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

Jing Zhang^1,2, Zefan Li^1,2, Yu Pang^2,3

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

Biorxiv : the Preprint Server for Biology

|March 13, 2023

Summary

Researchers developed a genetically encoded boronolectin biosensor for specific detection of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). This tool aids in studying cellular metabolism and glycosylation in live cells.

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Area of Science:

Biochemistry
Molecular Biology
Chemical Biology

Background:

Boronolectins are synthetic lectin mimics with boronic acid groups for recognizing diols like glycans.
Achieving specific recognition remains a significant challenge in boronolectin development.
Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) is crucial for metabolic sensing and cell signaling.

Approach:

Engineered a genetically encoded boronolectin by incorporating a noncanonical amino acid (p-boronophenylalanine) into a peptide sequence fused with a circularly permuted red fluorescent protein (cpRFP).
Developed a red fluorescent biosensor, bapaUGAc, capable of specifically binding UDP-GlcNAc.
Validated the sensor in vitro and in live mammalian cells, including expression in the endoplasmic reticulum (ER) and Golgi apparatus.

Key Points:

The genetically encoded bapaUGAc sensor demonstrates specific binding to UDP-GlcNAc.
The sensor successfully monitored UDP-GlcNAc levels in the ER and Golgi in response to metabolic changes and drug treatments.
Combined bapaUGAc with a green fluorescent sensor (UGAcS) for simultaneous monitoring of UDP-GlcNAc in ER and cytosol.

Conclusions:

This work facilitates the development of specific boronolectins for carbohydrate detection.
The genetically encoded bapaUGAc sensor is a valuable tool for glycobiology research and studying UDP-GlcNAc.
Enables real-time monitoring of crucial nucleotide sugar dynamics in cellular compartments.