Gradually glycosylated protein C mutants (Arg178Gln and Cys331Arg) are degraded by proteasome after mannose trimming
Gradually glycosylated protein C mutants (Arg178Gln and Cys331Arg) are degraded by proteasome after mannose trimming
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DOI：
10.1160/th04-07-0403
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发表时间：
2004-12-01
影响因子：
6.7
通讯作者：
Hirosawa, S
中科院分区：
文献类型：
--
作者：
Nakahara, M;Koyama, T;Hirosawa, S
未能获得正确三维结构的蛋白质保留在内质网（ER）中，并最终在细胞内降解。我们研究了突变蛋白的降解，使用天然存在的蛋白C（PC）突变体（Arg 178 Gln和Cys 331 Arg），导致先天性缺陷。用正常或突变表达载体转染中国仓鼠卵巢（CHO）细胞。在Asn 329处引入用于Winked糖基化的不寻常序列Asn-X-Cys的突变揭示了Cys 331处的突变，其可能阻止与Cys 345形成二硫键，导致在Asn 329处不添加Winked寡糖。具有4个糖基化位点的PC突变体在ER中逐渐糖基化，并且第四个糖基化位点不太容易糖基化，如血浆中PC所报告的那样。PC 178和PC 331突变体的半衰期分别约为5和4 h。PC突变体被降解，但降解被蛋白酶体抑制剂抑制。去除葡萄糖后对稠合寡糖的甘露糖修剪靶向PC突变体以供蛋白酶体降解。葡萄糖剪切的抑制也立即导致甘露糖剪切，导致PC突变体的加速降解。这些降解被甘露糖苷酶I抑制剂kifunensine抑制。这些结果表明，甘露糖苷酶I的甘露糖修剪的起始导致蛋白酶体介导的葡萄糖修剪或未修剪的PC突变体的降解。
Proteins that fail to attain their correct three-dimensional structure are retained in the endoplasmic reticulum (ER) and eventually degraded within the cells. We investigated the degradation of mutant proteins, using naturally occurring protein C (PC) mutants (Arg178Gln and Cys331Arg) which lead to congenital deficiencies. Chinese hamster ovary (CHO) cells were transfected with normal or mutant expression vectors. The introduction of mutation at Asn329 of an unusual sequence Asn-X-Cys for Winked glycosylation revealed that the mutation at Cys331, which may preclude a formation of disulfide bond with Cys345, resulted in no addition of Winked oligosaccharicles at Asn329. PC mutants with 4 glycosylation sites were gradually glycosylated in the ER, and the fourth glycosylation site is less accessible for glycosylation as reported for PC in plasma. The half lives of PC 178 and PC331 mutants were about 5 and 4 h, respectively. PC mutants were degraded, but the degradation was inhibited by inhibitors for proteasome. Mannose trimming of Winked oligosaccharides after glucose removal targeted PC mutants for degradation by proteasomes. And also the inhibition of glucose trimming immediately led to mannose trimming, resulting in the accelerated degradation of PC mutants. These degradations were inhibited by mannosidase I inhibitor, kifunensine. These results indicate that the initiation of mannose trimming by mannosidase I leads to the proteasome-mediated degradation of glucose-trimmed or untrimmed PC mutants.