Juliano Alves 1 , Jacquelyn Hennek 1,2 , Said A. Goueli 1,three and Hicham Zegzouti 1, 2Promega Corporation, R D Department, 2800 Woods Hollow Road, ETA Activator Storage & Stability Madison, WI 53719, USA; laurie.engel@promega (L.E.); juliano.alves@promega (J.A.); jhennek@exactsciences (J.H.); said.goueli@promega (S.A.G.) Exact Sciences Corporation, 5505 Endeavor Lane, Madison, WI 53719, USA Department of Pathology and Laboratory Medicine, University of Wisconsin College of Medicine and Public Well being, Madison, WI 53719, USA Correspondence: hicham.zegzouti@promegaCitation: Engel, L.; Alves, J.; Hennek, J.; Goueli, S.A.; Zegzouti, H. Utility of Bioluminescent Homogeneous Nucleotide Detection Assays in Measuring Activities of Nucleotide-Sugar Dependent Glycosyltransferases and Studying Their Inhibitors. Molecules 2021, 26, 6230. doi.org/10.3390/ moleculesAbstract: Regular glycosyltransferase (GT) activity assays usually are not easily configured for speedy detection nor for higher throughput screening since they depend on radioactive item isolation, the use of heterogeneous immunoassays or mass spectrometry. Within a standard glycosyltransferase biochemical reaction, two items are generated, a glycosylated item along with a nucleotide released from the sugar donor substrate. For that reason, an assay that detects the nucleotide could be universal to monitor the activity of diverse glycosyltransferases in vitro. Here we describe 3 homogeneous and bioluminescent glycosyltransferase activity assays based on UDP, GDP, CMP, and UMP detection. Each and every of these assays are performed in a one-step detection that relies on converting the nucleotide solution to ATP, then to bioluminescence Estrogen receptor Inhibitor drug employing firefly luciferase. These assays are very sensitive, robust and resistant to chemical interference. Different applications of those assays are presented, which includes studies around 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 also the study of their mode of action are described. We believe that the broad utility of those nucleotide assays will enable the investigation of a large number of GTs and may have a substantial effect on diverse areas of Glycobiology analysis. Keyword phrases: nucleotide assays; bioluminescence; sugar substrate; fucosyltransferase; OGT; inhibitorAcademic Editor: Stefan Janecek Received: 16 September 2021 Accepted: 12 October 2021 Published: 15 October1. Introduction Glycosyltransferases (GT) represent a sizable family of enzymes that belong to a welldefined enzymatic network that orchestrates the formation and maintenance of complicated carbohydrate structures located abundantly in all living organisms [1]. Working with activated sugars as donor substrates, glycosyltransferases transfer the sugar moiety to an array of acceptor substrates of several chemical natures, like proteins, lipids, sugars, nucleic acids, and modest molecules [2]. Essentially the most widespread donor substrates utilized by glycosyltransferases are nucleotide-activated sugars, including UDP-, GDP-, and CMP-sugars, but they also can use lipid sugar phosphates (e.g., dolichol phosphate sugar), and unsubstituted phosphates. Glycosyltransferases that use nucleotide-activated sugars are named Leloir enzymes, in honor of your 1970 chemistry Nobel Prize winner Luis F. Leloir, who found the first sugar nucleotide [3]. Due to the importance of the several oligosaccharide structures to cell f
