira R, Takeuchi K, Takasaki Y, et al.: "Relationships between autoantibody responses to deletion mutants of Ki antigen and clinical manifestations of lupus."J.Rheumatol. 30. 1208-1214 (2003)

Matsudaira R、Takeuchi K、Takasaki Y 等人：“Ki 抗原缺失突变体的自身抗体反应与狼疮临床表现之间的关系。”J.Rheumatol。

Matsushita M, Takasaki Y, Takeuchi K, Yamada H, Matsudaira R, Hashimoto H: "Autoimmune response to proteasome activator 28α in patients with connective tissue diseases."J Rheumatol. 31. 252-259 (2004)

Matsushita M、Takasaki Y、Takeuchi K、Yamada H、Matsudaira R、Hashimoto H：“结缔组织疾病患者对蛋白酶体激活剂 28α 的自身免疫反应。”J Rheumatol。 31. 252-259 (2004)