Amino acid conditions near the GPI anchor attachment site of prion protein for the conversion and the GPI anchoring.
Amino acid conditions near the GPI anchor attachment site of prion protein for the conversion and the GPI anchoring.
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DOI：
10.1016/j.bbrc.2009.12.128
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发表时间：
2010-01
影响因子：
3.1
通讯作者：
M. Hizume;A. Kobayashi;H. Mizusawa;T. Kitamoto
中科院分区：
文献类型：
--
作者：
M. Hizume;A. Kobayashi;H. Mizusawa;T. Kitamoto
PrP是一种糖基化磷脂酰肌醇(GPI)锚定蛋白，其C端GPI锚定信号序列(GPI-SS)在GPI锚定前被切割。然而，GPI-SS中GPI锚定连接位点(ω位点)附近的突变已在人类遗传性Prion疾病中被发现。此外，除了仓鼠外，PrP的ω位点还没有被鉴定出来，尽管已知在其他蛋白中的ω和ω+2位点有非常严格的氨基酸限制。为了研究PrP的ω位点附近的突变对转换和GPI锚定的影响，并发现小鼠PrP的ω位点，我们系统地创建了突变的小鼠PrP，在第228到240个氨基酸残基上有所有可能的单一氨基酸替换。我们将它们导入羊瘙痒病感染的小鼠神经母细胞瘤细胞，并检测每个突变PrP的转化效率和GPI锚定。ω位点附近的突变改变了转换效率和gpi锚定效率。特别是在小鼠PrP的第230和232位密码子上，氨基酸对转换和GPI锚定的限制是严重的，尽管它们没有其他GPI锚定蛋白那么严重。只有通过GPI锚点呈现在细胞表面的突变型PrP才具有转化能力。本研究表明，GPI-SS突变会影响GPI锚定和PrP的转换效率。我们首次阐明了小鼠PrP的ω位点和ω位点附近的氨基酸条件，以及GPI锚定。
Prion protein (PrP) is a glycosylphosphatidylinositol (GPI)-anchored protein, and the C-terminal GPI anchor signal sequence (GPI-SS) of PrP is cleaved before GPI anchoring. However, mutations near the GPI anchor attachment site (the ω site) in the GPI-SS have been recognized in human genetic prion diseases. Moreover, the ω site of PrP has not been identified except hamster, though it is known that amino acid restrictions are very severe at the ω and ω+2 sites in other GPI-anchored proteins. To investigate the effect of mutations near the ω site of PrP on the conversion and the GPI anchoring, and to discover the ω site of murine PrP, we systematically created mutant murine PrP with all possible single amino acid substitutions at every amino acid residue from codon 228 to 240. We transfected them into scrapie-infected mouse neuroblastoma cells and examined the conversion efficiencies and the GPI anchoring of each mutant PrP. Mutations near the ω site altered the conversion efficiencies and the GPI anchoring efficiencies. Especially, amino acid restrictions for the conversion and the GPI anchoring were severe at codons 230 and 232 in murine PrP, though they were less severe than in other GPI-anchored proteins. Only the mutant PrPs presented on a cell surface via a GPI anchor were conversion competent. The present study shows that mutations in the GPI-SS can affect the GPI anchoring and the conversion efficiency of PrP. We clarified for the first time the ω site of murine PrP and the amino acid conditions near the ω site for the conversion as well as GPI anchoring.