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Measurement of α(1-3)Fucosyltransferase Activity Using Scintillation Proximity
ANALYTICAL BIOCHEMISTRY ARTICLE NO.
255, 8 –12 (1998)
AB972449
Measurement of a(1-3)Fucosyltransferase Activity Using Scintillation Proximity Christopher M. Hood, Valerie A. Kelly, Michael I. Bird, and Christopher J. Britten1 Glycobiology Research Unit, Glaxo Wellcome Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, United Kingdom
Received May 5, 1997
The a3 fucosyltransferases are a family of glycosyltransferases involved in the addition of fucose onto glycoproteins and glycolipids. One of the best defined roles for the a3 fucosyltransferases is in the biosynthesis of the carbohydrate antigen sialyl Lewis X, the minimal ligand for the selectin family of adhesion molecules. We describe here the development of a singlestep assay for the measurement of a3 fucosyltransferase activity based on the principle of scintillation proximity. The fucosyltransferase catalyses the transfer of [3H]fucose, from GDP-[3H]fucose, onto the sugar chains of a glycoprotein acceptor noncovalently bound to a scintillant-impregnated microsphere (SPA bead). The resultant signal can be used as a measure of enzyme activity. Due to the nature of this assay no steps are required to separate unused substrate from product. Kinetic data from the assay compare favorably with those obtained from assays currently used for the a3 fucosyltransferases. This SPA-based assay appears generic for the a3 fucosyltransferases and readily adaptable for other glycosyltransferases. The particular advantage of the assay is anticipated to be found in the simple, routine testing of a large number of samples. © 1998 Academic Press
The extravasation of leukocytes, from the bloodstream to inflammatory sites within the tissue, is a complex, multistep process (1). The initial event in this process is the interaction between the circulating leukocyte and the activated endothelium, mediated by leukocyte-borne sialyl Lewis X and the endothelial adhesion molecules E- and P-selectin. Sialyl Lewis X (NeuAca(2-3)Galb(1-4)[Fuca(1-3)]GlcNAc-R) is a carbohydrate antigen that is synthesized through the action of a series of glycosyltransferases, with the termiTo whom correspondence should be addressed. Fax: 144-1438764818. E-mail: [email protected]. 1
8
nal step being the addition of a3-linked fucose (2). This addition is carried out by an a3 fucosyltransferase (a3 FucT). To date, five human a3 FucTs have been cloned and expressed and their basic characteristics described (3). The study of these enzymes has been dependent on a widely used assay that relies on the in vitro fucosylation of saccharide acceptors by the a3 fucosyltransferase. Along with the enzyme, radioactive GDP[3H]fucose is incubated with low-molecular-weight saccharide acceptors, glycoproteins, or glycolipids, and the transfer of [3H]fucose monitored by standard scintillation counting. These assays are laborious and require time-consuming steps, such as ion-exchange (4), hydrophobic chromatography (5), gel filtration (6), or thin-layer chromatography (7), for the separation of radioactive substrate and product. Here we describe a novel radioactive assay for the measurement of a3 fucosyltransferase activity that requires no separation of substrate and product and is based on the principle of scintillation proximity (8). Scintillation proximity assays (SPA)2 rely on the spatial proximity of a weak energy isotope, such as 3H b-particles, to a scintillant-impregnated microsphere. Only when the isotope is sufficiently close to the microsphere (SPA bead) will the bead emit light. This approach has been used extensively in the study of receptor–ligand interactions (9 –11). In this report we describe the development of an SPA for the a3 fucosyltransferases. The SPA beads are coated with a suitable glycoprotein acceptor, such as fetuin, and incubated with GDP-[3H]fucose and enzyme. Transfer of the [3H]fucose onto the glycoprotein, bound to the SPA bead, results in the emission of light from the bead. Unused radioactive substrate remains 2
Abbreviations used: SPA, scintillation proximity assays; SPA beads, scintillation proximity fluoromicrospheres; WGA, wheat germ agglutinin. 0003-2697/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
SCINTILLATION PROXIMITY ASSAY FOR a3 FUCOSYLTRANSFERASE
in solution but is not sufficiently close to the bead to trigger the light emission. It is this characteristic of the a3 fucosyltransferase SPA that removes the need for separation steps. The assay is suitably generic as the activity of a number of a3 fucosyltransferases, in addition to other glycosyltransferases, can be measured. The glycoprotein acceptor can also be modified to allow determination of acceptor specificity (e.g., fetuin, asialofetuin, a1 acid glycoprotein). This assay is single-step and homogeneous and is ideal for the routine assay of a large number of samples. MATERIALS AND METHODS
Materials Scintillation proximity fluoromicrospheres (SPA beads), precoated with wheat-germ agglutinin (WGA), were obtained from Amersham International (UK) and white clear-bottom microtitre plates from Wallac Oy (Finland). GDP-b-Fucose and fetuin, asialofetuin, and a1-acid glycoprotein were obtained from Sigma (UK). GDP-[3H]fucose (60 Ci/mmol) was obtained from Amersham International (Amersham, UK). Low-molecular-weight saccharide acceptors were from Dextra Laboratories (Reading, UK). Preparation of Glycoprotein-Coated SPA Beads Glycoproteins were immobilized on WGA-coated SPA beads by incubation of the beads (typically 500 mg) with a 10 mg/ml solution of glycoprotein, dissolved in 50 mM sodium cacodylate, pH 7.5, for 1 h at 37°C with occasional agitation. After 1 h, the beads were pelleted by centrifugation (2000g for 15 min at room temperature) and washed three times with 50 mM sodium cacodylate, pH 7.5. Beads were finally resuspended to a concentration of 40 mg of beads/ml in the same buffer containing 0.02% (w/v) sodium azide. Glycoprotein-coated SPA beads could be kept for several months at 4°C with negligible loss of activity. Preparation of a3 Fucosyltransferase Recombinant full-length human a1-3 fucosyltransferase (FucT-VI (12, 13)) was produced by infection of Trichoplusia ni cells with recombinant baculovirus. Cells expressing the active enzyme were solubilized in 1% (v/v) Triton X-100, 150 mM NaCl, 25% (v/v) glycerol, 10 mM MgCl2 buffered to pH 7.5 with 50 mM Tris-Cl by a 15-min incubation on ice followed by probe sonication (MSE Soniprep) for 4 3 15-s bursts at 4°C. Insoluble material was removed by centrifugation at 3000g for 5 min at 4°C. No a3 fucosyltransferase activity could be detected, using either the SPA or Dowex methods (see below), from mock-infected insect cells.
9
Protein concentrations were estimated using the Bio-Rad DC (Hemel Hempstead, UK) method, with human g-globulin as the standard protein. Assay of a3 Fucosyltransferase Using SPA
a3 Fucosyltransferase activity was assayed in a volume of 100 ml comprising 2.5 mM GDP-b-fucose, 100,000 cpm GDP-[3H]fucose, 4 mM MnCl2, 1 mM ATP, 50 mM sodium cacodylate, pH 6.5, the fetuin– SPA bead suspension (1 mg beads/assay), and varying amounts of enzyme. All incubations were performed in 96-well flat clear-bottomed plates (Wallac 1450-512). Enzyme and reagents were incubated for varying time points (see figure legends) at 37 or 22°C (room temperature). [3H]Fucose incorporation was measured using a Wallac Microbeta scintillation count plate reader (Model 1450). Results are generally expressed as net CPM, where the signal generated by the nonspecific interaction of GDP-[3H]fucose with the SPA bead (determined by duplicating the assay in the absence of enzyme) is subtracted from the signal generated in the presence of a3 fucosyltransferase. Signal-to-noise ratios were of the order 5 to 10:1. All results are representative of at least three experiments. Assay of a3 Fucosyltransferase Using Standard Dowex Assay
a3 Fucosyltransferase activity was assayed by a modification of Prieels et al. (4) as previously described (25). The assay medium contained 100 mM GDP-bfucose, 100,000 cpm GDP-[3H]fucose, 1 mM ATP, 4 mM MnCl2, buffered to pH 7.5 with 50 mM Hepes-NaOH. Acceptor saccharide (lacNAc: Galb(1-4)GlcNAc) was added to a final concentration of 5 mM. The reaction was started by the addition of enzyme (typically 10 mg protein) and transfer to a 37°C water bath. After 30 – 60 min at 37°C, the reaction was terminated by the addition of 1 ml of a Dowex slurry (Dowex 1-X8 (Cl2 form) 1:4 distilled water (w/v), vortexed, and centrifuged, and the radioactivity in the supernatant (600 ml) measured by liquid scintillation counting. This method of batch extraction using Dowex was found to remove .99% of the unused GDP-[3H]fucose. Nonspecific breakdown of GDP-fucose was assessed by duplicating the enzyme assay in the absence of acceptor saccharide. RESULTS AND DISCUSSION
The SPA developed for the measurement of a3 fucosyltransferase activity is illustrated schematically in Fig. 1. The fucose acceptor is bound to the SPA bead through the interaction of the carbohydrate chains on the glycoprotein acceptor and the WGA that is co-
10
HOOD ET AL.
FIG. 1. Schematic representation of the a3 fucosyltransferase SPA. Glycoprotein-coated SPA beads are incubated with the a3 fucosyltransferase and the radiolabeled substrate GDP-[3H]fucose. [3H]fucose transferred to the glycoprotein on the SPA bead, by the enzyme, excites the scintillant within the bead, causing it to emit light. Unused GDP-[3H]–fucose remains free in solution and is sufficiently distant from the bead to prevent scintillation. The carbohydrates . presented on the glycoprotein are represented as
valently coupled to the bead. The glycoproteins used in the development of this assay, namely fetuin and asialofetuin, bear N-linked carbohydrate chains containing the type II lactosamine core (Galb(1-4)GlcNAc) that is recognized by the a3 fucosyltransferases as a site for fucosylation (18, 19). The amount of fetuin that can be coated onto the WGA-SPA bead is limited and was determined empirically by incubating SPA beads with differing concentrations of fetuin. Coating of SPA beads was routinely performed for 1 h at 37°C. As shown in Fig. 2, the SPA beads become saturated at glycoprotein concentrations of less than 5 mg/ml. Despite increasing the concentration of fetuin four-fold, very little increase in fucosyltransferase activity was observed, suggesting that the
FIG. 2. Optimization of the concentration of fetuin. SPA beads were coated with varying amounts of fetuin (as described under Materials and Methods) and fucosylated with [3H]fucose by FucT-VI for 2 h at 37°C. Results are presented as Net CPM, with the CPM generated by nonspecific interaction of unused substrate (measured in the absence of enzyme) subtracted from the CPM obtained in the presence of enzyme. Beads were included at 1 mg/assay.
FIG. 3. Dependence of signal generation on enzyme concentration. Insect cells expressing FucT-VI were lysed in Triton X-100-containing buffer as described under Materials and Methods. Various amounts of cell lysate were incubated with fetuin-coated SPA beads and GDP-[3H]fucose for 2 h at 37°C. Results are expressed as Net CPM and are representative of three experiments.
beads cannot bind any more glycoprotein. To ensure consistent saturation of the beads, fetuin was coated at a concentration of 10 mg/ml. The signal generated is also dependent on the amount of fetuin-coated beads included in the assay. The rate of [3H]fucose incorporation was found to be linear up to 2 mg beads/assay, the highest concentration tested (data not shown). Although reducing the amount of SPA beads from 2 to 1 mg/assay resulted in a 50% decrease of the signal generated, the measured CPM were sufficiently high at 1 mg/assay to obviate the need for using greater quantities of beads. Background CPM, determined by duplicating the assay in the absence of enzyme, were typically 10-fold lower than those obtained in the presence of enzyme. This would suggest that the nonspecific generation of signal by GDP-[3H]fucose, being proximal to the beads, is measurable but low. The formation of radiolabeled product is proportional to the amount of enzyme included in the assay. As the beads can only be coated with a finite amount of fetuin (Fig. 2) it would be expected that only a finite amount of [3H]fucose could be transferred by the fucosyltransferase. Titration of the amount of enzyme (Fig. 3) shows that the maximum signal can be attained, with the beads becoming saturated with [3H]fucose as the enzyme concentration is increased. The rate at which the a3 fucosyltransferase will transfer [3H]fucose onto the fetuin-coated bead is timedependent. Under the assay conditions described, the rate of [3H]fucose incorporation mediated by FucT-VI is linear up to 180 min at 37°C (Fig. 4). Assays were routinely performed for 2 h at 37°C. Due to the different intrinsic catalytic activities of different enzymes,
SCINTILLATION PROXIMITY ASSAY FOR a3 FUCOSYLTRANSFERASE
FIG. 4. Time-dependence of [3H]fucose incorporation. Fetuincoated SPA beads were incubated with FucT-VI for up to 3 h at 37°C. Both the signal generated in the presence of enzyme (F) and the ‘‘blank’’ obtained in the absence of enzyme (E) are shown.
the dependence on time and temperature could be exploited to maximize the signal generated. Consequently, assays for both FucT-VII and FucT-III (not shown) were performed at 22°C (room temperature) for 16 h, as a short incubation at 37°C was insufficient for these enzymes to transfer enough [3H]fucose onto the SPA bead to generate significant CPM. The enzyme reaction could be terminated by the addition of an equal volume (100 ml) of 100% ethanol. The addition of solvent denatures the fucosyltransferase and prevents further fucosylation. The ability to stop the assay renders this SPA suitable for kinetic measurements. To demonstrate the consistency between the SPA and the more widely used Dowex-based assay (see Materials and Methods), the effect of a standard competitive inhibitor of the a3 fucosyltransferases was determined. The nucleotide diphosphate GDP is believed to compete with GDP-fucose for the active site of the fucosyltransferases and thus acts as a general inhibitor of this class of enzyme (20). Figure 5 shows the comparison of GDP inhibition using the two different assays. The concentration at which GDP causes 50% inhibition (IC50) of FucT-VI was determined to be 191 mM in the SPA and 214 mM in the Dowex assay. An identical amount of the FucT-VI preparation (10.5 mg) was used in both assays. The sensitivity of FucT-VI (25), along with the lack of sensitivity of FucT-VII (6), to the sulfhydryl reagent N-ethylmaleimide is also maintained (data not shown). Kinetic analysis of FucT-VI also shows the SPA to be comparable with other fucosyltransferase assays. A Km value for GDPfucose was determined to be 1.6 mM (Fig. 6), in line with values obtained for other a3 fucosyltransferases and other assay systems (5, 23, 26). This confirms the equivalence of the two assays.
11
FIG. 5. Comparison of the SPA and Dowex-based assay—inhibition of FucT-VI by GDP. The inhibitory effect of GDP on FucT-VI was determined using both the SPA and standard Dowex assays. For the SPA (F), enzyme was incubated with fetuin-coated SPA beads and varying concentrations of GDP for 2 h at 37°C. For the Dowex assay (E), reactions were performed with Galb(1-4)GlcNAc as the fucose acceptor, with varying concentrations of GDP, for 1 h at 37°C. The same concentration of FucT-VI was used in both the SPA and Dowex assay (10.5 mg/assay).
Unfortunately it is not possible to use this assay to determine the specific activity of the enzyme under investigation as the efficiency of the SPA scintillant is considerably lower than that usually found for scintillation fluid; direct comparisons cannot be made. However, this assay would be of use for the rapid testing of a large number of samples where the presence or ab-
FIG. 6. Determination of Km (GDP-fucose) for FucT-VI using the SPA. FucT-VI activity was measured after 2 h at 37°C with different concentrations of GDP-fucose. GDP-[3H]fucose was maintained at 100,000 cpm per assay. One milligram of fetuin-coated SPA beads was included in each assay point. Enzyme activity (arbitrary units) was determined by dividing the measured SPA-CPM by the amount of GDP-fucose (pmol). Inset, double-reciprocal plot of the Km determination.
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HOOD ET AL.
sence of a3 fucosyltransferase activity is the prime determinant. In conclusion, we describe the development of a novel radioactive method for the measurement of a3 fucosyltransferase activity. When considering the routine testing of a large number of samples, this method offers significant advantages over protocols currently employed. This SPA is simple, single-step, without the need for removal of unused substrate, and reproducible. Although the current Dowex-based methods are sensitive and useful for the testing of a small number of samples, the steps involved are laborious and timeconsuming. The nonradioactive assays that have also been developed for the measurement of a3 fucosyltransferase activity (23, 24) have the obvious advantage of not requiring radioactivity, but require a number of washing steps before product detection by antibody. The most recently described nonradioactive assay (24) is highly sensitive and can also be used for the detection of a3 fucosyltransferase activity in cancer cell lines. However, multiple washing and detection steps are required before enzyme activity can be determined. The SPA, despite being isotope-based, requires no such washes or special incubations. The described assay has been successfully used to measure the activity of a number of a3 fucosyltransferases (FucT-III, VI, and VII) in addition to an a6 sialyltransferase (data not shown). It clearly can be adapted easily for the measurement of other glycosyltransferase activities. The a3 fucosyltransferase SPA reported here is the most suitable method yet described for the routine testing of a large number of samples with the added advantage of possessing the potential for simple automation. ACKNOWLEDGMENTS We thank Dr. Nick Smithers and Miss Sara Witham for the supply of the recombinant a3 fucosyltransferases and Dr. Martyn Banks for advice on the SPA technology.
REFERENCES 1. McEver, R. P., Moore, K. L., and Cummings, R. D. (1995) J. Biol. Chem. 270, 11025–11028. 2. Holmes, E. H., Ostrander, G. K., and Hakomori, S-I. (1986) J. Biol. Chem. 261, 3737–3743. 3. Natsuka, S., and Lowe, J. B. (1994) Curr. Opin. Struct. Biol. 4, 683– 691.
4. Prieels, J. P., Monnom, D., Dolmans, M., Beyer, T. A., and Hill, R. L. (1981) J. Biol. Chem. 256, 10456 –10463. 5. de Vries, T., Srnka, C. A., Palcic, M. M., Swiedler, S. J., van den Eijnden, D. H., and Macher, B. A. (1995) J. Biol. Chem. 270, 8712– 8722. 6. Clarke, J. L., and Watkins, W. M. (1996) J. Biol. Chem. 271, 10317–10328. 7. Holmes, E. H., and Macher, B. A. (1993) Arch. Biochem. Biophys. 301, 190 –199. 8. Hart, H. E., and Greenwald, E. B. (1979) Mol. Immunol. 16, 265–267. 9. Udenfriend, S., Diekmann Gerber, L., Brink, L., and Spector, S. (1985) Proc. Natl. Acad. Sci. USA 82, 8672– 8676. 10. Pachter, J. A., Zhang, R. and Mayer-Ezell, R. (1995) Anal. Biochem. 230, 101–107. 11. Banks, M. N., Graber, P., Proudfoot, A. E. I., Arod, C. Y., Allet, B., Bernard, A. R., Sebille, E., McKinnon, M., Wells, T. N. C., and Solari, R. (1995) Anal. Biochem. 230, 321–328. 12. Weston, B. W., Smith, P. L., Kelly, R. J., and Lowe, J. B. (1992) J. Biol. Chem. 267, 24575–24584. 13. Kozsdin, K. L., and Bowen, B. R. (1992) Biochem. Biophys. Res. Commun. 187, 152–157. 14. Sasaki, K., Kurata, K., Funayama, K., Nagata, M., Watanabe, E., Ohta, S., Hanai, N., and Nishi, T. (1994) J. Biol. Chem. 269, 14730 –14737. 15. Natsuka, S., Gersten, K. M., Zenita, K., Kannagi, R., and Lowe, J. B. (1994) J. Biol. Chem. 269, 16789 –16794. 16. Kukowska-Latallo, J. F., Laersen, R. D., Nair, R. P., and Lowe, J. B. (1990) Genes Dev. 4, 1288 –1303. 17. Sueyoshi, S., Tsubo, S., Sawada-Hirai, R., Dang, U. N., Lowe, J. B., and Fukuda, M. N. (1994) J. Biol. Chem. 269, 32342– 32350. 18. Nilsson, B., Norden, N. E., and Svensson, S. (1979) J. Biol. Chem. 254, 4545– 4553. 19. Green, E. D., Adelt, G., Baenziger, J. U., Wilson, S., and van Halbeek, H. (1988) J. Biol. Chem. 263, 18253–18259. 20. Murray, B. W., Takayama, S., Schultz, J., and Wong, C-H. (1996) Biochemistry 35, 11183–11195. 21. Macher, B. A., Holmes, E. H., Swiedler, S. J., Stults, S. L., and Srnka, C. A. (1991) Glycobiology 1, 577–584. 22. Wagers, A. J., Lowe, J. B., and Kansas, G. S. (1996) Blood 88, 2125–2132. 23. Yan, L., Smith, D. F., and Cummings, R. D. (1994) Anal. Biochem. 223, 111–118. 24. Rabina, J., Smithers, N., Britten, C. J., and Renkonen, R. (1997) Anal. Biochem. 246, 71–78. 25. Britten, C. J., and Bird, M. I. (1997) Biochim. Biophys. Acta 1334, 57– 64. 26. Sarnesto, A., Kohlin, T., Hindsgaul, O., Vogele, K., BlaszczykThurin, M., and Thurin, J. (1992) J. Biol. Chem. 267, 2745–2752.
255, 8 –12 (1998)
AB972449
Measurement of a(1-3)Fucosyltransferase Activity Using Scintillation Proximity Christopher M. Hood, Valerie A. Kelly, Michael I. Bird, and Christopher J. Britten1 Glycobiology Research Unit, Glaxo Wellcome Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, United Kingdom
Received May 5, 1997
The a3 fucosyltransferases are a family of glycosyltransferases involved in the addition of fucose onto glycoproteins and glycolipids. One of the best defined roles for the a3 fucosyltransferases is in the biosynthesis of the carbohydrate antigen sialyl Lewis X, the minimal ligand for the selectin family of adhesion molecules. We describe here the development of a singlestep assay for the measurement of a3 fucosyltransferase activity based on the principle of scintillation proximity. The fucosyltransferase catalyses the transfer of [3H]fucose, from GDP-[3H]fucose, onto the sugar chains of a glycoprotein acceptor noncovalently bound to a scintillant-impregnated microsphere (SPA bead). The resultant signal can be used as a measure of enzyme activity. Due to the nature of this assay no steps are required to separate unused substrate from product. Kinetic data from the assay compare favorably with those obtained from assays currently used for the a3 fucosyltransferases. This SPA-based assay appears generic for the a3 fucosyltransferases and readily adaptable for other glycosyltransferases. The particular advantage of the assay is anticipated to be found in the simple, routine testing of a large number of samples. © 1998 Academic Press
The extravasation of leukocytes, from the bloodstream to inflammatory sites within the tissue, is a complex, multistep process (1). The initial event in this process is the interaction between the circulating leukocyte and the activated endothelium, mediated by leukocyte-borne sialyl Lewis X and the endothelial adhesion molecules E- and P-selectin. Sialyl Lewis X (NeuAca(2-3)Galb(1-4)[Fuca(1-3)]GlcNAc-R) is a carbohydrate antigen that is synthesized through the action of a series of glycosyltransferases, with the termiTo whom correspondence should be addressed. Fax: 144-1438764818. E-mail: [email protected]. 1
8
nal step being the addition of a3-linked fucose (2). This addition is carried out by an a3 fucosyltransferase (a3 FucT). To date, five human a3 FucTs have been cloned and expressed and their basic characteristics described (3). The study of these enzymes has been dependent on a widely used assay that relies on the in vitro fucosylation of saccharide acceptors by the a3 fucosyltransferase. Along with the enzyme, radioactive GDP[3H]fucose is incubated with low-molecular-weight saccharide acceptors, glycoproteins, or glycolipids, and the transfer of [3H]fucose monitored by standard scintillation counting. These assays are laborious and require time-consuming steps, such as ion-exchange (4), hydrophobic chromatography (5), gel filtration (6), or thin-layer chromatography (7), for the separation of radioactive substrate and product. Here we describe a novel radioactive assay for the measurement of a3 fucosyltransferase activity that requires no separation of substrate and product and is based on the principle of scintillation proximity (8). Scintillation proximity assays (SPA)2 rely on the spatial proximity of a weak energy isotope, such as 3H b-particles, to a scintillant-impregnated microsphere. Only when the isotope is sufficiently close to the microsphere (SPA bead) will the bead emit light. This approach has been used extensively in the study of receptor–ligand interactions (9 –11). In this report we describe the development of an SPA for the a3 fucosyltransferases. The SPA beads are coated with a suitable glycoprotein acceptor, such as fetuin, and incubated with GDP-[3H]fucose and enzyme. Transfer of the [3H]fucose onto the glycoprotein, bound to the SPA bead, results in the emission of light from the bead. Unused radioactive substrate remains 2
Abbreviations used: SPA, scintillation proximity assays; SPA beads, scintillation proximity fluoromicrospheres; WGA, wheat germ agglutinin. 0003-2697/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
SCINTILLATION PROXIMITY ASSAY FOR a3 FUCOSYLTRANSFERASE
in solution but is not sufficiently close to the bead to trigger the light emission. It is this characteristic of the a3 fucosyltransferase SPA that removes the need for separation steps. The assay is suitably generic as the activity of a number of a3 fucosyltransferases, in addition to other glycosyltransferases, can be measured. The glycoprotein acceptor can also be modified to allow determination of acceptor specificity (e.g., fetuin, asialofetuin, a1 acid glycoprotein). This assay is single-step and homogeneous and is ideal for the routine assay of a large number of samples. MATERIALS AND METHODS
Materials Scintillation proximity fluoromicrospheres (SPA beads), precoated with wheat-germ agglutinin (WGA), were obtained from Amersham International (UK) and white clear-bottom microtitre plates from Wallac Oy (Finland). GDP-b-Fucose and fetuin, asialofetuin, and a1-acid glycoprotein were obtained from Sigma (UK). GDP-[3H]fucose (60 Ci/mmol) was obtained from Amersham International (Amersham, UK). Low-molecular-weight saccharide acceptors were from Dextra Laboratories (Reading, UK). Preparation of Glycoprotein-Coated SPA Beads Glycoproteins were immobilized on WGA-coated SPA beads by incubation of the beads (typically 500 mg) with a 10 mg/ml solution of glycoprotein, dissolved in 50 mM sodium cacodylate, pH 7.5, for 1 h at 37°C with occasional agitation. After 1 h, the beads were pelleted by centrifugation (2000g for 15 min at room temperature) and washed three times with 50 mM sodium cacodylate, pH 7.5. Beads were finally resuspended to a concentration of 40 mg of beads/ml in the same buffer containing 0.02% (w/v) sodium azide. Glycoprotein-coated SPA beads could be kept for several months at 4°C with negligible loss of activity. Preparation of a3 Fucosyltransferase Recombinant full-length human a1-3 fucosyltransferase (FucT-VI (12, 13)) was produced by infection of Trichoplusia ni cells with recombinant baculovirus. Cells expressing the active enzyme were solubilized in 1% (v/v) Triton X-100, 150 mM NaCl, 25% (v/v) glycerol, 10 mM MgCl2 buffered to pH 7.5 with 50 mM Tris-Cl by a 15-min incubation on ice followed by probe sonication (MSE Soniprep) for 4 3 15-s bursts at 4°C. Insoluble material was removed by centrifugation at 3000g for 5 min at 4°C. No a3 fucosyltransferase activity could be detected, using either the SPA or Dowex methods (see below), from mock-infected insect cells.
9
Protein concentrations were estimated using the Bio-Rad DC (Hemel Hempstead, UK) method, with human g-globulin as the standard protein. Assay of a3 Fucosyltransferase Using SPA
a3 Fucosyltransferase activity was assayed in a volume of 100 ml comprising 2.5 mM GDP-b-fucose, 100,000 cpm GDP-[3H]fucose, 4 mM MnCl2, 1 mM ATP, 50 mM sodium cacodylate, pH 6.5, the fetuin– SPA bead suspension (1 mg beads/assay), and varying amounts of enzyme. All incubations were performed in 96-well flat clear-bottomed plates (Wallac 1450-512). Enzyme and reagents were incubated for varying time points (see figure legends) at 37 or 22°C (room temperature). [3H]Fucose incorporation was measured using a Wallac Microbeta scintillation count plate reader (Model 1450). Results are generally expressed as net CPM, where the signal generated by the nonspecific interaction of GDP-[3H]fucose with the SPA bead (determined by duplicating the assay in the absence of enzyme) is subtracted from the signal generated in the presence of a3 fucosyltransferase. Signal-to-noise ratios were of the order 5 to 10:1. All results are representative of at least three experiments. Assay of a3 Fucosyltransferase Using Standard Dowex Assay
a3 Fucosyltransferase activity was assayed by a modification of Prieels et al. (4) as previously described (25). The assay medium contained 100 mM GDP-bfucose, 100,000 cpm GDP-[3H]fucose, 1 mM ATP, 4 mM MnCl2, buffered to pH 7.5 with 50 mM Hepes-NaOH. Acceptor saccharide (lacNAc: Galb(1-4)GlcNAc) was added to a final concentration of 5 mM. The reaction was started by the addition of enzyme (typically 10 mg protein) and transfer to a 37°C water bath. After 30 – 60 min at 37°C, the reaction was terminated by the addition of 1 ml of a Dowex slurry (Dowex 1-X8 (Cl2 form) 1:4 distilled water (w/v), vortexed, and centrifuged, and the radioactivity in the supernatant (600 ml) measured by liquid scintillation counting. This method of batch extraction using Dowex was found to remove .99% of the unused GDP-[3H]fucose. Nonspecific breakdown of GDP-fucose was assessed by duplicating the enzyme assay in the absence of acceptor saccharide. RESULTS AND DISCUSSION
The SPA developed for the measurement of a3 fucosyltransferase activity is illustrated schematically in Fig. 1. The fucose acceptor is bound to the SPA bead through the interaction of the carbohydrate chains on the glycoprotein acceptor and the WGA that is co-
10
HOOD ET AL.
FIG. 1. Schematic representation of the a3 fucosyltransferase SPA. Glycoprotein-coated SPA beads are incubated with the a3 fucosyltransferase and the radiolabeled substrate GDP-[3H]fucose. [3H]fucose transferred to the glycoprotein on the SPA bead, by the enzyme, excites the scintillant within the bead, causing it to emit light. Unused GDP-[3H]–fucose remains free in solution and is sufficiently distant from the bead to prevent scintillation. The carbohydrates . presented on the glycoprotein are represented as
valently coupled to the bead. The glycoproteins used in the development of this assay, namely fetuin and asialofetuin, bear N-linked carbohydrate chains containing the type II lactosamine core (Galb(1-4)GlcNAc) that is recognized by the a3 fucosyltransferases as a site for fucosylation (18, 19). The amount of fetuin that can be coated onto the WGA-SPA bead is limited and was determined empirically by incubating SPA beads with differing concentrations of fetuin. Coating of SPA beads was routinely performed for 1 h at 37°C. As shown in Fig. 2, the SPA beads become saturated at glycoprotein concentrations of less than 5 mg/ml. Despite increasing the concentration of fetuin four-fold, very little increase in fucosyltransferase activity was observed, suggesting that the
FIG. 2. Optimization of the concentration of fetuin. SPA beads were coated with varying amounts of fetuin (as described under Materials and Methods) and fucosylated with [3H]fucose by FucT-VI for 2 h at 37°C. Results are presented as Net CPM, with the CPM generated by nonspecific interaction of unused substrate (measured in the absence of enzyme) subtracted from the CPM obtained in the presence of enzyme. Beads were included at 1 mg/assay.
FIG. 3. Dependence of signal generation on enzyme concentration. Insect cells expressing FucT-VI were lysed in Triton X-100-containing buffer as described under Materials and Methods. Various amounts of cell lysate were incubated with fetuin-coated SPA beads and GDP-[3H]fucose for 2 h at 37°C. Results are expressed as Net CPM and are representative of three experiments.
beads cannot bind any more glycoprotein. To ensure consistent saturation of the beads, fetuin was coated at a concentration of 10 mg/ml. The signal generated is also dependent on the amount of fetuin-coated beads included in the assay. The rate of [3H]fucose incorporation was found to be linear up to 2 mg beads/assay, the highest concentration tested (data not shown). Although reducing the amount of SPA beads from 2 to 1 mg/assay resulted in a 50% decrease of the signal generated, the measured CPM were sufficiently high at 1 mg/assay to obviate the need for using greater quantities of beads. Background CPM, determined by duplicating the assay in the absence of enzyme, were typically 10-fold lower than those obtained in the presence of enzyme. This would suggest that the nonspecific generation of signal by GDP-[3H]fucose, being proximal to the beads, is measurable but low. The formation of radiolabeled product is proportional to the amount of enzyme included in the assay. As the beads can only be coated with a finite amount of fetuin (Fig. 2) it would be expected that only a finite amount of [3H]fucose could be transferred by the fucosyltransferase. Titration of the amount of enzyme (Fig. 3) shows that the maximum signal can be attained, with the beads becoming saturated with [3H]fucose as the enzyme concentration is increased. The rate at which the a3 fucosyltransferase will transfer [3H]fucose onto the fetuin-coated bead is timedependent. Under the assay conditions described, the rate of [3H]fucose incorporation mediated by FucT-VI is linear up to 180 min at 37°C (Fig. 4). Assays were routinely performed for 2 h at 37°C. Due to the different intrinsic catalytic activities of different enzymes,
SCINTILLATION PROXIMITY ASSAY FOR a3 FUCOSYLTRANSFERASE
FIG. 4. Time-dependence of [3H]fucose incorporation. Fetuincoated SPA beads were incubated with FucT-VI for up to 3 h at 37°C. Both the signal generated in the presence of enzyme (F) and the ‘‘blank’’ obtained in the absence of enzyme (E) are shown.
the dependence on time and temperature could be exploited to maximize the signal generated. Consequently, assays for both FucT-VII and FucT-III (not shown) were performed at 22°C (room temperature) for 16 h, as a short incubation at 37°C was insufficient for these enzymes to transfer enough [3H]fucose onto the SPA bead to generate significant CPM. The enzyme reaction could be terminated by the addition of an equal volume (100 ml) of 100% ethanol. The addition of solvent denatures the fucosyltransferase and prevents further fucosylation. The ability to stop the assay renders this SPA suitable for kinetic measurements. To demonstrate the consistency between the SPA and the more widely used Dowex-based assay (see Materials and Methods), the effect of a standard competitive inhibitor of the a3 fucosyltransferases was determined. The nucleotide diphosphate GDP is believed to compete with GDP-fucose for the active site of the fucosyltransferases and thus acts as a general inhibitor of this class of enzyme (20). Figure 5 shows the comparison of GDP inhibition using the two different assays. The concentration at which GDP causes 50% inhibition (IC50) of FucT-VI was determined to be 191 mM in the SPA and 214 mM in the Dowex assay. An identical amount of the FucT-VI preparation (10.5 mg) was used in both assays. The sensitivity of FucT-VI (25), along with the lack of sensitivity of FucT-VII (6), to the sulfhydryl reagent N-ethylmaleimide is also maintained (data not shown). Kinetic analysis of FucT-VI also shows the SPA to be comparable with other fucosyltransferase assays. A Km value for GDPfucose was determined to be 1.6 mM (Fig. 6), in line with values obtained for other a3 fucosyltransferases and other assay systems (5, 23, 26). This confirms the equivalence of the two assays.
11
FIG. 5. Comparison of the SPA and Dowex-based assay—inhibition of FucT-VI by GDP. The inhibitory effect of GDP on FucT-VI was determined using both the SPA and standard Dowex assays. For the SPA (F), enzyme was incubated with fetuin-coated SPA beads and varying concentrations of GDP for 2 h at 37°C. For the Dowex assay (E), reactions were performed with Galb(1-4)GlcNAc as the fucose acceptor, with varying concentrations of GDP, for 1 h at 37°C. The same concentration of FucT-VI was used in both the SPA and Dowex assay (10.5 mg/assay).
Unfortunately it is not possible to use this assay to determine the specific activity of the enzyme under investigation as the efficiency of the SPA scintillant is considerably lower than that usually found for scintillation fluid; direct comparisons cannot be made. However, this assay would be of use for the rapid testing of a large number of samples where the presence or ab-
FIG. 6. Determination of Km (GDP-fucose) for FucT-VI using the SPA. FucT-VI activity was measured after 2 h at 37°C with different concentrations of GDP-fucose. GDP-[3H]fucose was maintained at 100,000 cpm per assay. One milligram of fetuin-coated SPA beads was included in each assay point. Enzyme activity (arbitrary units) was determined by dividing the measured SPA-CPM by the amount of GDP-fucose (pmol). Inset, double-reciprocal plot of the Km determination.
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HOOD ET AL.
sence of a3 fucosyltransferase activity is the prime determinant. In conclusion, we describe the development of a novel radioactive method for the measurement of a3 fucosyltransferase activity. When considering the routine testing of a large number of samples, this method offers significant advantages over protocols currently employed. This SPA is simple, single-step, without the need for removal of unused substrate, and reproducible. Although the current Dowex-based methods are sensitive and useful for the testing of a small number of samples, the steps involved are laborious and timeconsuming. The nonradioactive assays that have also been developed for the measurement of a3 fucosyltransferase activity (23, 24) have the obvious advantage of not requiring radioactivity, but require a number of washing steps before product detection by antibody. The most recently described nonradioactive assay (24) is highly sensitive and can also be used for the detection of a3 fucosyltransferase activity in cancer cell lines. However, multiple washing and detection steps are required before enzyme activity can be determined. The SPA, despite being isotope-based, requires no such washes or special incubations. The described assay has been successfully used to measure the activity of a number of a3 fucosyltransferases (FucT-III, VI, and VII) in addition to an a6 sialyltransferase (data not shown). It clearly can be adapted easily for the measurement of other glycosyltransferase activities. The a3 fucosyltransferase SPA reported here is the most suitable method yet described for the routine testing of a large number of samples with the added advantage of possessing the potential for simple automation. ACKNOWLEDGMENTS We thank Dr. Nick Smithers and Miss Sara Witham for the supply of the recombinant a3 fucosyltransferases and Dr. Martyn Banks for advice on the SPA technology.
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