Bioorganic & Medicinal Chemistry 22 (2014) 2571–2575
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
A fluorescence-based glycosyltransferase assay for high-throughput screening Jihye Ryu a, Min Sik Eom b, Wooseok Ko a, Min Su Han b,⇑, Hyun Soo Lee a,⇑ a b
Department of Chemistry, Sogang University, Seoul 121-742, Republic of Korea Department of Chemistry, Chung-Ang University, Seoul 156-756, Republic of Korea
a r t i c l e
i n f o
Article history: Received 19 December 2013 Revised 11 February 2014 Accepted 12 February 2014 Available online 2 March 2014 Keywords: Glycosyltransferase engineering Glycosyltransferase assay High-throughput screening ATP sensor
a b s t r a c t Glycosyltransferases catalyze the transfer of a monosaccharide unit from a nucleotide or lipid sugar donor to polysaccharides, lipids, and proteins in a stereospecific manner. Considerable effort has been invested in engineering glycosyltransferases to diversify sugar-containing drugs. An important requirement for glycosyltransferase engineering is the availability of a glycosyltransferase assay system for high-throughput screening of glycosyltransferase mutants. In this study, a general glycosyltransferase assay system was developed based on an ATP sensor. This system showed submicromolar sensitivity and compatibility with both purified enzymes and crude cell extracts. The assay system will be useful for glycosyltransferase engineering based on high-throughput screening, as well as for general glycosyltransferase assays and kinetics. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Carbohydrates are one of the major classes of biomolecules found in cells. They are involved in many important biological processes, including energy storage,1 cell–cell communication,2 immune response,3 inflammation and infection.4 Because of the importance of these biomolecules in biochemical and pharmaceutical sciences, much effort has been made to synthesize and modify glycans.5 A straightforward method for glycan synthesis is to prepare glycans by chemical synthesis. Although significant progress has been achieved with this method,6 the synthesis is still challenging owing to the requirement for stringent regio- and stereochemical control of the glycosyl transfer reactions. In biological systems, glycosyl transfer reactions are catalyzed by enzymes, called glycosyltransferases (GTs).7 These enzymes catalyze the transfer of a monosaccharide unit from a nucleotide or lipid sugar donor to polysaccharides, lipids, and proteins in a regio- and stereospecific manner. However, the utility of enzymatic glycan synthesis is also restricted by the limited substrate specificity of GTs.8 Recently, GTs has been engineered to catalyze glycosyl transfer reactions for diverse acceptor and donor substrates.9,10 In one example, a sialyltransferase mutant library was generated, and a variant that could transfer a sugar unit to a fluorescently labeled ⇑ Corresponding authors. Tel.: +82 2 820 5198; fax: +82 2 705 7893 (M.S.H.); tel.: +82 2 705 7958; fax: +82 2 705 7893 (H.S.L.). E-mail addresses: [email protected] (M.S. Han), [email protected] (H.S. Lee). http://dx.doi.org/10.1016/j.bmc.2014.02.027 0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
acceptor sugar with a 400-fold increased catalytic activity was found using a fluorescence-activated cell sorter (FACS).9 Another example used a simple, in vitro high-throughput screening system based on a fluorescent acceptor substrate to expand the specificity of the oleandomycin GT (OleD) for a glycosyl donor.10 Although in both cases a variety of glycans were enzymatically prepared by engineering GTs, their screening methods were based on specific fluorescent substrates and, therefore, cannot be generally applied to other GTs that use different glycosyl acceptor substrates. Although GT assays based on antibodies, radioisotopes, pH sensors, phosphatases, and fluorescent sensors are available,11–15 these assays are not suitable for GT engineering by high-throughput screening. Recently, a xanthene-based nucleoside diphosphate (NDP) sensor was used for a GT assay for high-throughput screening and showed high sensitivity and compatibility with crude cell extracts.16 Even though this GT assay system is useful and can be applied to various GTs, a critical limitation of the method is that the xanthene-based sensor requires a time-consuming multistep synthesis (twelve steps), which often prevents researchers from using this method. Herein, we report the development of a general GT assay based on a fluorescent adenosine triphosphate (ATP) sensor that can be readily prepared by synthesis. As this GT assay is highly sensitive, amenable to high-throughput screening for GT engineering, and can be applied to most Leloir-type GTs, the assay is anticipated to be useful for general GT research and especially, GT engineering.
2572
J. Ryu et al. / Bioorg. Med. Chem. 22 (2014) 2571–2575
2. Results and discussion In Leloir-type GT reactions, where sugar nucleotide derivatives are used as the glycosyl donors, a common byproduct is NDP. Therefore, when developing a new GT assay, NDP is a good target for measuring the enzymatic activity of GTs. Recently, a fluorescence-based ATP sensor was reported in which an anthracene-based Zn(II) complex ([Zn2(9,10-bis[(2,2-dipicolylamino)methyl]anthracene)]4+, Ant-Zn) and pyrocatechol violet were used.17 Because this sensor is based on fluorescence quenching and restoration, it shows low background signal and high sensitivity for ATP and ADP. Although the selectivity of the sensor for ADP over ADP-sugar, which is crucial for a GT assay, was not reported, it was expected that the sugar moiety attached to the phosphate would significantly reduce its binding affinity for the sensor.16,18,19 Based on this idea, a GT assay system was designed as shown in Figure 1. First, the sensor was tested for selectivity for NDP over NDP-sugar. Ant-Zn was prepared according to the literature,20 and thymidine diphosphate (TDP) and TDP-glucose were tested in this experiment.21 A solution containing Ant-Zn (5 lM) and pyrocatechol violet (10 lM) in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 20 mM, pH 7.0) was titrated with TDP and TDP-glucose, and fluorescence was measured at 413 nm with excitation at 380 nm. A steep increase in fluorescence was observed with increasing concentrations of TDP, while little fluorescence was seen with TDP-glucose (Fig. 2). As expected, Ant-Zn showed high sensitivity (apparent dissociation constant, K0 d = 0.19 lM) for TDP and excellent selectivity over TDP-glucose (K0 d >20 lM). Next, the sensor was evaluated in an in vitro enzymatic assay. OleD wild type (WT) and two variants (ASP and TDP16) were used in this assay, and TDP-glucose and 2-naphthol were used as the glycosyl donor and glycosyl acceptor. OleD WT and the variants are good target GTs for the development of a GT assay because they are known to have different GT activity with different donors and acceptors.10,22,23 The ASP variant was generated by using a fluorescence-based directed evolution strategy, the variant showed a broad substrate specificity for various glycosyl acceptors and donors,10,22 and the TDP16 variant was found to have improved GT activity for TDP-sugar.23 The GT reaction was performed by adding one of the OleDs (5 lM) to a solution containing 1 mM TDP-glucose, 1 mM MgCl2, and 1 mM 2-naphthol in 10 mM Tris (pH 8.0). The reaction was quenched with methanol, followed by addition of the mixture (5 lL) to an assay solution containing
Figure 2. Titration of Ant-Zn and pyrocatechol violet with TDP ( ) and TDP-glucose (TDP-G, ). Each data point represents the average of three experiments. Measurement conditions: 20 mM HEPES (pH 7.0), 5 lM Ant-Zn, and 10 lM pyrocatechol violet; excitation at 380 nm, emission at 413 nm.
5 lM Ant-Zn and 10 lM pyrocatechol in 20 mM HEPES (pH 7.0), and fluorescence was measured. In this experiment, TDP16 showed a strong, time-dependent increase in fluorescence, while OleD WT and ASP showed little or no increase in fluorescence (Fig. 3). These results are in agreement with a previous study,23 confirming that the fluorescence changes observed correlate with the enzymatic activity of the OleDs. In the absence of an enzyme or acceptor, little or no fluorescence increase was detected, demonstrating that the fluorescence increase was caused by the production of TDP from the GT reaction. The transfer of glucose from TDP-glucose to 2-naphthol was further validated by high performance liquid chromatography (HPLC) and mass analysis of the products from the GT reaction (Fig. 4). For TDP16, the most active variant, steady-state kinetic parameters were determined by performing the assay in a 96-well plate format. Initial reaction velocity was measured with different concentrations of 2-naphthol at a fixed concentration of TDP-glucose (1 mM), providing an apparent KM of 0.39 ± 0.090 mM and a kcat of 0.25 ± 0.002 min1 (Fig. S1). In order to apply this assay to high-throughput screening for GT engineering, it is necessary for the assay to be compatible with crude cell extracts. This compatibility was tested by carrying out the GT assay described above with crude cell extracts from OleD-expressing bacterial cells, instead of purified enzymes. When
Figure 1. Schematic representation of the GT assay mechanism of Ant-Zn. The fluorescence of Ant-Zn is quenched by pyrocatechol violet, and the replacement of the quencher by NDP restores the fluorescence. Measurement of this fluorescence allows quantification of the amount of NDP produced from the GT reactions.
J. Ryu et al. / Bioorg. Med. Chem. 22 (2014) 2571–2575
Figure 3. GT assay results for OleD-WT and two variants with Ant-Zn and pyrocatechol violet. Fluorescence was measured at 413 nm with excitation at 380 nm. Each data point represents the average of three experiments. GT reaction conditions: 10 mM Tris (pH 8.0), 1 mM TDP-glucose, 1 mM MgCl2, 1 mM 2-naphthol, and 5 lM OleD, room temperature; (+), in the presence of 2-naphthol; (), in the absence of 2-naphthol; the control contains no enzyme. Assay conditions: 10 lL of the reaction mixture (5 lL of the GT reaction mixture and 5 lL of methanol) was added to 190 lL of the assay solution containing 20 mM HEPES (pH 7.0), 5 lM Ant-Zn, and 10 lM pyrocatechol.
different amounts of the cell extracts from TDP16-expressing cells were used to optimize the assay condition, the best signal was obtained from 2 to 5 lL of cell extract in 50 lL of the GT reaction
2573
Figure 5. GT assay results with different amounts of cell extracts prepared from TDP16-expressing bacterial cells. Fluorescence was measured at 413 nm with excitation at 380 nm. Each data point represents the average of three experiments. GT reaction conditions: 10 mM Tris (pH 8.0), 1 mM TDP-glucose, 1 mM MgCl2, 1 mM 2-naphthol, and the indicated volume of lysate (in 50 lL of total reaction volume), room temperature.
mixture (Fig. 5). When more cell extract was used, some components of the cell extract, such as proteins, interfered with Ant-Zn, resulting in a decrease in fluorescence intensity.24 Next, the sensor was tested to ascertain whether it could measure GT activity with crude cell lysates. OleD WT, ASP, and TDP16 were expressed in bacterial cells, from which cell lysates were prepared, and the quantity
Figure 4. HPLC analysis of the product from the GT reaction catalyzed by OleD-TDP16. A, TDP; B, TDP-glucose; C, 2-naphthol; D, reaction mixture; E, control containing no enzyme. O-glucosyl-2-naphthol was analyzed by LC–MS: calculated for C16H18O6 (M+Na) 329.1, observed, 329.2.
2574
J. Ryu et al. / Bioorg. Med. Chem. 22 (2014) 2571–2575
containing 5 lM Ant-Zn and 10 lM pyrocatechol violet in HEPES (20 mM, pH 7.0). TDP or TDP-glucose at different concentrations (0.25, 0.50, 0.75, 1.00, 2.00, 3.00, 4.00, 5.00, 7.50, 10.00, 12.50, 15.00 lM, final concentrations) was added to the assay solution (200 lL, final volume) and the fluorescence was measured at 413 nm with excitation at 380 nm. 4.3. Enzymatic assays OleD-WT or one of the variants (5 lM, final concentration) was added to the reaction buffer containing 10 mM Tris (pH 8.0), 1 mM TDP-glucose, 1 mM MgCl2, 1 mM 2-naphthol, and the mixture (100 lL) was incubated at room temperature. The reaction was quenched by adding the same volume (100 lL) of methanol, the mixture (10 lL) was added to the assay solution (190 lL), and the fluorescence was measured as described for the TDP binding assay. 4.4. Crude cell extract assays Figure 6. GT assay results with crude cell extracts from OleD WT- or variantsexpressing cells. The assay was performed in 96-well plates and fluorescence was measured by a fluorescence reader at 413 nm with excitation at 380 nm. GT reaction conditions: 10 mM Tris (pH 8.0), 1 mM TDP-glucose, 1 mM MgCl2, 1 mM 2-naphthol, and 10% crude cell extracts (10 lL for 100 lL GT reaction), room temperature; (+), in the presence of 2-naphthol; (), in the absence of 2-naphthol; the blank contains the cell extract from cells expressing a blank vector.
of each OleD variant in the cell lysates was estimated by SDS–PAGE analysis to ensure that each lysate contained identical quantities of the OleD variants (Fig. S2). These extracts were used in the GT assay in a 96-well plate format, and the results are shown in Figure 6. As observed in the enzymatic assay, TDP16 was the most active variant and the others showed little GT activity, verifying that the assay system can be applied to screen GT variants with cell extracts in a high-throughput format. 3. Conclusions In summary, a general GT assay system was developed based on a synthetically accessible ATP sensor. Because the sensor is based on fluorescence quenching and restoration, and has high selectivity for NDP over NDP-sugar, the assay system displayed low background signal and high sensitivity (submicromolar). In addition, because the sensor is compatible with crude cell extracts, enzymatic activity of GTs in cell extracts can be measured. Therefore, the GT assay system will be useful for GT engineering, as well as for general GT assays and kinetics. 4. Materials and methods 4.1. General All chemicals and DNA oligomers were obtained from commercial sources and used without further purification. Fluorescence was measured on an F-7000 fluorescence spectrophotometer (Hitachi High-Technologies Corporation, Tokyo Japan) and Victor X5 multilabel plate reader (PerkinElmer, Massachusetts USA). 4.2. TDP binding assay Complex Ant-Zn stock solution was prepared by mixing 500 lL of Ant (1 mM) in 40% DMSO in water, 1000 lL of Zn(ClO4)2 (1 mM) in water and 8.5 mL of water to afford 50 lM Ant-Zn stock solution. Assay solution was prepared by adding the complex stock solution (1 mL) and 100 lM pyrocatechol violet (1 mL) in water to 8 mL of 25 mM HEPES (pH 7.0) to make 10 mL assay solution
Cells from OleD-expressing bacterial cell cultures (25 mL) were harvested by centrifugation (4000 rpm) and frozen at 80 °C. The frozen cell pellets were thawed on ice, resuspended in the lysis buffer (2 mL) containing 50 mM Tris (pH 8.0), lysozyme (1 mg/mL, 50 kU/mL) and benzonaze (125 U/mL), and incubated on ice for 1 h. Removal of the cell debris by centrifugation (12,000 rpm) afforded crude cell extracts. OleD assays with crude cell extracts were carried out as described for the assays with purified enzymes by adding crude cell extracts instead of purified enzymes to the reaction buffer. Acknowledgements This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Korea government (MSIP) (NRF-2013M2B 2A4040238, NRF-2013R1A2A2A01007000); the Converging Research Center Program through the Ministry of Education, Science and Technology (2013K000326). We would like to thank Jon S. Thorson for providing the plasmids. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.2014.02.027. References and notes 1. (a) Woods, S. C.; Seeley, R. J.; Porte, D., Jr.; Schwartz, M. W. Science 1998, 280, 1378; (b) Flatt, J.-P. Am. J. Clin. Nutr. 1995, 61, 952S. 2. Crocker, P. R.; Feizi, T. Curr. Opin. Struct. Biol. 1996, 6, 679. 3. (a) Brown, G. D.; Gordon, S. Immunity 2003, 19, 311; (b) Cambi, A.; Koopman, M.; Figdor, C. G. Cell. Microbiol. 2005, 7, 481. 4. (a) Lowe, J. B. Curr. Opin. Cell Biol. 2003, 15, 531; (b) Sacks, D.; Kamhawi, S. Annu. Rev. Microbiol. 2001, 55, 453. 5. (a) Davis, B. G. Pure Appl. Chem. 2009, 81, 285; (b) Seeberger, P. H.; Werz, D. B. Nature 2007, 446, 1046; (c) Pratt, M. R.; Bertozzi, C. R. Chem. Soc. Rev. 2005, 34, 58; (d) Hoelemann, A.; Seeberger, P. H. Curr. Opin. Biotechnol. 2004, 15, 615. 6. Zhu, X.; Schmidt, R. R. Angew. Chem., Int. Ed. 2009, 48, 1900. 7. Sinnott, M. L. Chem. Rev. 1990, 90, 1171. 8. Wagner, G. K.; Pesnot, T.; Field, R. A. Nat. Prod. Rep. 2009, 26, 1172. 9. Aharoni, A.; Thieme, K.; Chiu, C. P.; Buchini, S.; Lairson, L. L.; Chen, H.; Strynadka, N. C.; Wakarchuk, W. W.; Withers, S. G. Nat. Methods 2006, 3, 609. 10. Williams, G. J.; Zhang, C.; Thorson, J. S. Nat. Chem. Biol. 2007, 10, 657. 11. Wagner, G. K.; Pesnot, T. ChemBioChem 2010, 11, 1939. 12. Palcic, M. M.; Sujino, K. Trends Glycosci. Glycotechnol. 2001, 72, 361. 13. Wu, Z. L.; Ethen, C. M.; Prather, B.; Machacek, M.; Jiang, W. Glycobiology 2011, 21, 727. 14. Chen, X.; Jou, M. J.; Yoon, J. Org. Lett. 2009, 11, 2181. 15. Wongkongkatep, J.; Miyahara, Y.; Ojida, A.; Hamachi, I. Angew. Chem., Int. Ed. 2006, 45, 665.
J. Ryu et al. / Bioorg. Med. Chem. 22 (2014) 2571–2575 16. Lee, H. S.; Thorson, J. S. Anal. Biochem. 2011, 418, 85. 17. Jang, H. H.; Yi, S.; Kim, M. H.; Kim, S.; Lee, N. H.; Han, M. S. Tetrahedron Lett. 2009, 50, 6241. 18. Ojida, A.; Takashima, I.; Kohira, T.; Nonaka, H.; Hamachi, I. J. Am. Chem. Soc. 2008, 130, 12095. 19. Kim, S. K.; Lee, D. H.; Hong, J.-I.; Yoon, J. Acc. Chem. Res. 2009, 42, 23. 20. Ojida, A.; Mito-oka, Y.; Inoue, M.; Hamachi, I. J. Am. Chem. Soc. 2002, 124, 6256. 21. TDP and TDP-glucose were used instead of UDP and UDP-glucose because UDPglucose was hydrolyzed by OleD-ASP in the absence of a glycosyl acceptor, which caused significant background fluorescence.
2575
22. Gantt, R. W.; Goff, R. D.; Williams, G. J.; Thorson, J. S. Angew. Chem., Int. Ed. 2008, 47, 8889. 23. Williams, G. J.; Yang, J.; Zhang, C.; Thorson, J. S. ACS Chem. Biol. 2011, 6, 95. 24. We divided the cell lysate into large-molecule and small-molecule parts, and examined them to ascertain which caused a decrease in the fluorescence. We observed a significant decrease in the fluorescence owing to the large-molecule fraction while the small-molecule fraction did not alter the fluorescence.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
A fluorescence-based glycosyltransferase assay for high-throughput screening Jihye Ryu a, Min Sik Eom b, Wooseok Ko a, Min Su Han b,⇑, Hyun Soo Lee a,⇑ a b
Department of Chemistry, Sogang University, Seoul 121-742, Republic of Korea Department of Chemistry, Chung-Ang University, Seoul 156-756, Republic of Korea
a r t i c l e
i n f o
Article history: Received 19 December 2013 Revised 11 February 2014 Accepted 12 February 2014 Available online 2 March 2014 Keywords: Glycosyltransferase engineering Glycosyltransferase assay High-throughput screening ATP sensor
a b s t r a c t Glycosyltransferases catalyze the transfer of a monosaccharide unit from a nucleotide or lipid sugar donor to polysaccharides, lipids, and proteins in a stereospecific manner. Considerable effort has been invested in engineering glycosyltransferases to diversify sugar-containing drugs. An important requirement for glycosyltransferase engineering is the availability of a glycosyltransferase assay system for high-throughput screening of glycosyltransferase mutants. In this study, a general glycosyltransferase assay system was developed based on an ATP sensor. This system showed submicromolar sensitivity and compatibility with both purified enzymes and crude cell extracts. The assay system will be useful for glycosyltransferase engineering based on high-throughput screening, as well as for general glycosyltransferase assays and kinetics. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Carbohydrates are one of the major classes of biomolecules found in cells. They are involved in many important biological processes, including energy storage,1 cell–cell communication,2 immune response,3 inflammation and infection.4 Because of the importance of these biomolecules in biochemical and pharmaceutical sciences, much effort has been made to synthesize and modify glycans.5 A straightforward method for glycan synthesis is to prepare glycans by chemical synthesis. Although significant progress has been achieved with this method,6 the synthesis is still challenging owing to the requirement for stringent regio- and stereochemical control of the glycosyl transfer reactions. In biological systems, glycosyl transfer reactions are catalyzed by enzymes, called glycosyltransferases (GTs).7 These enzymes catalyze the transfer of a monosaccharide unit from a nucleotide or lipid sugar donor to polysaccharides, lipids, and proteins in a regio- and stereospecific manner. However, the utility of enzymatic glycan synthesis is also restricted by the limited substrate specificity of GTs.8 Recently, GTs has been engineered to catalyze glycosyl transfer reactions for diverse acceptor and donor substrates.9,10 In one example, a sialyltransferase mutant library was generated, and a variant that could transfer a sugar unit to a fluorescently labeled ⇑ Corresponding authors. Tel.: +82 2 820 5198; fax: +82 2 705 7893 (M.S.H.); tel.: +82 2 705 7958; fax: +82 2 705 7893 (H.S.L.). E-mail addresses: [email protected] (M.S. Han), [email protected] (H.S. Lee). http://dx.doi.org/10.1016/j.bmc.2014.02.027 0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
acceptor sugar with a 400-fold increased catalytic activity was found using a fluorescence-activated cell sorter (FACS).9 Another example used a simple, in vitro high-throughput screening system based on a fluorescent acceptor substrate to expand the specificity of the oleandomycin GT (OleD) for a glycosyl donor.10 Although in both cases a variety of glycans were enzymatically prepared by engineering GTs, their screening methods were based on specific fluorescent substrates and, therefore, cannot be generally applied to other GTs that use different glycosyl acceptor substrates. Although GT assays based on antibodies, radioisotopes, pH sensors, phosphatases, and fluorescent sensors are available,11–15 these assays are not suitable for GT engineering by high-throughput screening. Recently, a xanthene-based nucleoside diphosphate (NDP) sensor was used for a GT assay for high-throughput screening and showed high sensitivity and compatibility with crude cell extracts.16 Even though this GT assay system is useful and can be applied to various GTs, a critical limitation of the method is that the xanthene-based sensor requires a time-consuming multistep synthesis (twelve steps), which often prevents researchers from using this method. Herein, we report the development of a general GT assay based on a fluorescent adenosine triphosphate (ATP) sensor that can be readily prepared by synthesis. As this GT assay is highly sensitive, amenable to high-throughput screening for GT engineering, and can be applied to most Leloir-type GTs, the assay is anticipated to be useful for general GT research and especially, GT engineering.
2572
J. Ryu et al. / Bioorg. Med. Chem. 22 (2014) 2571–2575
2. Results and discussion In Leloir-type GT reactions, where sugar nucleotide derivatives are used as the glycosyl donors, a common byproduct is NDP. Therefore, when developing a new GT assay, NDP is a good target for measuring the enzymatic activity of GTs. Recently, a fluorescence-based ATP sensor was reported in which an anthracene-based Zn(II) complex ([Zn2(9,10-bis[(2,2-dipicolylamino)methyl]anthracene)]4+, Ant-Zn) and pyrocatechol violet were used.17 Because this sensor is based on fluorescence quenching and restoration, it shows low background signal and high sensitivity for ATP and ADP. Although the selectivity of the sensor for ADP over ADP-sugar, which is crucial for a GT assay, was not reported, it was expected that the sugar moiety attached to the phosphate would significantly reduce its binding affinity for the sensor.16,18,19 Based on this idea, a GT assay system was designed as shown in Figure 1. First, the sensor was tested for selectivity for NDP over NDP-sugar. Ant-Zn was prepared according to the literature,20 and thymidine diphosphate (TDP) and TDP-glucose were tested in this experiment.21 A solution containing Ant-Zn (5 lM) and pyrocatechol violet (10 lM) in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 20 mM, pH 7.0) was titrated with TDP and TDP-glucose, and fluorescence was measured at 413 nm with excitation at 380 nm. A steep increase in fluorescence was observed with increasing concentrations of TDP, while little fluorescence was seen with TDP-glucose (Fig. 2). As expected, Ant-Zn showed high sensitivity (apparent dissociation constant, K0 d = 0.19 lM) for TDP and excellent selectivity over TDP-glucose (K0 d >20 lM). Next, the sensor was evaluated in an in vitro enzymatic assay. OleD wild type (WT) and two variants (ASP and TDP16) were used in this assay, and TDP-glucose and 2-naphthol were used as the glycosyl donor and glycosyl acceptor. OleD WT and the variants are good target GTs for the development of a GT assay because they are known to have different GT activity with different donors and acceptors.10,22,23 The ASP variant was generated by using a fluorescence-based directed evolution strategy, the variant showed a broad substrate specificity for various glycosyl acceptors and donors,10,22 and the TDP16 variant was found to have improved GT activity for TDP-sugar.23 The GT reaction was performed by adding one of the OleDs (5 lM) to a solution containing 1 mM TDP-glucose, 1 mM MgCl2, and 1 mM 2-naphthol in 10 mM Tris (pH 8.0). The reaction was quenched with methanol, followed by addition of the mixture (5 lL) to an assay solution containing
Figure 2. Titration of Ant-Zn and pyrocatechol violet with TDP ( ) and TDP-glucose (TDP-G, ). Each data point represents the average of three experiments. Measurement conditions: 20 mM HEPES (pH 7.0), 5 lM Ant-Zn, and 10 lM pyrocatechol violet; excitation at 380 nm, emission at 413 nm.
5 lM Ant-Zn and 10 lM pyrocatechol in 20 mM HEPES (pH 7.0), and fluorescence was measured. In this experiment, TDP16 showed a strong, time-dependent increase in fluorescence, while OleD WT and ASP showed little or no increase in fluorescence (Fig. 3). These results are in agreement with a previous study,23 confirming that the fluorescence changes observed correlate with the enzymatic activity of the OleDs. In the absence of an enzyme or acceptor, little or no fluorescence increase was detected, demonstrating that the fluorescence increase was caused by the production of TDP from the GT reaction. The transfer of glucose from TDP-glucose to 2-naphthol was further validated by high performance liquid chromatography (HPLC) and mass analysis of the products from the GT reaction (Fig. 4). For TDP16, the most active variant, steady-state kinetic parameters were determined by performing the assay in a 96-well plate format. Initial reaction velocity was measured with different concentrations of 2-naphthol at a fixed concentration of TDP-glucose (1 mM), providing an apparent KM of 0.39 ± 0.090 mM and a kcat of 0.25 ± 0.002 min1 (Fig. S1). In order to apply this assay to high-throughput screening for GT engineering, it is necessary for the assay to be compatible with crude cell extracts. This compatibility was tested by carrying out the GT assay described above with crude cell extracts from OleD-expressing bacterial cells, instead of purified enzymes. When
Figure 1. Schematic representation of the GT assay mechanism of Ant-Zn. The fluorescence of Ant-Zn is quenched by pyrocatechol violet, and the replacement of the quencher by NDP restores the fluorescence. Measurement of this fluorescence allows quantification of the amount of NDP produced from the GT reactions.
J. Ryu et al. / Bioorg. Med. Chem. 22 (2014) 2571–2575
Figure 3. GT assay results for OleD-WT and two variants with Ant-Zn and pyrocatechol violet. Fluorescence was measured at 413 nm with excitation at 380 nm. Each data point represents the average of three experiments. GT reaction conditions: 10 mM Tris (pH 8.0), 1 mM TDP-glucose, 1 mM MgCl2, 1 mM 2-naphthol, and 5 lM OleD, room temperature; (+), in the presence of 2-naphthol; (), in the absence of 2-naphthol; the control contains no enzyme. Assay conditions: 10 lL of the reaction mixture (5 lL of the GT reaction mixture and 5 lL of methanol) was added to 190 lL of the assay solution containing 20 mM HEPES (pH 7.0), 5 lM Ant-Zn, and 10 lM pyrocatechol.
different amounts of the cell extracts from TDP16-expressing cells were used to optimize the assay condition, the best signal was obtained from 2 to 5 lL of cell extract in 50 lL of the GT reaction
2573
Figure 5. GT assay results with different amounts of cell extracts prepared from TDP16-expressing bacterial cells. Fluorescence was measured at 413 nm with excitation at 380 nm. Each data point represents the average of three experiments. GT reaction conditions: 10 mM Tris (pH 8.0), 1 mM TDP-glucose, 1 mM MgCl2, 1 mM 2-naphthol, and the indicated volume of lysate (in 50 lL of total reaction volume), room temperature.
mixture (Fig. 5). When more cell extract was used, some components of the cell extract, such as proteins, interfered with Ant-Zn, resulting in a decrease in fluorescence intensity.24 Next, the sensor was tested to ascertain whether it could measure GT activity with crude cell lysates. OleD WT, ASP, and TDP16 were expressed in bacterial cells, from which cell lysates were prepared, and the quantity
Figure 4. HPLC analysis of the product from the GT reaction catalyzed by OleD-TDP16. A, TDP; B, TDP-glucose; C, 2-naphthol; D, reaction mixture; E, control containing no enzyme. O-glucosyl-2-naphthol was analyzed by LC–MS: calculated for C16H18O6 (M+Na) 329.1, observed, 329.2.
2574
J. Ryu et al. / Bioorg. Med. Chem. 22 (2014) 2571–2575
containing 5 lM Ant-Zn and 10 lM pyrocatechol violet in HEPES (20 mM, pH 7.0). TDP or TDP-glucose at different concentrations (0.25, 0.50, 0.75, 1.00, 2.00, 3.00, 4.00, 5.00, 7.50, 10.00, 12.50, 15.00 lM, final concentrations) was added to the assay solution (200 lL, final volume) and the fluorescence was measured at 413 nm with excitation at 380 nm. 4.3. Enzymatic assays OleD-WT or one of the variants (5 lM, final concentration) was added to the reaction buffer containing 10 mM Tris (pH 8.0), 1 mM TDP-glucose, 1 mM MgCl2, 1 mM 2-naphthol, and the mixture (100 lL) was incubated at room temperature. The reaction was quenched by adding the same volume (100 lL) of methanol, the mixture (10 lL) was added to the assay solution (190 lL), and the fluorescence was measured as described for the TDP binding assay. 4.4. Crude cell extract assays Figure 6. GT assay results with crude cell extracts from OleD WT- or variantsexpressing cells. The assay was performed in 96-well plates and fluorescence was measured by a fluorescence reader at 413 nm with excitation at 380 nm. GT reaction conditions: 10 mM Tris (pH 8.0), 1 mM TDP-glucose, 1 mM MgCl2, 1 mM 2-naphthol, and 10% crude cell extracts (10 lL for 100 lL GT reaction), room temperature; (+), in the presence of 2-naphthol; (), in the absence of 2-naphthol; the blank contains the cell extract from cells expressing a blank vector.
of each OleD variant in the cell lysates was estimated by SDS–PAGE analysis to ensure that each lysate contained identical quantities of the OleD variants (Fig. S2). These extracts were used in the GT assay in a 96-well plate format, and the results are shown in Figure 6. As observed in the enzymatic assay, TDP16 was the most active variant and the others showed little GT activity, verifying that the assay system can be applied to screen GT variants with cell extracts in a high-throughput format. 3. Conclusions In summary, a general GT assay system was developed based on a synthetically accessible ATP sensor. Because the sensor is based on fluorescence quenching and restoration, and has high selectivity for NDP over NDP-sugar, the assay system displayed low background signal and high sensitivity (submicromolar). In addition, because the sensor is compatible with crude cell extracts, enzymatic activity of GTs in cell extracts can be measured. Therefore, the GT assay system will be useful for GT engineering, as well as for general GT assays and kinetics. 4. Materials and methods 4.1. General All chemicals and DNA oligomers were obtained from commercial sources and used without further purification. Fluorescence was measured on an F-7000 fluorescence spectrophotometer (Hitachi High-Technologies Corporation, Tokyo Japan) and Victor X5 multilabel plate reader (PerkinElmer, Massachusetts USA). 4.2. TDP binding assay Complex Ant-Zn stock solution was prepared by mixing 500 lL of Ant (1 mM) in 40% DMSO in water, 1000 lL of Zn(ClO4)2 (1 mM) in water and 8.5 mL of water to afford 50 lM Ant-Zn stock solution. Assay solution was prepared by adding the complex stock solution (1 mL) and 100 lM pyrocatechol violet (1 mL) in water to 8 mL of 25 mM HEPES (pH 7.0) to make 10 mL assay solution
Cells from OleD-expressing bacterial cell cultures (25 mL) were harvested by centrifugation (4000 rpm) and frozen at 80 °C. The frozen cell pellets were thawed on ice, resuspended in the lysis buffer (2 mL) containing 50 mM Tris (pH 8.0), lysozyme (1 mg/mL, 50 kU/mL) and benzonaze (125 U/mL), and incubated on ice for 1 h. Removal of the cell debris by centrifugation (12,000 rpm) afforded crude cell extracts. OleD assays with crude cell extracts were carried out as described for the assays with purified enzymes by adding crude cell extracts instead of purified enzymes to the reaction buffer. Acknowledgements This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Korea government (MSIP) (NRF-2013M2B 2A4040238, NRF-2013R1A2A2A01007000); the Converging Research Center Program through the Ministry of Education, Science and Technology (2013K000326). We would like to thank Jon S. Thorson for providing the plasmids. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.2014.02.027. References and notes 1. (a) Woods, S. C.; Seeley, R. J.; Porte, D., Jr.; Schwartz, M. W. Science 1998, 280, 1378; (b) Flatt, J.-P. Am. J. Clin. Nutr. 1995, 61, 952S. 2. Crocker, P. R.; Feizi, T. Curr. Opin. Struct. Biol. 1996, 6, 679. 3. (a) Brown, G. D.; Gordon, S. Immunity 2003, 19, 311; (b) Cambi, A.; Koopman, M.; Figdor, C. G. Cell. Microbiol. 2005, 7, 481. 4. (a) Lowe, J. B. Curr. Opin. Cell Biol. 2003, 15, 531; (b) Sacks, D.; Kamhawi, S. Annu. Rev. Microbiol. 2001, 55, 453. 5. (a) Davis, B. G. Pure Appl. Chem. 2009, 81, 285; (b) Seeberger, P. H.; Werz, D. B. Nature 2007, 446, 1046; (c) Pratt, M. R.; Bertozzi, C. R. Chem. Soc. Rev. 2005, 34, 58; (d) Hoelemann, A.; Seeberger, P. H. Curr. Opin. Biotechnol. 2004, 15, 615. 6. Zhu, X.; Schmidt, R. R. Angew. Chem., Int. Ed. 2009, 48, 1900. 7. Sinnott, M. L. Chem. Rev. 1990, 90, 1171. 8. Wagner, G. K.; Pesnot, T.; Field, R. A. Nat. Prod. Rep. 2009, 26, 1172. 9. Aharoni, A.; Thieme, K.; Chiu, C. P.; Buchini, S.; Lairson, L. L.; Chen, H.; Strynadka, N. C.; Wakarchuk, W. W.; Withers, S. G. Nat. Methods 2006, 3, 609. 10. Williams, G. J.; Zhang, C.; Thorson, J. S. Nat. Chem. Biol. 2007, 10, 657. 11. Wagner, G. K.; Pesnot, T. ChemBioChem 2010, 11, 1939. 12. Palcic, M. M.; Sujino, K. Trends Glycosci. Glycotechnol. 2001, 72, 361. 13. Wu, Z. L.; Ethen, C. M.; Prather, B.; Machacek, M.; Jiang, W. Glycobiology 2011, 21, 727. 14. Chen, X.; Jou, M. J.; Yoon, J. Org. Lett. 2009, 11, 2181. 15. Wongkongkatep, J.; Miyahara, Y.; Ojida, A.; Hamachi, I. Angew. Chem., Int. Ed. 2006, 45, 665.
J. Ryu et al. / Bioorg. Med. Chem. 22 (2014) 2571–2575 16. Lee, H. S.; Thorson, J. S. Anal. Biochem. 2011, 418, 85. 17. Jang, H. H.; Yi, S.; Kim, M. H.; Kim, S.; Lee, N. H.; Han, M. S. Tetrahedron Lett. 2009, 50, 6241. 18. Ojida, A.; Takashima, I.; Kohira, T.; Nonaka, H.; Hamachi, I. J. Am. Chem. Soc. 2008, 130, 12095. 19. Kim, S. K.; Lee, D. H.; Hong, J.-I.; Yoon, J. Acc. Chem. Res. 2009, 42, 23. 20. Ojida, A.; Mito-oka, Y.; Inoue, M.; Hamachi, I. J. Am. Chem. Soc. 2002, 124, 6256. 21. TDP and TDP-glucose were used instead of UDP and UDP-glucose because UDPglucose was hydrolyzed by OleD-ASP in the absence of a glycosyl acceptor, which caused significant background fluorescence.
2575
22. Gantt, R. W.; Goff, R. D.; Williams, G. J.; Thorson, J. S. Angew. Chem., Int. Ed. 2008, 47, 8889. 23. Williams, G. J.; Yang, J.; Zhang, C.; Thorson, J. S. ACS Chem. Biol. 2011, 6, 95. 24. We divided the cell lysate into large-molecule and small-molecule parts, and examined them to ascertain which caused a decrease in the fluorescence. We observed a significant decrease in the fluorescence owing to the large-molecule fraction while the small-molecule fraction did not alter the fluorescence.
