EDEM2 stably disulfide-bonded to TXNDC11 catalyzes the first mannose trimming step in mammalian glycoprotein ERAD
EDEM2 stably disulfide-bonded to TXNDC11 catalyzes the first mannose trimming step in mammalian glycoprotein ERAD
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DOI：
10.7554/elife.53455
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发表时间：
2020-02-17
期刊：
影响因子：
7.7
通讯作者：
Mori, Kazutoshi
中科院分区：
文献类型：
--
作者：
George, Ginto;Ninagawa, Satoshi;Mori, Kazutoshi
N-聚糖的顺序甘露糖修剪（Man（9）GlcNAc（2）-> Man（8）GlcNAc（2）-> Man（7）GlcNAc（2））促进错误折叠的糖蛋白（gpERAD）的内质网相关降解。我们在人HCT 116细胞中的基因敲除实验表明，EDEM 2是第一步所必需的。然而，先前显示纯化的EDEM 2在体外对Man（9）GlcNAc（2）没有表现出α 1，2-甘露糖苷酶活性。在这里，我们发现，EDEM 2是稳定的二硫键连接到TXNDC 11，内质网蛋白含有五个硫氧还蛋白（Trx）样结构域。存在于EDEM 2的甘露糖苷酶同源结构域之外的C558与Trx 5中的C692连接，Trx 5仅包含TXNDC 11中的CXXC基序。这种共价键合对于HCT 116细胞中的甘露糖修剪和随后的gpERAD是必不可少的。此外，从转染的HCT 116细胞纯化的EDEM 2-TXNDC 11复合物在体外将Man（9）GlcNAc（2）转化为Man（8）GlcNAc（2）（异构体B）。我们的研究结果确立了EDEM 2作为gpERAD启动子的作用，并代表了EDEM家族蛋白体外甘露糖苷酶活性的首次明确证明。
Sequential mannose trimming of N-glycan (Man(9)GlcNAc(2) -> Man(8)GlcNAc(2) -> Man(7)GlcNAc(2)) facilitates endoplasmic reticulum-associated degradation of misfolded glycoproteins (gpERAD). Our gene knockout experiments in human HCT116 cells have revealed that EDEM2 is required for the first step. However, it was previously shown that purified EDEM2 exhibited no alpha 1,2-mannosidase activity toward Man(9)GlcNAc(2) in vitro. Here, we found that EDEM2 was stably disulfide-bonded to TXNDC11, an endoplasmic reticulum protein containing five thioredoxin (Trx)-like domains. C558 present outside of the mannosidase homology domain of EDEM2 was linked to C692 in Trx5, which solely contains the CXXC motif in TXNDC11. This covalent bonding was essential for mannose trimming and subsequent gpERAD in HCT116 cells. Furthermore, EDEM2-TXNDC11 complex purified from transfected HCT116 cells converted Man(9)GlcNAc(2) to Man(8)GlcNAc(2)(isomerB) in vitro. Our results establish the role of EDEM2 as an initiator of gpERAD, and represent the first clear demonstration of in vitro mannosidase activity of EDEM family proteins.