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自身抗体识别选择性结合dsDNA末端的70 kD/80 kD蛋白（p70/p80）异源二聚体。Ku抗原与DNA依赖性蛋白激酶（DNA-PK）的结合。用聚合酶链反应（PCR）扩增p80（aa 696-732）和p545 -609的DNA片段，并进行单链构象多态性（SSCP）分析。虽然正常人和患者之间的基因组表位结构没有差异，但1例着色性干皮病患者和1例正常人显示p70基因的多态性结构。p70似乎由至少两个独立的基因编码。这些结果表明存在编码Ku抗原的基因家族。 ...更多信息 从抗Ku抗体与HeLa细胞Ku抗原免疫沉淀物中提取的DNA片段，亚克隆到M13载体上，并测定其核苷酸序列。当检测30个DNA克隆时，（平均长度196 bp），八聚体样序列[ATTT（G/T）（C/T）（A/T）T]和转铁蛋白受体元件样序列[GAAGTNA（C/G）]出现在37和18处，与DNA序列及DNA末端结合。当在等渗缓冲液中提取HeLa细胞作为抗原源时。该蛋白被鉴定为DNA-PK的催化亚基p350，因为用抗Ku沉淀的350 kD蛋白被DNA-PK p350的超免疫兔血清识别。Ku和p350之间的结合需要双链DNA的存在下，被0.5M NaCl可逆地解离。DNA-PK的酶活性需要Ku和DNA同时存在，表明Ku抗原与p350结合形成DNA-PK全酶，并作为DNA-PK的激活亚基。少

项目成果

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”。

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)。

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。”

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。”