Borchelt D R, Scott M, Taraboulos A, Stahl N, Prusiner S B
Department of Neurology, University of California, San Francisco 94143.
J Cell Biol. 1990 Mar;110(3):743-52. doi: 10.1083/jcb.110.3.743.
Both the cellular and scrapie isoforms of the prion protein (PrP) designated PrPc and PrPSc are encoded by a single-copy chromosomal gene and appear to be translated from the same 2.1-kb mRNA. PrPC can be distinguished from PrPSc by limited proteolysis under conditions where PrPC is hydrolyzed and PrPSc is resistant. We report here that PrPC can be released from the surface of both normal-control and scrapie-infected murine neuroblastoma (N2a) cells by phosphatidylinositol-specific phospholipase C (PIPLC) digestion and it can be selectively labeled with sulfo-NHS-biotin, a membrane impermeant reagent. In contrast, PrPSc was neither released by PIPLC nor labeled with sulfo-NHS-biotin. Pulse-chase experiments showed that [35S]methionine was incorporated almost immediately into PrPC while incorporation into PrPSc molecules was observed only during the chase period. While PrPC is synthesized and degraded relatively rapidly (t1/2 approximately 5 h), PrPSc is synthesized slowly (t1/2 approximately 15 h) and appears to accumulate. These results are consistent with several observations previously made on rodent brains where PrP mRNA and PrPC levels did not change throughout the course of scrapie infection, yet PrPSc accumulated to levels exceeding that of PrPC. Our kinetic studies demonstrate that PrPSc is derived from a protease-sensitive precursor and that the acquisition of proteinase K resistance results from a posttranslational event. Whether or not prolonged incubation periods, which are a cardinal feature of prion diseases, reflect the slow synthesis of PrPSc remains to be established.
朊病毒蛋白（PrP）的细胞型和瘙痒病型分别称为PrPc和PrPSc，它们均由单拷贝染色体基因编码，且似乎是从同一2.1 kb的mRNA翻译而来。在PrPc被水解而PrPSc具有抗性的条件下，通过有限的蛋白酶解作用可区分PrPc和PrPSc。我们在此报告，通过磷脂酰肌醇特异性磷脂酶C（PIPLC）消化，PrPc可从正常对照和瘙痒病感染的小鼠神经母细胞瘤（N2a）细胞表面释放，并且它可以用磺基-NHS-生物素选择性标记，磺基-NHS-生物素是一种不能透过膜的试剂。相比之下，PrPSc既不能被PIPLC释放，也不能被磺基-NHS-生物素标记。脉冲追踪实验表明，[35S]甲硫氨酸几乎立即掺入PrPc，而仅在追踪期才观察到其掺入PrPSc分子。虽然PrPc合成和降解相对较快（半衰期约5小时），但PrPSc合成缓慢（半衰期约15小时）且似乎会积累。这些结果与先前在啮齿动物大脑上的一些观察结果一致，在瘙痒病感染过程中，PrP mRNA和PrPc水平没有变化，但PrPSc积累到超过PrPc的水平。我们的动力学研究表明，PrPSc来源于蛋白酶敏感的前体，并且对蛋白酶K抗性的获得是翻译后事件的结果。朊病毒疾病的一个主要特征是潜伏期延长，这是否反映了PrPSc的缓慢合成仍有待确定。
