Utility of Bioluminescent Homogeneous Nucleotide Detection Assays in Measuring Activities of Nucleotide-Sugar Dependent Glycosyltransferases and Studying Their Inhibitors

Abstract

Traditional glycosyltransferase (GT) activity assays are not easily configured for rapid detection nor for high throughput screening because they rely on radioactive product isolation, the use of heterogeneous immunoassays or mass spectrometry. In a typical glycosyltransferase biochemical reaction, two products are generated, a glycosylated product and a nucleotide released from the sugar donor substrate. Therefore, an assay that detects the nucleotide could be universal to monitor the activity of diverse glycosyltransferases in vitro. Here we describe three homogeneous and bioluminescent glycosyltransferase activity assays based on UDP, GDP, CMP, and UMP detection. Each of these assays are performed in a one-step detection that relies on converting the nucleotide product to ATP, then to bioluminescence using firefly luciferase. These assays are highly sensitive, robust and resistant to chemical interference. Various applications of these assays are presented, including studies on the specificity of sugar transfer by diverse GTs and the characterization of acceptor substrate-dependent and independent nucleotide-sugar hydrolysis. Furthermore, their utility in screening for specific GT inhibitors and the study of their mode of action are described. We believe that the broad utility of these nucleotide assays will enable the investigation of a large number of GTs and may have a significant impact on diverse areas of Glycobiology research.

Glycosyltransferase 8 domain-containing protein 1 (GLT8D1) is a UDP-dependent galactosyltransferase

“…Here, GLT8D1 appeared to hydrolyze UDP-Gal, UDP-Xyl, UDP-GalNAc, UDP-GlcA, UDP-GalA, UDP-Arap and UDP-Araf in the presence of Mn 2+ , but the activity values were low compared with controls of well-described glycosyltransferases B4GALT1 and B4GALT1-Y289L. Some GTs are acceptor-dependent enzymes, such as, fucosyltransferase 7 39 , which could be the case of GLT8D1, and only assays in the presence of the proper acceptor substrate would demonstrate activity. Other reports from the literature showed UDP-Gal hydrolysis by full-length GLT8D1 9 , 15 , and also UDP-Glc hydrolysis 15 but the activity values also appear low.…”

“…Many GTs are capable of hydrolyzing their sugar nucleotide donors in the absence of any acceptor, which is particularly useful to characterize activities when the acceptor substrates are unknown 38 , 39 . Here, GLT8D1 appeared to hydrolyze UDP-Gal, UDP-Xyl, UDP-GalNAc, UDP-GlcA, UDP-GalA, UDP-Arap and UDP-Araf in the presence of Mn 2+ , but the activity values were low compared with controls of well-described glycosyltransferases B4GALT1 and B4GALT1-Y289L.…”

Structure-function and engineering of plant UDP-glycosyltransferase

“… Screening target Method Application scenarios Screening sample Reference Products Transfer a radiolabeled sugar to the acceptor, and then remove the unreacted sugar donors and detect the labeled products All UGTs Purified protein, cell lysate [77] Products Utilize cell-permeable fluorescence-labeled acceptors to generate cell-impermeable fluorescence-labeled product, and then screen by FACS. Specific UGTs Whole cell [78] , [79] UDP Synthesize ATP with UDP, then transfer ATP into bioluminescence signal with UDP-Glo™ All UGTs Purified protein [80] UDP Synthesize Glucose-6-phospate with UDP step by step, then measure Glucose-6-phospate with NADP and resazurin. All UGTs Purified protein [81] , [82] UDP Utilize OleD to catalyze production of colorimetric 2-chloro-4-nitrophenolate with UDP All UGTs Purified protein, cell lysate [41] , [83] , [84] , [85] UDP Use rYND1 to hydrolyze UDP into UMP and Pi, then use a phosphorus molybdenum blue chromogenic reaction to detect Pi.…”

“…These analytical methods were normally based on fluorescent, luminescent, or colorimetric signals ( Table 2 ). Some of the reported methods, such as the UDP-Glo™ assay [80] and the NADP-resazurin assay [81] , require purified UGT protein as they are sensitive to background signals from living cells or cell lysates. While these methods are suitable for screening chemical inhibitors targeting specific UGT proteins, they do not meet the high-throughput requirements for the selection of novel UGTs from mutation libraries.…”

Glycosyltransferases: Mining, engineering and applications in biosynthesis of glycosylated plant natural products

“…Some progress has been made recently, such as the development of a high-throughput screening method by using mass spectrometry [ 122 ]. Another publication has recently highlighted the potential for a fluorescence-based method to universally monitor the activity of GTs by detecting the nucleotides generated in a biochemical reaction [ 123 ].…”