Role of Anti-ORP150 autoantibody in transplanted heart

150kDa的氧调节蛋白（ORP150）最初被确定为缺氧星形胶质细胞中的一种新的应激蛋白，其氨基酸序列表明其可能参与细胞蛋白质运输。Northern blot分析显示其在肾组织中表达丰富，免疫组化分析显示ORP150抗原在肾小管上皮细胞中表达增强。ORP150在上皮内的免疫强度与Henle厚升肢小管上皮标志物tam - horsfall蛋白的免疫强度共定位，提示ORP150可能在小管上皮内的蛋白转运中起作用。在代表肾上皮的MDCK细胞中，缺氧暴露导致ORP150抗原升高，同时抗ORP150抗体免疫沉淀的gp80 （8OkDa糖蛋白）抗原升高。在ORP150反义转化模拟细胞中，OPR150的表达受到强烈抑制，缺氧后内质网更需要GP-80抗原。代谢标记一致显示，缺氧条件下反义转化细胞中gp80成熟延迟，而缺氧条件下野生型细胞中gp80成熟形态缺失，说明ORP150在蛋白质转运中，特别是在缺氧条件下的转运。ORP150对固定化ATP-琼脂糖的亲和色谱分析表明，ORP150对ATP的亲和性高于MDCK细胞中其他分子伴侣GRP78/Bip和GRP94。进一步，atp水解分析。为了确定0RP150在细胞缺氧适应过程中的作用，我们构建了一个稳定转染过表达ORPl50反义RNA的细胞系。在稳定过表达ORPl50反义RNA的人胚胎肾（HEK）细胞中，ORP150抗原和转录本在常氧和缺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。由于缺氧介导的葡萄糖调节蛋白（GRP） 78和GRP94的增强得以维持，ORP150在反义转染物中的表达抑制是选择性的。然而，与野生型和义转染的HEK细胞相比，反义ORP150转染在缺氧条件下表现出较低的活力。相比之下，ORP150水平的降低对其他刺激（包括氧自由基和砷酸钠）诱导的细胞毒性没有影响。尽管缺氧时细胞ATP含量相似，但比较ORP150反义转染物和will型HEK细胞，基于DNA断裂和核形态的改变，ORP150表达的抑制与细胞凋亡加速有关。缺氧介导的细胞死亡在反义HEK转染中没有引起半胱天冬酶活性或细胞质细胞色素c抗原的增加。在HEK细胞中，staurosporine是一种公认的凋亡诱导剂

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