Genetically Encoded Green Fluorescent Biosensors for Monitoring UDP-GlcNAc in Live Cells

Abstract

Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) is a nucleotide sugar used by glycosyltransferases to synthesize glycoproteins, glycosaminoglycans, glycolipids, and glycoRNA. UDP-GlcNAc also serves as the donor substrate for forming O-GlcNAc, a dynamic intracellular protein modification involved in diverse signaling and disease processes. UDP-GlcNAc is thus a central metabolite connecting nutrition, metabolism, signaling, and disease. There is a great interest in monitoring UDP-GlcNAc in biological systems. Here, we present the first genetically encoded, green fluorescent UDP-GlcNAc sensor (UGAcS), an optimized insertion of a circularly permuted green fluorescent protein (cpGFP) into an inactive mutant of an Escherichia coli UDP-GlcNAc transferase, for ratiometric monitoring of UDP-GlcNAc dynamics in live mammalian cells. Although UGAcS responds to UDP-GlcNAc quite selectively among various nucleotide sugars, UDP and uridine triphosphate (UTP) interfere with the response. We thus developed another biosensor named UXPS, which is responsive to UDP and UTP but not UDP-GlcNAc. We demonstrated the use of the biosensors to follow UDP-GlcNAc levels in cultured mammalian cells perturbed with nutritional changes, pharmacological inhibition, and knockdown or overexpression of key enzymes in the UDP-GlcNAc synthesis pathway. We further utilized the biosensors to monitor UDP-GlcNAc concentrations in pancreatic MIN6 β-cells under various culture conditions.