Role of Anti-ORP150 autoantibody in transplanted heart

150 kDa的氧调节蛋白（Oxygen-regulated protein，ORP 150）最初被鉴定为缺氧星形胶质细胞中的一种新的应激蛋白，其氨基酸序列提示其可能参与细胞内蛋白质的转运。北方印迹分析显示其转运蛋白在肾组织中大量表达，免疫组化分析显示其在肾小管上皮细胞中表达增强。上皮中的ORP 150免疫强度与Tamm-Horsfall蛋白共定位，Tamm-Horsfall蛋白是Henle氏粗升支中肾小管上皮的标记物，表明ORP 150在肾小管上皮中的蛋白运输中可能起作用。在代表肾上皮细胞的MDCK细胞中，缺氧暴露导致ORP 150抗原的增加，以及抗ORP 150抗体免疫沉淀的GP 80（80 kDa糖蛋白）抗原的增加。在OPR 150反义寡核苷酸MOCK细胞中，OPR 150的表达被强烈抑制，GP-80抗原保留在OPR 150反义寡核苷酸MOCK细胞中。 ...更多信息 结果表明，缺氧条件下，反义寡核苷酸细胞中GP 8 O成熟延迟，而野生型细胞中成熟的GP 8 O缺失，表明ORP 150在蛋白质转运中起重要作用，尤其是在缺氧条件下。ORP 150对固定化ATP-琼脂糖的亲和层析分析表明，ORP 150对ATP的亲和性高于在MDCK细胞中起作用的其他分子伴侣GRP 78/Bip和GRP 94。此外，ATP水解分析。为了确定ORP 150对缺氧适应的细胞过程的贡献，建立了稳定转染以过表达ORP 150反义RNA的细胞系。在稳定过表达ORP 150反义RNA的人胚肾（HEK）细胞中，ORP 150抗原和转录物在常氧和缺氧中被抑制至低水平，而野生型细胞显示出在缺氧的情况下ORP 150的诱导。反义转染子中ORP 150表达的抑制是选择性的，因为低氧介导的葡萄糖调节蛋白（GRP）78和GRP 94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ORP 150转染子在缺氧时显示出降低的活力。相反，降低水平的ORP 150对其他刺激物（包括氧自由基和砷酸钠）诱导的细胞毒性没有影响。虽然细胞的ATP含量是相似的缺氧，比较ORP 150反义转染和将型HEK细胞，ORP 150表达的抑制与加速凋亡，DNA片段化和核形态的变化的基础上。缺氧介导的细胞死亡反义HEK转染没有引起caspase活性或细胞质细胞色素c抗原的增加。一种公认的HEK细胞凋亡诱导剂，星形孢菌素，

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