Role of Anti-ORP150 autoantibody in transplanted heart

抗ORP150自身抗体在移植心脏中的作用

• 批准号: 09044298
• 负责人: TOHYAMA Masaya
• 金额: $ 7.04万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09044298/
• 关键词: Oxygen regulated protein; stress response; hypoxia; molecular chaperone; Glucose regulated protein; Cardiac transpantation; ischemia; tragsgenic mouse; Oxygen; ストレス蛋白; 小胞体; 虚血; 低酸素; グリア細胞; 神経細胞; 虚血耐性

The 150kDa Oxygen-regulated protein (ORP150) was initially identified as a novel stress protein in hypoxic astrocytes and its amino acid sequence suggest its possible participation in cellular protein transport. Northern blot analysis showed abundant expression of its transciipts the kidney, and immunohistochemical analysis suggested the enhanced expression of ORP150 antigen in renal tubular epitheliurm. The ORP150 immunointensity in epithelium was colocalized with that of Tamm-Horsfall protein, a marker of tubular epithelium in thick ascending limb of Henle, suggesting a possible role of ORP150 in protein transport in tublar epithellum. In MDCK cells, a cell line representing the renal epitheliurm, exposure to hypoxia resulted in the increase of ORP150 antigen, as well as the incresae of GP8O (8OkDa glycoprotein) antigen immunoprecipitated by anti-ORP150 antibody. In the ORP150 antisense transformant MOCK cells, where the expression of OPR150 is strongly supressed, GP-80 antigen retai … More ned in the ER after the exposure to hypoxia Consistently, metabolic labelling showed the delay of GP8O maturation in antisense transformant cells in hypoxia, whereas matured form of GP8O was defected in wild type cells under hypoxia, indicating the role of ORP150 in protein transport, especially in hypoxia. The affinity chromatographic analysis of ORP150 to immobilized ATP-agarose suggested a higher affinity of ORP150 to ATP than those of GRP78/Bip and GRP94, other molecular chaperons functioning in the MDCK cells. Further, the ATP-hydrolysis analysis. showed the ORP150 can release GP8O in a lower ATP concentration To determine the contribution of 0RP150 to cellular processes underlying adaptation to hypoxia, a cell line stably-transfected to overexpress ORPl50 antisense RNA was created. In human embryonic kidney (HEK) cells stably overexpressing ORPl50 antisense RNA, ORP150 antigen and transcripts were suppressed to low levels in normoxia and hypoxia, whereas wild-type cells showed induction of ORP150 with oxygen deprivation. Inhibition of ORP150 expression in antisense transfectants was selective, as hypoxia-mediated enhancement of glucose-regulated protein (GRP) 78 and GRP94 were maintained. However, antisense ORP150 transfectants displayed reduced viability when subjected to hypoxia, compared with wild-type and sense transfected HEK cells. In contrast, diminished levels of ORP150 had no effect on cytotoxicity induced by other stimuli, including oxygen-free radicals and sodium arsenate. Although cellular ATP content was similar in hypoxia, comparing ORP150 antisense transfectants and will-type HEK cells, suppression of ORP150 expression was associated with accelerated apoptosis, based on DNA fragmentation and changes in nuclear morphology. Hypoxia-mediated cell death in antisense HEK transfectants did not cause an increase in caspase activity or in cytoplasmic cytochrome c antigen. A well-recognized inducer of apoptosis in HEK cells, staurosporine, caused increas Less

150kDa 氧调节蛋白 (ORP150) 最初被鉴定为缺氧星形胶质细胞中的一种新型应激蛋白，其氨基酸序列表明其可能参与细胞蛋白转运。 Northern印迹分析显示其转录本在肾脏中大量表达，免疫组织化学分析表明肾小管上皮中ORP150抗原的表达增强。上皮中的 ORP150 免疫强度与 Tamm-Horsfall 蛋白的免疫强度共定位，Tamm-Horsfall 蛋白是 Henle 升肢粗管状上皮的标记物，表明 ORP150 在管状上皮中蛋白质转运中可能发挥作用。在代表肾上皮细胞的 MDCK 细胞中，暴露于缺氧会导致 ORP150 抗原增加，以及抗 ORP150 抗体免疫沉淀的 GP8O（8OkDa 糖蛋白）抗原增加。在 ORP150 反义转化体 MOCK 细胞中，OPR150 的表达受到强烈抑制，暴露于缺氧后，GP-80 抗原保留在内质网中。一致地，代谢标记显示反义转化体细胞在缺氧时 GP8O 成熟延迟，而成熟形式的 GP8O 在缺氧下野生型细胞中缺陷，表明缺氧的作用 ORP150 参与蛋白质运输，尤其是在缺氧情况下。 ORP150 与固定化 ATP-琼脂糖的亲和色谱分析表明，ORP150 与 ATP 的亲和力高于 GRP78/Bip 和 GRP94（在 MDCK 细胞中发挥作用的其他分子伴侣）。进一步，ATP-水解分析。显示ORP150可以在较低的ATP浓度下释放GP8O。为了确定0RP150对适应缺氧的细胞过程的贡献，创建了稳定转染以过表达ORP150反义RNA的细胞系。在稳定过表达ORP150反义RNA的人胚胎肾(HEK)细胞中，ORP150抗原和转录物在常氧和缺氧下被抑制至低水平，而野生型细胞在缺氧时表现出ORP150的诱导。反义转染子中 ORP150 表达的抑制是选择性的，因为缺氧介导的葡萄糖调节蛋白 (GRP) 78 和 GRP94 的增强得以维持。然而，与野生型和正义转染的 HEK 细胞相比，反义 ORP150 转染子在缺氧时表现出活力降低。相比之下，ORP150水平降低对其他刺激（包括氧自由基和砷酸钠）诱导的细胞毒性没有影响。虽然细胞 ATP 含量在缺氧条件下相似，但比较 ORP150 反义转染子和 will 型 HEK 细胞，根据 DNA 断裂和核形态的变化，ORP150 表达的抑制与细胞凋亡加速相关。反义 HEK 转染子中缺氧介导的细胞死亡不会导致 caspase 活性或细胞质细胞色素 c 抗原的增加。星形孢菌素是 HEK 细胞中公认的细胞凋亡诱导剂，可导致细胞凋亡增加 Less

项目成果

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Tsukamoto Y.、Kuwabara K.、Okawa S. 和 Kitamura Y.：“150 kDa 氧调节蛋白 (ORP150) 在人类动脉粥样硬化斑块中表达，使单核吞噬细胞能够承受暴露于缺氧和修饰 LDL 时的细胞应激。”

Yan S.D., Zhu Y., Zhu A., Fu J., Zhu H., Zhu Y., Gbson L., Collison K., Mohanna Al., Ogawa S,.Roher A., Clarke SG., and Stern D.: "Role of ERAB/L-3 Hydroxyacyl-coenzyme A dehydrogenase type II activity in Ab-induced cytotoxicity." J.Biol.Chem. (in press).

Yan S.D.、Zhu Y.、Zhu A.、Fu J.、Zhu H.、Zhu Y.、Gbson L.、Collison K.、Mohanna Al.、Okawa S、Roher A.、Clarke SG. 和 Stern D

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Yamashita T、Shimada S、takagi T、Tohyama M：“脑肽/组氨酸转运蛋白的克隆和功能表达。”

Yan S.D., Saido T., Tohyama T., Ogawa S., Roher A., and Stern D.: "An intracelluar protein that binds amyloid-beta protein and mediates reurotoxicity in Alzheimer's dicasc." Nature. 389. 689-692 (1997)

Yan S.D.、Saido T.、Tohyama T.、Okawa S.、Roher A. 和 Stern D.：“一种细胞内蛋白，可结合淀粉样蛋白 - β 蛋白并介导阿尔茨海默病二倍体中的肾毒性。”

Imuta et al.: "Induction of 72 kDa inducible heat shock protein(HSP72)in cultured rat astrocytes after energy depletion." J.Neurochem.70. 550-556 (1998)

Imuta 等人：“能量耗尽后，在培养的大鼠星形胶质细胞中诱导 72 kDa 诱导型热休克蛋白 (HSP72)。”