Function of the DNA-terminal binding protein regognized by anti-Ku antibodies and its etiologic significance in collagen disease

抗Ku抗体识别的DNA末端结合蛋白的功能及其在胶原病中的病因学意义

• 批准号: 04670395
• 负责人: MIMORI Tsuneyo
• 金额: $ 1.34万
• 依托单位: Keio University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04670395/
• 关键词: Autoantibody; Ku antigen; DNA-terminal binding protein; DNA-dependent protein kinase; gene polymorphism; autoimmune disease; collagen disease; overlap syndrome; DNA依存性プロテインキナーゼ; オクタマー配列; DNA依存性蛋白キナーゼ; 自己抗原; PCR; PCR-SSCP

Anti-Ku autoantibodies in patients with PSS-PM overlap syndrome recognize a 70kD/80kD protein (p70/p80) heterodimer which selectively binds to terminal region of dsDNA.I this project, I have investigated 1) genomic structure and polymorphism of epitope regions of the Ku antigen, 2) structure of DNA that binds to the Ku antigen, and 3) association between the Ku antigen and DNA-dependent protein kinase (DNA-PK).Structure of genomic DNAs that encode for epitopes of p70 (aa 545-609) and p80 (aa 696-732) was examined by single-strand conformation polymorphism (SSCP) after those DNA regions were amplified by polymerase chain reaction (PCR). Although there was no difference in genomic epitope structure between normals and patients, one patient with Xeroderma pigmentosum and one normal subject showed polymorphic structures of the p70 gene. p70 appeared to be ecoded by at least two independent genes. These results suggest a presence of a gene family encoding the Ku antigen.DNAs that were extra … More cted from immunoprecipitates between anti-Ku antobodies and the Ku antigen from HeLa cells were subcloned into M13 vector and their nucleotide sequences were determined. When 30 DNA clones were examined (mean length 196bp), the octamer-like sequence [ATTT (G/T) (C/T) (A/T) T] and the transferrin receptor element-like sequence [GAAGTNA (C/G)] appeared at 37 and 18 binds specific DNA sequences as well as DNA termini.Anti-Ku antibodies precipitated a 350kD protein besides of p70/p80, when HeLa cells were extracted in an isotonic buffer as antigen source. This protein was identified as the catalytic subunit p350 of DNA-PK,since the 350kD protein precipitated with anti-Ku was recognized by a hyperimmune rabbit serum to DNA-PK p350. Binding between the Ku and p350 required the presence of dsDNA and was dissociated reversibly by 0.5M NaCl. Enzymatic activity of DNA-PK required the presence of both Ku and DNA.These results indicate that the Ku antigen binds p350 to form the DNA-PK holoenzyme and acts as an activation subunit of DNA-PK. Less

PSS-PM重叠综合征患者的抗KU自身抗体识别70KD/80KD蛋白（p70/p80）异二聚体，有选择地与DSDNA的终末区域进行选择性结合，我投资了1），我投资了1）基因组结构和基因组结构和ku antig antig antig antignignignign and dna的基因组结构，dna，2） 3) association between the Ku antigen and DNA-dependent protein kinase (DNA-PK).Structure of genomic DNAs that encode for epitopes of p70 (aa 545-609) and p80 (aa 696-732) was examined by single-strand conference polymorphism (SSCP) after those DNA regions were amplified by polymerase chain reaction (PCR).尽管正常人和患者之间的基因组表位结构没有差异，但一名患者患有心排骨色素和一个正常受试者，显示了p70基因的多态性结构。 P70在编辑上至少由两个独立基因编辑。这些结果表明，编码ku抗原的基因家族存在额外的dnas，这些基因来自抗ku抗原之间的免疫沉淀物和来自HELA细胞的KU抗原的免疫沉淀更多，并确定了其核苷酸序列。当检查30个DNA克隆（平均长度196BP）时，八聚体样序列[attt（g/t）（c/t）（c/t）（a/t）t]和转移蛋白受体元件样序列[Gaagtna（c/g）]出现在37和18的特定DNA序列和DNA termini.anti-Ku antii antii anties anties anties anties Anties ant a 350 kn 3550 P70/P80，将HeLa细胞作为抗原源提取时。该蛋白被鉴定为DNA-PK的催化亚基P350，因为用抗KU沉淀的350kD蛋白通过高免疫兔血清识别为DNA-PK P350。 KU和P350之间的结合需要DSDNA的存在，并通过0.5m的NaCl可逆地解离。 DNA-PK的酶促活性需要KU和DNA的存在。这些结果表明，KU抗原结合p350形成DNA-PK全酶，并充当DNA-PK的激活亚基。较少的

项目成果

三森経世ほか: "膠原病の血清学的診断" 総合臨床. 43. 1102-1105 (1994)

Tsuneyo Mimori 等人：“胶原病的血清学诊断”《一般临床实践》43. 1102-1105 (1994)。

Watanabe F,Mimori T et al: "Molecular propaties, substrate specificity and regulation of DNA-dependent protein kinase." Biochim Biophys Acta. 1223. 255-260 (1994)

Watanabe F、Mimori T 等人：“DNA 依赖性蛋白激酶的分子特性、底物特异性和调节”。

M.Hirakata, T.Mimori,et al: "Autoantibodies reactive with snRNP and scRNP in Japanese patients with polymyositis." Arthritis Rheum.35. 449-456 (1992)

M.Hirakata、T.Mimori 等人：“日本多发性肌炎患者的自身抗体与 snRNP 和 scRNP 发生反应。”

Tsuzaka K,Mimori T et al: "Nonprecipitating IgG or IgM anti‐Sm antibody:Clinical significance and changes in immunoglobulin class." J.Rheumatol.20. 822-830 (1993)

Tsuzaka K、Mimori T 等人：“非沉淀 IgG 或 IgM 抗 Sm 抗体：免疫球蛋白类别的临床意义和变化。J.Rheumatol.20 (1993)。

Kuwana M,Mimori T et al: "Autoantigenic epitopes on DNA topoisomerase I:Clinical and immunogenetic associations in systemic sclerosis." Arthritis Rheum.36. 1406-1413 (1993)

Kuwana M、Mimori T 等人：“DNA 拓扑异构酶 I 上的自身抗原表位：系统性硬化症的临床和免疫遗传学关联。”