Role of Anti-ORP150 autoantibody in transplanted heart

150 kDa氧调节蛋白(ORP150)最初被鉴定为低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的蛋白质运输。Northern印迹分析显示其在肾小管上皮细胞中大量表达，免疫组织化学分析显示ORP150抗原在肾小管上皮细胞中表达增强。肾小管上皮细胞中ORP150的表达强度与Henle升支粗大段肾小管上皮细胞的标志物Tamm-Horsfall蛋白共存，提示ORP150可能在肾小管上皮细胞的蛋白转运中起作用。在代表肾上皮细胞系的MDCK细胞中，低氧可导致ORP150抗原表达增加，并使抗ORP150抗体免疫沉淀的GP8O(8OkDa糖蛋白)抗原表达增加。在OPR150表达被强烈抑制的反义转化子模拟细胞中，GP-80抗原表达…缺氧后内质网中的NED持续增加，代谢标记显示反义转化子细胞在低氧条件下GP8O成熟延迟，而野生型细胞在低氧条件下GP8O成熟形式缺失，提示ORP150在蛋白质运输中的作用，特别是在低氧条件下。ORP150对固定化的ATP-琼脂糖亲和层析分析表明，ORP150对ATP的亲和力高于与MDCK细胞中其他分子伴侣GRP78/Bip和GRP94的亲和力。进一步，进行了三磷酸腺苷的水解性分析。显示ORP150可以在较低的ATP浓度下释放GP8O，以确定0RP150在细胞缺氧适应过程中的贡献，建立了稳定表达ORPl50反义RNA的细胞系。在稳定过表达ORP50反义RNA的人胚胎肾(HEK)细胞中，ORP150抗原和转录本在常氧和低氧条件下被抑制到低水平，而野生型细胞在缺氧条件下表现出ORP150的诱导。由于维持了低氧对葡萄糖调节蛋白(GRP)78和GRP94表达的增强，因此对反义转染体中ORP150表达的抑制是有选择性的。然而，与野生型和正义转染型HEK细胞相比，反义ORP150转染体在低氧条件下显示出活力降低。相反，ORP150水平的降低对包括氧自由基和砷酸钠在内的其他刺激诱导的细胞毒性没有影响。尽管在低氧条件下，细胞的ATP含量与ORP150反义转基因细胞和Will-型HEK细胞相似，但基于DNA片段化和核形态的变化，ORP150表达的抑制与细胞凋亡的加速有关。低氧介导的反义HEK细胞死亡不会引起caspase活性或胞浆细胞色素c抗原的增加。一种公认的HEK细胞凋亡诱导剂，星形孢子素，引起的增量较少

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