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

Abstract

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.

Development, Characterization, and Structural Analysis of a Genetically Encoded Red Fluorescent Peroxynitrite Biosensor

“…Screening the libraries for increased brightness at 37 °C led to the identification of an enhanced cpmApple mutant (ecpApple), which is three mutations (S9G, E135K, and M206V) away from cpmApple (Figure S1). Next, taking inspiration from pnGFP and pnGFP-Ultra, we introduced p BoF to the chromophore-forming tyrosine residue of ecpApple via genetic code expansion. The codon of residue 176 of ecpApple was mutated to TAG (amber codon), and an amber suppression plasmid, pEvol- p BoF, which expresses orthogonal, p BoF-specific tRNA and aminoacyl-tRNA synthetase in Escherichia coli, was used to incorporate p BoF in response to the amber codon. , Unfortunately, our prepared protein (ecpApple-Y176B) did not show robust fluorescence responses to peroxynitrite.…”

“…The generation of ecpApple from R-GECO1 was described elsewhere . Next, overlap extension polymerase chain reactions (PCRs) were used to introduce the TAG amber codon to specific residue positions of ecpApple.…”

“…The generation of ecpApple from R-GECO1 was described elsewhere. 36 Next, overlap extension polymerase chain reactions (PCRs) were used to introduce the TAG amber codon to specific residue positions of ecpApple. To introduce the TAG codon to residue 14, oligos ecpApple-S14TAG-F and cpRFP_R (Table S2) were first used to amplify a fragment from ecpApple; next, oligos cpRFP_F and cpRFP_R were used to further amplify and extend the fragment recovered from the previous reaction.…”

Illuminating Anions in Biology with Genetically Encoded Fluorescent Biosensors

“…Expanding the color palette of anion-sensitive FP biosensors is key to enable multiplex imaging. Along these lines, to generate a red-shifted biosensor for multicolor imaging of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) between the cytosol, ER, and Golgi apparatus, Zhang et al first engineered the reporter domain cpmApple to improve its brightness resulting in ecpApple [66]. To recognize UDP-GlcNAc, two lectin peptides were inserted into the b-bulge of ecpApple, and further supported by a nearby noncanonical amino acid p-boronophenylalanine in the FP, resulting in bapaUGAc.…”

“…To recognize UDP-GlcNAc, two lectin peptides were inserted into the b-bulge of ecpApple, and further supported by a nearby noncanonical amino acid p-boronophenylalanine in the FP, resulting in bapaUGAc. As a proof of concept, bapaUGAc was targeted to the ER and paired with the green UDP-GlcNAc biosensor UGAcS in the cytosol of HEK293T cells [66,67]. Dual-color imaging with both biosensors showed the conversion of exogenously supplemented glucosamine (GlcN) to UDP-GlcNAc in the cytosol and eventual transport to the ER (Figure 3b).…”