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首页> 外文期刊>Molecular and Cellular Biology >Biogenesis and transmembrane orientation of the cellular isoform of the scrapie prion protein [published errratum appears in Mol Cell Biol 1987 May;7(5):2035]
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Biogenesis and transmembrane orientation of the cellular isoform of the scrapie prion protein [published errratum appears in Mol Cell Biol 1987 May;7(5):2035]

机译：瘙痒病病毒蛋白的细胞同工型的生物发生和跨膜方向[发表的勘误出现在Mol Cell Biol 1987年5月; 7（5）：2035]

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Considerable evidence suggests that the scrapie prion protein (PrP) is a component of the infectious particle. We studied the biogenesis and transmembrane orientation of an integral-membrane form of PrP in a cell-free transcription-linked translation-coupled translocation system programmed with a full-length PrP cDNA cloned behind the SP6 promoter. Translation of SP6 transcripts of the cDNA or of native mRNA from either normal or infected hamster brain in the absence of dog pancreas membranes resulted in the synthesis of a single PrP immunoreactive polypeptide (each polypeptide was the same size; Mr, 28,000), as predicted from the known sequence of the coding region. In the cotranslational presence of membranes, two additional forms were observed. Using peptide antisera specific to sequences from the amino- or the carboxy-terminal domain of PrP together with proteinase K or endoglycosidase H digestion or both, we showed that one of these forms included an integrated and glycosylated form of PrP (Mr = 33,000) which spans the bilayer twice, with domains of both the amino and carboxy termini in the extracytoplasmic space. By these criteria, the other form appeared to be an unglycosylated intermediate of similar transmembrane orientation. The PrP cell-free translation products did not display resistance to proteinase K digestion in the presence of nondenaturing detergents. These results suggest that the PrP cell-free translation products most closely resemble the normal cellular isoform of the protein, since its homolog from infected brain was proteinase K resistant. The implications of these findings for PrP structure and function are discussed.
机译：大量证据表明，瘙痒病病毒蛋白（PrP）是感染性颗粒的组成部分。我们研究了PrP的完整膜形式的生物发生和跨膜方向在无细胞的转录连接翻译偶联易位系统中编程，该系统由克隆在SP6启动子后面的全长PrP cDNA编程。在没有狗胰膜的情况下，来自正常或受感染的仓鼠大脑的cDNA或天然mRNA的SP6转录物的翻译导致了单个PrP免疫反应性多肽的合成（每个多肽大小相同; Mr，28,000）来自编码区的已知序列。在膜的共翻译存在下，观察到两种其他形式。使用对PrP的氨基末端或羧基末端结构域序列具有特异性的肽段抗血清，以及蛋白酶K或糖苷内切酶H消化或两者同时进行，我们显示这些形式之一包括整合的糖基化PrP（Mr = 33,000），其中跨越双层两次，在胞外空间具有氨基和羧基末端的结构域。根据这些标准，另一种形式似乎是类似跨膜取向的未糖基化的中间体。在非变性去污剂存在下，无PrP细胞的翻译产物对蛋白酶K的消化没有抵抗力。这些结果表明，PrP无细胞翻译产物与蛋白质的正常细胞亚型最相似，因为它来自感染脑的同源物对蛋白酶K具有抗性。讨论了这些发现对PrP结构和功能的影响。

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