Cellular responses to the expression of unstable secretory proteins in the filamentous fungus Aspergillus oryzae
Cellular responses to the expression of unstable secretory proteins in the filamentous fungus Aspergillus oryzae
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DOI：
10.1007/s00253-016-8086-3
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发表时间：
2017-01
影响因子：
5
通讯作者：
Jun-ichi Yokota;Daisuke Shiro;Mizuki Tanaka;Yasumichi Onozaki;O. Mizutani;Dararat R. Kakizono;Sakurako Ichinose;Tomoko Shintani;K. Gomi;T. Shintani
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文献类型：
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作者：
Jun-ichi Yokota;Daisuke Shiro;Mizuki Tanaka;Yasumichi Onozaki;O. Mizutani;Dararat R. Kakizono;Sakurako Ichinose;Tomoko Shintani;K. Gomi;T. Shintani
丝状真菌由于其分泌大量水解酶的能力，经常被用作重组蛋白生产的细胞工厂。然而，即使使用强转录启动子，非真菌蛋白质的产量通常远低于真菌蛋白质的产量。最近的分析表明，某些非真菌分泌蛋白的表达诱导未折叠蛋白反应（UPR），这表明它们被认为是丝状真菌中的折叠缺陷蛋白。然而，最近发现，即使是高表达的内源性分泌蛋白也能引起UPR。这些发现提出了一个问题，即丝状真菌的质量控制机制是否选择性地识别蛋白质的未折叠或错误折叠状态。在这项研究中，真菌分泌蛋白质（1，2-α-D-甘露糖苷酶; MsdS）的突变，降低其热稳定性在不同水平上表达在Aspergillussp.中。我们发现，在中等的表达水平，野生型MsdS分泌到培养基中，而突变体没有。在与缺失的thehrdA基因，这是参与内质网相关的降解途径，突变MsdS有具体增加的水平在细胞内部分，但不分泌的菌株。当过表达时，突变蛋白分泌到培养基中的程度与野生型蛋白相似;然而，突变体经历了高糖基化并诱导了UPR。α-淀粉酶（A.）减轻突变型MsdS过表达引起的UPR诱导。这些发现表明，错误折叠的MsdS和未折叠的α-淀粉酶种类可能协同作用于UPR诱导。
Filamentous fungi are often used as cell factories for recombinant protein production because of their ability to secrete large quantities of hydrolytic enzymes. However, even using strong transcriptional promoters, yields of nonfungal proteins are generally much lower than those of fungal proteins. Recent analyses revealed that expression of certain nonfungal secretory proteins induced the unfolded protein response (UPR), suggesting that they are recognized as proteins with folding defects in filamentous fungi. More recently, however, even highly expressed endogenous secretory proteins were found to evoke the UPR. These findings raise the question of whether the unfolded or misfolded state of proteins is selectively recognized by quality control mechanisms in filamentous fungi. In this study, a fungal secretory protein (1,2-α-D-mannosidase; MsdS) with a mutation that decreases its thermostability was expressed at different levels inAspergillus oryzae. We found that, at moderate expression levels, wild-type MsdS was secreted to the medium, while the mutant was not. In the strain with a deletion for thehrdAgene, which is involved in the endoplasmic reticulum-associated degradation pathway, mutant MsdS had specifically increased levels in the intracellular fraction but was not secreted. When overexpressed, the mutant protein was secreted to the medium to a similar extent as the wild-type protein; however, the mutant underwent hyperglycosylation and induced the UPR. Deletion of α-amylase (the most abundant secretory protein inA. oryzae) alleviated the UPR induction by mutant MsdS overexpression. These findings suggest that misfolded MsdS and unfolded species of α-amylase might act synergistically for UPR induction.