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Genetically Encoded Boronolectin as a Specific Red Fluorescent UDP-GlcNAc Biosensor.

Jing Zhang1,2, Zefan Li1,2, Yu Pang2,3

  • 1Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, 1340 Jefferson Park Ave, Charlottesville, Virginia 22908, United States.

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|July 22, 2023
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Summary

Scientists developed a specific boronolectin biosensor using a genetically encoded protein. This tool precisely detects uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) in live cells, advancing glycobiology research.

Keywords:
boronolectindiol-containing moleculesglycobiologylectin mimicsred fluorescent UDP-GlcNAc biosensor

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Glycobiology

Background:

  • Boronolectins are synthetic lectin mimics for diol recognition, but achieving specificity is challenging.
  • Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) is crucial in metabolic sensing and cell signaling, particularly in glycosylation within the ER and Golgi.

Purpose of the Study:

  • To engineer a genetically encoded boronolectin biosensor with high specificity for UDP-GlcNAc.
  • To characterize the biosensor's function in vitro and in live mammalian cells, including its localization in the ER and Golgi.

Main Methods:

  • Incorporation of a noncanonical amino acid (p-boronophenylalanine) into a hybrid protein with peptide sequences and a circularly permuted red fluorescent protein.
  • Genetic encoding and expression of the boronic acid- and peptide-assisted UDP-GlcNAc sensor (bapaUGAc) in mammalian cells.
  • In vitro and in vivo characterization, including monitoring UDP-GlcNAc levels in response to metabolic changes and co-expression with another UDP-GlcNAc sensor (UGAcS).

Main Results:

  • Successful development of a genetically encoded boronolectin, bapaUGAc, capable of specifically binding UDP-GlcNAc.
  • Validation of bapaUGAc in live mammalian cells, demonstrating its ability to detect UDP-GlcNAc in the ER and Golgi.
  • Simultaneous monitoring of UDP-GlcNAc levels in the ER and cytosol using bapaUGAc in conjunction with UGAcS.

Conclusions:

  • The genetically encoded bapaUGAc sensor provides a specific and versatile tool for studying UDP-GlcNAc.
  • This work facilitates the development of novel boronolectins for carbohydrate detection and advances research in glycobiology and metabolic sensing.