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.
Highlights
Glycosyltransferases (GT) represent a large family of enzymes that belong to a welldefined enzymatic network that orchestrates the formation and maintenance of complex carbohydrate structures found abundantly in all living organisms [1]
Depending on the biological event to be investigated, the assay can be configured to detect variable amounts of the enzyme, luciferin [39], and ATP itself as the other substrate of luciferase
Profiling GT Substrate Selectivity with Nucleotide Detection. Because these assays can detect the activity of any nucleotide-sugar-dependent glycosyltransferase that produces the corresponding nucleotide, regardless of the acceptor substrate chemical structure, they could potentially provide a powerful strategy for specifying the nature of donor and acceptor substrates used by putative GT enzymes or validate the acceptor selectivity of known GTs
Summary
Glycosyltransferases (GT) represent a large family of enzymes that belong to a welldefined enzymatic network that orchestrates the formation and maintenance of complex carbohydrate structures found abundantly in all living organisms [1]. There is no robust assay that can be used to characterize the family of phosphoglycosyltransferases due to their nature of being localized in the membrane, the difficulties associated with their expression and purification, and the challenge of synthesizing labeled versions of their substrate to use in activity analysis [28] These assays have been used successfully to characterize glycosyltransferase activities, most still suffer from a variety of limitations that make them difficult to address all the needs of GT activity determination without relying on lengthy protocols, use of hazardous radiochemicals, special reagent synthesis, or the requirement of specialized detection instruments. We demonstrate the application of this same platform to develop luciferasebased nucleotide assays for glycosyltransferase activity detection, and we demonstrate their utility in studying the specificity of transfer of different sugars to different acceptors by glycosyltransferases from different families These bioluminescent assays were shown to be adequate for determining enzyme kinetic parameters, such as Km for donor and acceptor substrates, and for identifying GT small molecule modulators. We demonstrate that this generic GT assay platform can be used to characterize GTs from different families, such as GlcNAc transferases, fucosyltransferases, sialyltransferases, and the hard to analyze phosphoglycosyltransferases
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