Differential release of cellular and scrapie prion proteins from cellular membranes by phosphatidylinositol-specific phospholipase C.
Differential release of cellular and scrapie prion proteins from cellular membranes by phosphatidylinositol-specific phospholipase C.
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DOI：
10.1021/bi00474a028
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发表时间：
1990-06
期刊：
影响因子：
2.9
通讯作者：
N. Stahl;D. Borchelt;S. Prusiner
中科院分区：
文献类型：
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作者：
N. Stahl;D. Borchelt;S. Prusiner
瘙痒病蛋白PrPSc的异常亚型既是一种宿主来源的蛋白，也是引起瘙痒病的感染源的一种成分。PrPSc和正常的细胞亚型PrPC具有不同的物理性质，显然是由翻译后事件产生的。两种PrP异构体在羧基末端被糖基肌醇磷脂共价修饰。利用从正常和瘙痒病感染的仓鼠脑组织中分离出来的细胞制备的样品，我们发现大多数PrPC是由磷脂酰肌醇特异性磷脂酶C(PIPLC)从膜中释放出来的，而PrPSc是抵抗释放的。相反，纯化的变性PrP27-30(它是在纯化过程中通过氨基末端的蛋白质分解从PrPSc中形成的)被PIPLC完全切割。将细胞制剂与蛋白酶K孵育后，PrPSc裂解形成PrP27-30，表明PrPSc可被添加的酶所访问。我们还开发了一种涉及生物素化的方案，给出了暴露在细胞外部的蛋白质部分的定量估计。使用这一策略，我们发现细胞制剂中的很大一部分PrPSc与膜非意味着的生物素化试剂反应。PrPSc的替代膜锚定、糖肌醇磷脂锚定对PIPLC的不可及或与其他细胞成分的结合是否是导致PrPSc蛋白从细胞中差异释放的原因仍有待确定。
The abnormal isoform of the scrapie prion protein PrPSc is both a host-derived protein and a component of the infectious agent causing scrapie. PrPSc and the normal cellular isoform PrPC have different physical properties that apparently arise from a posttranslational event. Both PrP isoforms are covalently modified at the carboxy terminus by a glycoinositol phospholipid. Using preparations of dissociated cells derived from normal and scrapie-infected hamster brain tissue, we find that the majority of PrPC is released from membranes by phosphatidylinositol-specific phospholipase C (PIPLC), while PrPSc is resistant to release. In contrast, purified denatured PrP 27-30 (which is formed from PrPSc during purification by proteolysis of the amino terminus) is completely cleaved by PIPLC. Incubation of the cell preparations with proteinase K cleaves PrPSc to form PrP 27-30, demonstrating that PrPSc is accessible to added enzymes. We have also developed a protocol involving biotinylation that gives a quantitative estimate of the fraction of a protein exposed to the cell exterior. Using this strategy, we find that a large portion of PrPSc in the cell preparations reacts with a membrane-impermeant biotinylation reagent. Whether alternative membrane anchoring of PrPSc, inaccessibility of the glycoinositol phospholipid anchor to PIPLC, or binding to another cellular component is responsible for the differential release of prion proteins from cells remains to be determined.