INTRACELLULAR ACCUMULATION OF THE CELLULAR PRION PROTEIN AFTER MUTAGENESIS OF ITS ASN-LINKED GLYCOSYLATION SITES
INTRACELLULAR ACCUMULATION OF THE CELLULAR PRION PROTEIN AFTER MUTAGENESIS OF ITS ASN-LINKED GLYCOSYLATION SITES
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DOI：
10.1093/glycob/1.1.101
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发表时间：
1990-01-01
期刊：
影响因子：
4.3
通讯作者：
PRUSINER S B
中科院分区：
文献类型：
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作者：
ROGERS M;TARABOULOS A;PRUSINER S B
朊病毒蛋白（PrPC）的细胞同种型是一种唾液酸糖蛋白，通过糖基磷脂酰肌醇锚几乎完全结合在质膜的外表面上。叙利亚仓鼠PrPC的推导氨基酸序列确定了在氨基酸181-183（Asn-Ile-Thr）和197-199（Asn-Phe-Thr）处添加Asn连接碳水化合物的两个潜在位点。我们已经改变了这些网站，用丙氨酸取代苏氨酸残基和表达的突变体蛋白质瞬时在CV1细胞利用诱变载体与T7启动子位于上游的PrP基因。通过用重组牛痘病毒感染来提供T7 RNA聚合酶。与野生型（wt）PrP相比，3种突变蛋白（PrPAla 183、PrPAla 199和PrPAla 183/199）具有降低的相对分子量。去糖基化以及合成衣霉素的存在下，降低了所有的PrP物种的相对分子量的双突变体PrPAla 183/199。我们的研究结果表明，这两个单一的网站突变朊病毒蛋白在非突变位点糖基化，他们建议，这两个潜在的网站Asn连接的糖基化利用野生型朊病毒。免疫荧光研究表明，虽然野生型PrPC定位于细胞表面，所有的突变体PrP分子积累细胞内。PrPAla 183的积累位点可能在中间高尔基体堆叠之前，因为该蛋白质不获得对糖苷内切酶H的抗性。突变型PrPC物种的细胞内位置是否与朊病毒蛋白（PrPSc）的羊瘙痒病亚型相同仍有待确定。
The cellular isoform of the prion protein (PrPC) is a sialoglycoprotein bound almost exclusively on the external surface of the plasma membrane by a glycosyl phosphatidylinositol anchor. The deduced amino acid sequence of Syrian hamster PrPC identifies two potential sites for the addition of Asn-linked carbohydrates at amino acids 181-183 (Asn-Ile-Thr) and 197-199 (Asn-Phe-Thr). We have altered these sites by replacing the threonine residues with alanine and expressed the mutant proteins transiently in CV1 cells utilizing a mutagenesis vector with the T7 promoter located upstream from the PrP gene. The T7 RNA polymerase was supplied by infection with recombinant vaccinia virus. The 3 mutant proteins (PrPAla183, PrPAla199 and PrPAla183/199) have a reduced relative molecular weight compared to wild-type (wt) PrP. Deglycosylation as well as synthesis in the presence of tunicamycin reduced the relative molecular weight of all the PrP species to that of the double mutant PrPAla183/199. Our results indicate that both single-site mutant prion proteins are glycosylated at non-mutated sites and they suggest that both potential sites for Asn-linked glycosylation are utilized in wt PrPC. Immunofluorescence studies demonstrate that while wt PrPC localizes to the cell surface, all the mutant PrP molecules accumulate intracellularly. The site of accumulation of PrPAla183 is probably prior to the mid-Golgi stack since this protein does not acquire resistance to endoglycosidase H. Whether the intracellular locations of the mutant PrPC species are the same as those identified for the scrapie isoform of the prion protein (PrPSc) remains to be established.