The introduction of an N-glycosylation site into prochymosin greatly enhances its production and secretion by Pichia pastoris.
The introduction of an N-glycosylation site into prochymosin greatly enhances its production and secretion by Pichia pastoris.
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在凝乳酶原中引入 N-糖基化位点大大增强了毕赤酵母的生产和分泌
DOI：
10.1186/s12934-022-01904-3
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发表时间：
2022-08-30
影响因子：
6.4
通讯作者：
中科院分区：
文献类型：
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N -糖基化是最重要的翻译后修饰之一。许多研究表明，N -糖基化对酵母细胞中异源糖蛋白的分泌水平有显著影响。然而，到目前为止，很少有研究对N -糖基化影响异源糖蛋白分泌的细胞内机制给出清晰统一的解释。毕赤酵母是生产异源蛋白的重要微生物细胞工厂。研究N -糖基化对异源蛋白分泌水平的影响具有重要意义。骆驼凝乳酶是一种在奶酪制造业中具有较高应用潜力的糖蛋白。我们已经在毕赤酵母GS115中表达了骆驼凝乳酶原，但较低的分泌水平限制了其工业应用。本研究试图通过N -糖基化提高凝乳酶原的分泌水平，并探索N -糖基化影响分泌的分子机制。
在凝乳酶原前肽的第34个氨基酸处添加一个N -糖基化位点显著提高了其在毕赤酵母中的分泌。N -糖基化提高了凝乳酶原的热稳定性，且不影响其酶活性。免疫沉淀结合质谱（IP - MS）分析表明，与野生型凝乳酶原（chy）相比，与N -糖基化突变体（chy34）相互作用的蛋白质数量减少，并且在chy34 - GS115细胞中所有差异相互作用蛋白（DIPs）均下调。内质网中的DIPs主要集中在错误折叠蛋白途径。在该途径的5种DIPs中，BiP的过表达显著提高了chy的分泌。可能的错误折叠蛋白识别元件UDP - 葡萄糖：糖蛋白葡萄糖基转移酶1和2（UGGT1/2）的敲除对酵母细胞的生长和凝乳酶原的分泌没有影响。
总之，N -糖基化通过调节细胞内相互作用的蛋白质增加了毕赤酵母中凝乳酶原的分泌。我们的研究结果可能有助于阐明N -糖基化影响分泌的分子机制，并为提高毕赤酵母中异源糖蛋白的分泌提供一种新的研究方法。
网络版包含补充材料，可在10.1186/s12934 - 022 - 01904 - 3获取。
Background
N-glycosylation is one of the most important post-translational modifications. Many studies have shown that N-glycosylation has a significant effect on the secretion level of heterologous glycoproteins in yeast cells. However, there have been few studies reporting a clear and unified explanation for the intracellular mechanism that N-glycosylation affect the secretion of heterologous glycoproteins so far. Pichia pastoris is an important microbial cell factory producing heterologous protein. It is of great significance to study the effect of N-glycosylation on the secretion level of heterologous protein. Camel chymosin is a glycoprotein with higher application potential in cheese manufacturing industry. We have expressed camel prochymosin in P. pastoris GS115, but the lower secretion level limits its industrial application. This study attempts to increase the secretion level of prochymosin through N-glycosylation, and explore the molecular mechanism of N-glycosylation affecting secretion.
Results
Adding an N-glycosylation site at the 34th amino acid of the propeptide of prochymosin significantly increased its secretion in P. pastoris. N-glycosylation improved the thermostability of prochymosin without affecting the enzymatic activity. Immunoprecipitation coupled to mass spectrometry (IP-MS) analysis showed that compared with the wild prochymosin (chy), the number of proteins interacting with N-glycosylated mutant (chy34) decreased, and all differential interacting proteins (DIPs) were down-regulated in chy34-GS115 cell. The DIPs in endoplasmic reticulum were mainly concentrated in the misfolded protein pathway. Among the five DIPs in this pathway, overexpression of BiP significantly increased the secretion of chy. The knockout of the possible misfolded protein recognition elements, UDP-glycose:glycoprotein glucosyltransferase 1 and 2 (UGGT1/2) had no effect on the growth of yeast cells and the secretion of prochymosin.
Conclusions
In conclusion, N-glycosylation increased the secretion of prochymosin in P. pastoris trough the adjustment of intracellular interacted proteins. The results of our study may help to elucidate the molecular mechanism of N-glycosylation affecting secretion and provide a new research method to improve the secretion of heterologous glycoprotein in P. pastoris.
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影响因子：
2.9
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通讯作者：
Martínez-Martínez I