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GDP-Glo™ Glycosyltransferase Assay Instructions for Use of Products VA1090, VA1091 and VA1092

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GDP-Glo™ Glycosyltransferase Assay Instructions for Use of Products VA1090, VA1091 and VA1092 TECHNICAL MANUAL GDP-Glo™ Glycosyltransferase Assay Instructions for Use of Products VA1090, VA1091 and VA1092 10/17 TM505 GDP-Glo™ Glycosyltransferase Assay All technical literature is available at: www.promega.com/protocols/ Visit the web site to verify that you are using the most current version of this Technical Manual. E-mail Promega Technical Services if you have questions on use of this system: [email protected] 1. Description .........................................................................................................................................1 2. Product Components and Storage Conditions ........................................................................................6 3. Preparing for the GDP-Glo™ Glycosyltransferase Assay .........................................................................9 3.A. Preparing the GDP Detection Reagent ..........................................................................................9 3.B. Generating a Standard Curve for GDP ........................................................................................ 10 4. GDP-Glo™ Glycosyltransferase Assay Protocols ................................................................................... 11 4.A. GDP-Glo™ Glycosyltransferase Assay Protocol ............................................................................ 11 4.B. Optimizing Glycosyltransferase Reaction Conditions ................................................................... 12 4.C. Determining Km Value for FUT7 Substrate GDP-Fucose ............................................................... 15 5. General Considerations ..................................................................................................................... 18 6. References ........................................................................................................................................ 19 7. Composition of Buffers and Solutions ................................................................................................. 20 8. Related Products ............................................................................................................................... 20 1. Description The GDP-Glo™ Glycosyltransferase Assay(a) is a bioluminescent assay for detecting the activity of glycosyltransferases that use GDP-sugars as donor substrates and release GDP as a product. Glycosylating reactions catalyzed by glycosyltransferases (GTs) are central to many biological processes, including cell-cell interactions, cell signaling and bacterial cell wall biosynthesis (1). Because of the importance of this class of enzymes, there is a need for biochemical assays to monitor their activity, their mode of regulation, and to search for their selective and potent inhibitors. Glycosyltransferases transfer sugar from a nucleotide-glycosyl donor to an acceptor molecule. GDP-sugar is one of the most utilized sugar donors for glycosylating enzymes after UDP-sugars (e.g., GDP-fucose and GDP-mannose; 2). In a glycosyltransferase reaction, the GDP moiety is released as a product. Therefore, an assay that detects GDP as product of these reactions would be suitable for monitoring the activity of all the GDP-sugar-utilizing glycosyltransferases. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 1 www.promega.com TM505 · 10/17 1. Description (continued) The GDP-Glo™ Glycosyltransferase Assay is a homogeneous, one-step-reagent-addition method to rapidly detect GDP formation in glycosyltransferase reactions. After the glycosyltransferase reaction, an equal volume of GDP Detection Reagent is added to simultaneously convert the GDP product to ATP and generate light in a luciferase reaction. The light generated is detected using a luminometer (Figure 1). Luminescence can be correlated to GDP concentration by using a GDP standard curve. The light output is proportional to the concentration of GDP from low nM to 25µM (Figure 2, Panel A). The assay is easy to use and highly sensitive (Figure 2, Panel B), two features that are desirable and essential for measuring the activity of different GDP-sugar-utilizing glycosyltransferases such as fucosyltransferases (Figure 3). Therefore, the GDP detection assay uses less enzyme in glycosyltransferase reactions. This assay is fast and simple (Figure 4). The GDP-Glo™ Assay is performed in a single well of a multiwell plate and can be used to detect glycosyltransferase activity in as little as a 5µl reaction. GDP Detection Assay OH HO O HO O GDP Detection Glycosyltransferase OH Reagent + GDP Light One-step reaction OH converts GDP to ATP HO and generates light. O 14115MA GDP Glycosyltransferase HO O Reaction products substrate OH GDP-sugar substrate GDP-forming reaction Figure 1. GDP-Glo™ Glycosyltransferase Assay principle. The assay is performed in one step. After the glycosyltransferase reaction, GDP Detection Reagent is added to convert GDP to ATP and measure the newly synthesized ATP using a luciferase/luciferin reaction. The light generated correlates to the amount of GDP produced by the glycosyltransferase, which indicates glycosyltransferase activity. The GDP-Glo™ Glycosyltransferase Assay relies on the properties of a proprietary thermostable luciferase (Ultra-Glo™ Recombinant Luciferase) that is formulated to generate a stable glow-type luminescent signal and improve performance across a wide range of assay conditions. The signal produced by the luciferase reaction initiated by adding the GDP Detection Reagent is stable for more than 3 hours (Figure 2, Panel C). This extended stability eliminates the need for a luminometer equipped with injectors and allows batch-mode processing of multiple plates. Furthermore, the combination of Ultra-Glo™ Recombinant Luciferase (3) and proprietary formulation of the GDP Detection Reagent results in luminescence that is much less susceptible to interference from library compounds than other luciferase- or fluorescence- based assays (4). In addition to providing biochemical values (e.g., Km of GDP-sugars or Km of acceptor substrates) comparable to those reported in the literature, the GDP-Glo™ Glycosyltransferase Assay can be used in screening for specific glycosyltransferase inhibitors and the study of their mode of action. 2 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 TM505 · 10/17 www.promega.com A. 9 × 106 8 × 106 7 × 106 6 × 106 r2 = 0.9945 5 × 106 4 × 106 3.5 × 105 3.0 × 105 2.5 × 105 r2 = 0.9999 3 × 106 2.0 × 105 Luminescence (RLU) 1.5 × 105 1.0 × 105 6 2 × 10 0.5 × 105 Luminescence (RLU) 0 0 0.2 0.4 0.6 0.8 1.0 6 1 × 10 GDP (µM) 0 0510 15 20 25 30 GDP (µM) B. GDP (µM) 25 12.5 6.25 3.13 1.56 0.78 0.39 0.20 0.10 0.05 0.02 0 Signal-to-BackgroundSignal-to-Background Ratio 335,1245,124 19,75819,758 110,8980,898 5,6825,682 2,962,9655 1,4571,457 771515 369 1818339966446611 atat 60 minutesminutes C. 140 12.5µM GDP 6.3µM GDP 120 3.1µM GDP 1.6µM GDP 100 0.8µM GDP 80 0.4µM GDP 0.2µM GDP 60 0.1µM GDP 0.05µM GDP 40 0.02µM GDP ercent Signal Remaining P 20 0 1 hour 3 hours 14116MA Time Figure 2. Linearity and sensitivity of the GDP-Glo™ Glycosyltransferase Assay. Panel A. GDP standard curve was prepared over the indicated range of GDP concentrations in 25μl of 1X glycosyltransferase reaction buffer in a solid white 96-well plate. (Standard curve preparation is described in Section 3.B.) GDP-Glo™ Glycosyltransferase Assay was performed using 25μl of GDP Detection Reagent at room temperature as described in Section 4. Luminescence was recorded using a GloMax® 96 Microplate Luminometer (Cat.# E6501). Values represent the mean of four replicates. Panel B. Luminescence was measured 1 hour after adding the GDP Detection Reagent, and signal-to-background ratios calculated for each concentration of the GDP standard curve. Panel C. To determine signal stability, luminescence was recorded at 1 hour and 3 hours after adding the GDP Detection Reagent. Values represent the mean of two replicates. The signal-to-background ratio did not change over the time measured (data not shown). RLU = relative light units. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 3 www.promega.com TM505 · 10/17 1. Description (continued) A. B. 6 × 106 8 × 106 GDP-Fucose + α-Lactose GDP-Fucose + Acetyl Lactosamine 5 × 106 6 × 106 4 × 106 3 × 106 4 × 106 2 × 106 Luminescence (RLU) Luminescence (RLU) 2 × 106 1 × 106 0 0 0.1 110100 1,000 0.01 0.1 110100 1,000 Fucosyltransferase 2 (ng) Fucosyltransferase 3 (ng) C. 1.5 × 107 GDP-Fucose + Fetuin 1.3 × 107 1.0 × 107 15ng FUT7 Signal-to-background ratio of 35 0.75 × 107 0.50 × 107 Luminescence (RLU) 0.25 × 107 0 14117MA 0.1 110100 1,000 Fucosyltransferase 7 (ng) Figure 3. Detection of the activity of various GDP-sugar-utilizing enzymes. Panel A. FUT2, (R&D Systems Cat.# 7770-GT) was titrated in 1X FUT2 reaction buffer the presence of 40µM of Ultra Pure GDP-Fucose (Cat.# VA1097) and 10mM α-Lactose (Sigma Cat.# L2643) as an acceptor substrate. Panel B. FUT3 (R&D Systems Cat.# 4950-GT) was titrated in 1X FUT3 reaction buffer in the presence of 40µM of Ultra Pure GDP-Fucose and 100mM Acetyl Lactosamine (Carbosynth Cat.# OA08244), as an acceptor substrate. Panel C. FUT7, (R&D Systems Cat.# 6409-GT) was titrated in 1X FUT7 reaction buffer the presence of 40µM of Ultra Pure GDP-Fucose and 20µM Fetuin
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