Genetically Encoded Boronolectin as a Specific Red Fluorescent UDP-GlcNAc Biosensor
Genetically Encoded Boronolectin as a Specific Red Fluorescent UDP-GlcNAc Biosensor
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基因编码的硼凝集素作为特定红色荧光 UDP-GlcNAc 生物传感器
DOI：
10.1101/2023.03.01.530644
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Hui
中科院分区：
文献类型：
--
作者：
Jing Zhang;Zefan Li;Yu Pang;Yichong Fan;Hui
合成的含硼酸官能团的凝集素模拟物，用于可逆识别含二醇的分子，如糖链和核糖核苷酸，引起了人们的极大兴趣。然而，获得特异性仍然是一个巨大的挑战。在这里，我们提出了一种基因编码的硼油凝集素，它是一种杂交蛋白，由非规范氨基酸(NCAA)对硼苯丙氨酸(PBoF)、天然凝集素衍生的肽序列和环状排列的红色荧光蛋白(CpRFP)组成。基因的可编码性使得一种简单的蛋白质工程过程可以获得一种红色荧光生物传感器，该传感器可以与尿苷二磷酸N-乙酰氨基葡萄糖(UDP-GlcNAc)特异性结合，UDP-GlcNAc是一种参与代谢传感和细胞信号传递的重要核苷酸糖。我们在体外和活的哺乳动物细胞中进一步鉴定了得到的硼酸和多肽辅助的UDP-GlcNAc传感器(BapaUGAc)。由于内质网(ER)和高尔基体中的UDP-GlcNAc在内质网(ER)和高尔基体中的糖基化生物分子中起着至关重要的作用，我们在内质网和高尔基体中进行了遗传表达，并验证了该传感器对代谢干扰和药物抑制的响应。此外，我们将bapaUGAc与最近报道的基于另一种传感机制的绿色荧光UDP-GlcNAc传感器UGAcS相结合，以同时监测内质网和细胞质中UDP-GlcNAc水平的变化。我们希望我们的工作能够促进未来碳水化合物专用硼烷凝集素的开发。此外，这种新开发的基因编码的bapaUGAc传感器将是研究UDP-GlcNAc和糖生物学的有价值的工具。
There is great interest in developing boronolectins, which are synthetic lectin mimics containing a boronic acid functional group for reversible recognition of diol-containing molecules, such as glycans and ribonucleotides. However, it remains a significant challenge to gain specificity. Here, we present a genetically encoded boronolectin, which is a hybrid protein consisting of a noncanonical amino acid (ncAA) p-boronophenylalanine (pBoF), natural-lectin-derived peptide sequences, and a circularly permuted red fluorescent protein (cpRFP). The genetic encodability permitted a straightforward protein engineering process to derive a red fluorescent biosensor that can specifically bind uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), an important nucleotide sugar involved in metabolic sensing and cell signaling. We further characterized the resultant boronic acid-and peptide-assisted UDP-GlcNAc sensor (bapaUGAc) both in vitro and in live mammalian cells. Because UDP-GlcNAc in the endoplasmic reticulum (ER) and Golgi apparatus plays essential roles in glycosylating biomolecules in the secretory pathway, we genetically expressed bapaUGAc in the ER and Golgi and validated the sensor for its responses to metabolic disruption and pharmacological inhibition. In addition, we combined bapaUGAc with UGAcS, a recently reported green fluorescent UDP-GlcNAc sensor based on an alternative sensing mechanism, to monitor UDP-GlcNAc level changes in the ER and cytosol simultaneously. We expect our work to facilitate the future development of specific boronolectins for carbohydrates. In addition, this newly developed genetically encoded bapaUGAc sensor will be a valuable tool for studying UDP-GlcNAc and glycobiology.
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影响因子：
15
作者：
Chen ZJ;Tian Z;Kallio K;Oleson AL;Ji A;Borchardt D;Jiang DE;Remington SJ;Ai HW
通讯作者：
Ai HW
影响因子：
15
作者：
Li, Minyong;Lin, Na;Huang, Zhen;Du, Lupei;Altier, Craig;Fang, Hao;Wang, Binghe
通讯作者：
Wang, Binghe
影响因子：
16.6
作者：
Brustad, Eric;Bushey, Mark L.;Schultz, Peter G.
通讯作者：
Schultz, Peter G.
影响因子：
6.8
作者：
Parker, Joanne L.;Newstead, Simon
通讯作者：
Newstead, Simon
尿苷二磷酸 N-乙酰基-D-葡萄糖胺的酶法分析。
影响因子：
2.9
作者：
Namboori,SeemaC;Graham,DavidE
通讯作者：
Graham,DavidE