Zhang Jing, Li Zefan, Pang Yu, Fan Yichong, Ai Hui-Wang
Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, 1340 Jefferson Park Ave, Charlottesville, Virginia, 22908, USA.
Center for Membrane and Cell Physiology, University of Virginia School of Medicine, Charlottesville, Virginia, 22908, USA.
bioRxiv. 2023 Mar 1:2023.03.01.530644. doi: 10.1101/2023.03.01.530644.
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.
开发硼凝集素引起了人们极大的兴趣，硼凝集素是一种合成的凝集素模拟物，含有硼酸官能团，可用于可逆识别含二醇的分子，如聚糖和核糖核苷酸。然而，获得特异性仍然是一个重大挑战。在这里，我们展示了一种基因编码的硼凝集素，它是一种杂合蛋白，由非天然氨基酸（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。基因可编码性允许进行直接的蛋白质工程过程，以获得一种红色荧光生物传感器，该传感器可以特异性结合尿苷二磷酸N-乙酰葡糖胺（UDP-GlcNAc），这是一种参与代谢传感和细胞信号传导的重要核苷酸糖。我们进一步在体外和活的哺乳动物细胞中对所得的硼酸和肽辅助UDP-GlcNAc传感器（bapaUGAc）进行了表征。由于内质网（ER）和高尔基体中的UDP-GlcNAc在分泌途径中对生物分子进行糖基化方面起着至关重要的作用，我们在ER和高尔基体中基因表达了bapaUGAc，并验证了该传感器对代谢破坏和药理抑制的反应。此外，我们将bapaUGAc与UGAcS（一种最近报道的基于另一种传感机制的绿色荧光UDP-GlcNAc传感器）相结合，以同时监测ER和细胞质中UDP-GlcNAc水平的变化。我们期望我们的工作能够促进未来针对碳水化合物的特异性硼凝集素的开发。此外，这种新开发的基因编码bapaUGAc传感器将成为研究UDP-GlcNAc和糖生物学的有价值工具。
