2Institute for Molecular Bioscience, University of Queensland, Australia, 4072, and 3Alchemia Pty Ltd, PO Box 6242, Upper Mt Gravatt, Brisbane, Australia 4122
Received on November 27, 2000; revised on January 30, 2001; accepted on February 6, 2001.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: biosensor/glycosyltransferase activity/lectin/sialyltransferase
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The increasing interest in glycobiology and the potential biological roles of glycosyltransferases has necessitated the development of practical and fast methods for analysis of the kinetics and substrate specificities of these enzymes. Traditionally the analysis of glycosyltransferase activity has involved the use of radioactive transfer assays (Rearick et al., 1979; Beyer et al., 1981
; Weinstein et al., 1982
). These assays require the use of labeled substrates, along with the separation of the unused labeled compound from the products, prior to the determination of enzyme activity. Sample handling problems were solved in part by the method developed by Laroy et al. (1997)
, which converted the assays to a microtiter plate format. In this method the labeled product is precipitated and unused labeled components are removed by filtration on glass fiber supports. Laroy et al. (1997)
report that this style of assay detected as little as 0.1 µU of enzyme activity in 2 h. However, this assay cannot be used to distinguish between different kinds of sialyltransferase activity and still requires a radioactively labeled donor substrate.
Solid-phase absorbance and light-based assay methods have been developed for assessing sialyltransferase activities in pure and crude samples (Mattox et al., 1992; Yeh and Cummings, 1996
). These methods are free of radioactivity, use light-based signal amplification, and can be performed in a microtiter plate format with sensitivity down to 0.2 µU enzyme activity.
Here we report the development of an assay that uses the technique of real-time biospecific interaction analysis where measurements are based on surface plasmon resonance (SPR). Sample handling and detection are automated in the BIAcore 1000 instrument (BIAcore AB, Sweden). The instrument consists of a robotic injection system, an integrated fluidics controller, and a diode array optical detector. The sensor chip surface is a carboxymethylated dextran hydrogel matrix. The hydrogel acts as a medium for immobilization of ligands, in this case the lectin Sambucus nigra agglutinin (SNA), which can be done using a variety of chemistries. In general the hydrogel matrix does not interfere with the interaction between the immobilised ligand and the analyte in free solution. The BIAcore instrument uses SPR to investigate interactions that occur at the sensor chip surface. SPR is a phenomenon that occurs when light is reflected off thin metal films, in this case gold. A fraction of the light energy incident at a sharply defined angle can interact with the delocalised electrons in the metal film (plasmon), thus reducing the reflected light intensity. The precise angle of incidence at which this occurs is determined by a number of factors, but in the BIAcore instruments the principal determinant becomes the refractive index close to the surface of the gold sensor chip surface.
Target molecules are immobilized on the sensor chip surface and can interact with analyte molecules in the mobile phase running along a flow cell. If binding occurs to the immobilized target the local refractive index changes, leading to a change in SPR angle. The change in SPR can be monitored in real-time by detecting changes in the intensity of the reflected light, producing a sensorgram. The rates of change of the SPR signal can be analyzed to yield apparent rate constants for the association and dissociation phases of the reaction. The size of the change in SPR signal is directly proportional to the mass change at the sensor chip surface as a result of binding interactions between the immobilized target and analyte (Jonsson, 1992).
The increase in relative mass at the sensor chip surface is expressed as response units (RUs); a 1000-RU signal is equivalent to a change in mass of approximately 1 ng mm2 on the chip surface. The instrument generates a plot of RU versus time, the sensorgram. It is therefore possible to follow and study on and off rates for interacting species as well as effects of pH, ionic strength, and inhibitors on binding events.
The use of SPR techniques for the characterization of lectincarbohydrate binding specificities and kinetics has recently been reported by Haseley et al. (1999). They reported that SNA immobilized to the carboxymethyl dextran sensor chip surface binds both oligosaccharides and glycoproteins containing the NeuAc-
2-6Gal epitope moiety. They found that the association constants (KA) for various epitope ranged between 3.4 x 107 M1 for a multibranched oligosaccharide and 7.80 x 105 M1 for human transferrin. Hutchinson (1994)
used SPR techniques to characterize the oligosaccharide structures on bovine fetuin. Bovine fetuin was immobilized on the sensor chip surface and digested in situ using specific glycosidases. After each deglycosylation step the immobilized fetuin was analyzed with a panel of lectins. These studies indicated that the binding of lectins to specific oligosaccharides could be used as a sensitive method for the detection of product formation by glycosyltransferases. We have developed these methods specifically to study the activity of Galß1-4GlcNAc
2,6-sialyltransferase (EC 2.4.99.1) (ST6Gal I).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Background binding associated with the components of the assay mixture was accounted for by setting up a matrix of assays that covered all possible combinations of mixture components. Analysis of this suite of assays indicated that low levels of background binding were associated with asialofetuin (ASF) in the reaction mixture (data not shown). ASF is produced by the mild acid hydrolysis of fetuin to remove sialic acid residues (Manzi and Varki, 1992). This method is not quantitative, and batch-to-batch variation in the levels of background binding atributable to ASF can be expected. The inclusion of the appropriate controls in each experimental set allows the subtraction of background binding levels from the raw data.
Calibration of the SNA surface
Figure 1 shows the linearity of the detector response with increasing concentrations of assay product. Serial dilutions of assay mixtures, diluted with HBS pH 7.4 running buffer, were injected over the immobilized SNA. The assay mixtures, including 0.05 mU rat liver Galß1-4GlcNAc 2,6-sialyltransferase (EC 2.4.99.1) and 100 µg ASF, were incubated at 37°C for 5.5 h. The assay conditions are such that there is enough enzyme to sialylate all the available Gal residues on the ASF (estimated to be 150 nmole Gal per mg ASF) (Scudder and Chantler, 1981
; Gross et al., 1989
; Gross and Brossmer, 1995
) The binding responses for these injections were plotted against the concentration of fetuin in each sample. Using this data, the sensor chip surface can be calibrated for a known level of sialyltransferase activity and the calibration curve can then be used to estimate the activity in samples. When the conversion of ASF to fetuin is in the range of 0.195 (4 nM) to 0.781 µg (18 nM) the RU bound (
RU) values approach linearity (Figure 1). The equation for the linear regression line shown in Figure 1 is y = 146.9 ± 9.3x 6.1 ± 3.4. Detectable
RU is observed when the initial 0.05 mU assay is diluted 1:25,600 with HBS pH 7.4. This is equivalent to an assay containing 1 x 109 units of sialyltransferase activity. At higher concentrations of fetuin the SNA on the surface of the chip becomes saturated and the curve is no longer linear. Haseley et al. (1999)
used concentrations of fetuin in the range of 0.3110 µM to determine binding kinetics for the SNAfetuin interaction using a BIAcore 2000. They noted that if the lectin surface was saturated, any further increase in the concentration of the ligand reduced the observed response, presumably as a result of interactions between the bound and unbound ligand. To avoid complicating the analysis of the bound assay reaction product on the immobilized SNA, all reaction mixtures in this study were diluted with HBS so that the final concentration of the reaction product was within the range of 0.195 µg (4 nM) to 0.781 µg (18nM) which was found to be linear.
|
Assay reaction progress curve
The practicality of this method was also demonstrated in assays designed to determine the kinetics of product formation. Experiments comparing the activity of a recombinant human ST6Gal I (Halliday and Franks, unpublished data) and the commercially available rat liver ST6Gal I (EC 2.4.99.1) were performed. Details of the assay mixtures prepared are described under Materials and methods. Injections of the diluted assay mix were performed sequentially from the same assay vial every 20.25 min (Figure 2). T = 0 was taken as the time when the enzyme sample was added to the assay mixture. The length of the injection (association), dissociation, and regeneration steps of each biosensor cycle determines the time interval between injections. There is no observable difference between the reaction progress curves obtained for the rat liver ST6Gal I and the recombinant human ST6Gal I (Figure 2). The linear regression plot of the kinetics of the reaction during the first 80 min of the assay (Figure 2), shows that the initial phase of the reaction approached linearity for both the rat liver ST6Gal I (y = 0.025 ± 0.0007x + 23.09 ± 2.1) and the recombinant human ST6Gal I (y = 0.025 ± 0.001x + 30.3 ± 4.8). Each time SNA is immobilized on a new sensor chip flow cell, the absolute RU values detected change. Provided appropriate calibrations and controls are included, it is possible to compare data obtained from different flow cell surfaces.
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The preliminary studies of the kinetic parameters of ST6Gal I activity presented here suggest that the method can be further developed to allow detailed studies of Km and Vmax for a variety of donor and acceptor substrates. The Km values of between 29 and 35 µM for CMP-Neu5Ac derived from the experiments presented here fall between the values of 5.3 µM (Weinstein et al., 1982) and 120 µM (Gross et al., 1989
) reported in the literature. As mentioned, the use of specific oligossacharide acceptors in kinetics experiments will give more precise quantitiative results. The glycoprotein ASF used in the experiments presented here provides a heterogeneous population of ologosaccharide acceptor sites that may be sialylated at different rates by the enzyme, which can complicate analysis of results. To ameliorate the effects of heterogeneity of ASF acceptor sites in assays, care was taken to use the same batch of ASF in all experiments. In addition these reaction mixtures contained a single sialyltransferase, ST6Gal I. These precautions effectively ensured that the available acceptor sites and rates of transfer to those sites remained constant in all assays. Digestion of the reaction product with specific neuraminidases showed there was no detectable
2,3 or
2,8 sialyltransferase activity in the reaction mixture (data not shown).
In particular it may be possible to use this method to analyse small quantities of substrates extracted from cellular material, therefore allowing determinations of kinetic values that more closely approximate in vivo values. However, in these assays specific acceptor oligosaccharides would need to be used to ensure that the activitiy of a specific sialyltransferase was being detected.
It is a relatively simple process to develop new assays for different glycosyltransferases provided that suitable lectins or antibodies are available to immobilise on the surface of the BIAcore chip. For example Hutchinson (1994) used SPR to study the carbohydrate structures on bovine fetuin. That study reports that fetuin was immobilized on the carboxymethyl dextran and sequentially digested with glycosidases. After each digestion step the immobilized glycoprotein was probed sequentially with a panel of specific lectins to determine which carbohydrate epitopes had been exposed. Using these types of methods it is possible to do sequential glycosidase digestions followed by detection of released carbohydrate epitopes using immobilized lectins in a semi-automated manner.
Based on the definition that 1.0 U sialyltransferase transfers 1 µmol of CMP-NeuAc transferred to an acceptor substrate per minute at 37°C (Weinstein et al., 1982) we can assume that 1.0 U ST6Gal I will sialylate 1.0 µmol of Gal per min. It is reported that there are approximately 150 nmol available Gal sites per mg ASF (Scudder and Chantler, 1981
; Gross et al., 1989
; Gross and Brossmer, 1995
). In an assay mixture that contains 100 µg of ASF there are approximately 15 nmol of Gal sites. Using the assay methods described in this article we are able to detect product formation on as little as 0.08 µg of ASF, which is approximately 3.6 pmoles NeuAc-
2-6Gal-R. This is equates to approximately 0.4 µU of ST6Gal I activity.
This biosensor method is still several hundred-fold less sensitive than the method using CMP-9-fluoresceinyl-NeuAc as the donor, which can detect 0.001 µU of sialyltransferase activity (Gross et al., 1990). However, the use of CMP-9-fluoresceinyl-NeuAc as a donor requires a time-consuming separation step, and different kinetic parameters are obtained when compared to the same assay conducted with radioactively labeled CMP-NeuAc (Gross and Brossmer, 1988
). In addition, the large fluoresceinyl group on the donor molecule could interfere with the binding and transfer to some substrates, making interpretation of comparative kinetic data difficult.
In summary, we have developed an easy, quick, and sensitive method for studying ST6Gal I activity that could be applied to a number of different glycosyltransferase assays or different enzyme species, provided a suitable detection surface is available. The assay mixture is left intact and is easily collected for further analysis of product and enzymes. Aside from the initial expense of the instrumentation, further costs and disposal of wastes are considerably reduced when compared to other methods. In our minds the best application of this method is in the monitoring of sialyltransferase activity in the expression and purification of recombinant human ST6Gal I. The relatively rapid assay time and the small volumes required mean that expression and purification optimization on a small scale can be completed quickly. This will be particularly useful in the expression and characterization of mutants where only small quantities of recombinant enzymes are produced. Using conventional radioassay methods, such studies would take significantly longer when separation and scintillation counting time are accounted for.
The increasing repertoire of specific lectins and other carbohydrate-recognizing receptors, as well as advances in instrument design and sensor chip surfaces, should lead to the development of biosensor-based methods for additional applications in glycobiology.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The running buffer used for BIAcore experiments was 10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% (v/v) P-20 (HBS). Unless otherwise stated, all sialyltransferase assays were performed in 35 mM MES buffer, pH 6.50, 10 mM MnCl2, 0.1% Triton X-100.
Immobilization
SNA was immobilized on the sensor surface using NHS-EDC amine coupling chemistry. Chemical activation of the carboxymethyl dextran sensor chip surface (CM5 sensor chip) was accomplished by injecting 30 µl of 0.1 M NHS mixed with 0.1 M EDC in a 1:1 ratio at 1 µl min1. The protein was dissolved in 10 mM sodium acetate pH 4.0 at a concentration of 200 µg ml1 and 30 µl was injected across the activated surface at 1 µl min1. Injecting 35 µl of 1.0 M ethanolamine in water at 5 µl min1 capped residual activated esters on the surface. Prior to any further injections the surface was washed by six 20-µl pulses of 0.05 M HCl at 20 µl min1 to remove noncovalently attached SNA from the hydrogel matrix. The running buffer used for all steps was HBS pH 7.4.
Sialyltransferase assays
Assays were performed in 35 mM MES buffer pH 6.50, 10 mM MnCl2, 0.1% Triton X-100 with substrate concentrations as follows: ASF 100 µg, CMP-NeuAc 150 µM, including 9 nM 14C-CMP-NeuAc in some assays, in a total volume of 40 µl. Typically sialyltransferase reactions were stopped by adding 10 µl of 0.1M CTP, which acts as an inhibitor of enzyme activity. Details of specific sialyltransferase assay mixtures are given in individual figure legends. The assay mixtures were diluted 1:50 in HBS unless otherwise stated. Fifteen to 40 µl of diluted sample was injected across the SNA surface at either 10 or 20 µl min1. A report point was taken 60 s after the end of the injection. This value represents the level of transfer in the assay and is shown as RU bound (RU). The SNA surface was regenerated using 10-µl pulses of 0.02 M HCl injected at 10 µl min1; this disrupts the binding between the NeuAc
2-6Gal-R product and the SNA, allowing the product to be washed off the sensor surface without removing the SNA.
The stability of the SNA surface to repeated injections was studied by performing multiple injection cycles of a standard assay as described above at a 1:50 dilution with HBS buffer. Starting baseline and bound product data were extracted and plotted as a function of cycle number (results not shown).
Calibration of the SNA surface
Duplicate assay mixtures containing 0.05 mU rat liver Galß1-4GlcNAc 2,6-sialyltransferase (EC 2.4.99.1) and 100 µg ASF were incubated at 37°C for 5.5 h. The assay conditions are such that this amount of enzyme will sialylate all the available Gal residues on the ASF (estimated to be 150 nmole Gal per mg ASF) (Scudder and Chantler, 1981
; Gross et al., 1989
; Gross and Brossmer, 1995
). At the end of the reaction period the assays were stopped by the addition of 0.1 M CTP and diluted 1:50 with HBS followed by serial 1:2 dilutions with HBS. This gave a set of samples containing the equivalent of 50, 25, 12.5, 6.25, down to 0.048 µg ml1 of fetuin. These diluted samples were then injected in duplicate across the SNA surface to generate a calibration curve. All data analyses and curve fitting were performed using GraphPad Prism.
Assay reaction progress curve
The time-course of reaction product formation was determined using conditions similar to those described above. Assays containing either 0.3 mU commercially available rat liver ST6Gal I or 0.3 mU of rhST6Gal I, 300 µg ASF, 128 µM CMP-NeuAc were started by the addition of the enzyme. At about 20-min intervals, a sample from the assay was removed and diluted 1:50 using the instrument robotics unit. A 40-µl injection of this diluted sample was then performed at 10 µl min1 with regeneration conditions as described above.
Comparison of the traditional radioactive transfer assay with the bisensor-based assay
To compare results from standard radioactive assays with data obtained from the BIAcore, a time-course experiment was performed where assays were run using 14C-labeled CMP-NeuAc. The assay mixture contained 0.05 mU rat liver ST6Gal I, 50 µg ASF, 130 µM CMP-NeuAc including CMP-[4,5,6,7,8,9-14C]NeuAc to give a final specific activity of 5000 c.p.m./nmol in 35 mM MES buffer pH 6.50, 10 mM MnCl2, 0.1% (w/v) Triton X-100. Activity was measured at 20-min intervals by taking an aliquot of the reaction mixture and stopping the reaction by the addition of 10 µl 0.1 M CTP in water. Aliquots were frozen until analysis. Half of each aliquot was injected onto a Pharmacia HR10/10 column packed with Spehadex G25 superfine and product separated from substrates by elution with 50 mM sodium phosphate buffer pH 7.4 at 1.5 ml min1. Fractions of 0.3 ml were collected and 2.5 ml of scintillant was added to each. After thorough mixing, each fraction was counted in a Wallac RackBeta Liquid Scintillation Counter. The enzyme activity was expressed as % transfer, which can be calculated by dividing the CPM bound to the acceptor by the total CPM present in the assay mixture. In parallel, the other half of the assay mixture was diluted 1:50 with HBS and analyzed using the BIAcore method described above. All assays were done in duplicate and percent transfer and RUs bound (RU) were plotted as a function of assay duration.
Determination of apparent Km
A series of sialyltransferase assays were performed to determine if the biosensor-based method described above could be used to derive kinetic values for CMP-Neu5Ac. Assay reaction mixes contained 2.5 µg µl1 ASF, 10 mM MnCl2, and 0.1%Tx-100 in 35 mM MES pH 6.5, the concentration of CMP-Neu5Ac used ranged between 12.5 µM and 200 µM. In all experiment 0.15 mU of rat liver Galß1-4GlcNAc 2,6-sialyltransferase (EC 2.4.99.1) (Calbiochem) was added to the reaction mixture to give a final reaction mixture volume of 100 µl. Addition of the enzyme was designated as T = 0 min. At T = 5, 10, 15, 20, and 25 min, 10 µl of the reaction mixture was removed from the reaction and diluted 1:100 with HBS running buffer. Diluted aliquots were frozen until they were analyzed on the BIAcore as described above.
Biosensor sialyltransferase assays used to monitor the expression and purification of recombinant human ST6Gal I (rhST6Gal I)
To facilitate the rapid screening of multiple samples from expression and purification optimisation experiments the sialyltransferase assay was adapted to a microtitre plate format. Assay mix was made such that 3.75 µl could be added to 6.25 µl of cell culture medium or cell lysate containing rhST6Gal I (Halliday and Franks, unpublished data) to give a final concentration of 35 mM MES pH 6.5, 0.1% Triton X-100, 2.5 µg ml1 ASF, 150 µM CMP-Neu5Ac, and 5 mM MnCl2 in a total volume of 10 µl. Reactions were incubated for up to 2 h at 37°C and were stopped by the addition of 490 µl of HBS. Comparisons between (1) assay reactions stopped by the addition of 0.1 M CTP and then diluted with HBS and (2) those stopped by dilution alone were performed. The two sets of diluted assays were analysed twice with an interval of 24 h between each analysis. There was no observable difference between samples stopped by CTP and dilution versus dilution alone (results not shown). The diluted reaction mixture was injected over the flow cell surface as described above. In all experiments 0.01 and/or 0.05 mU of rat liver ST6Gal I (Calbiochem) was included as a positive control. Negative controls included cell culture media or cell lysate from uninfected or untransfected cell lines and assay mixture without exogenous ST6Gal I added.
![]() |
Abbreviations |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Brockhausen, I., Schutzbach, J., and Kuhns, W. (1998) Glycoproteins and their relationship to human disease. Acta Anatom., 161, 3678.[ISI]
Gross, H.J., and Brossmer, R. (1988) Enzymatic introduction of a fluorescent sialic acid into oligosaccharide chains of glycoproteins. Eur. J. Biochem., 177, 583589.[Abstract]
Gross, H.J., and Brossmer, R. (1995) Enzymatic transfer of sialic acids modified at c-5 employing four different sialyltransferases. Glycoconj. J., 12, 739746.[ISI][Medline]
Gross, H.J., Rose, U., Krause, J.M., Paulson, J.C., Schmid, K., Feeney, R.E., and Brossmer, R. (1989) Transfer of synthetic sialic acid analogues to N- and O-linked glycoprotein glycans using four different mammalian sialyltransferases. Biochemistry, 28, 73867392.[ISI][Medline]
Gross, H.J., Sticher, U., and Brossmer, R. (1990) A highly sensitive fluorometric assay for sialyltransferase activity using CMP-9-fluoresceinyl-NeuAc as donor. Anal. Biochem., 186, 127134.[ISI][Medline]
Haseley, S., Talaga, P., Kamerling, J., and Vliegenthart, J. (1999) Characterization of the carbohydrate binding specificity and kinetic parameters of lectins by using Surface Plasmon Resonance. Anal. Biochem., 274, 203210.[ISI][Medline]
Hutchinson, A.M. (1994) Characterization of glycoprotein oligosaccharides using Surface Plasmon Resonance. Anal. Biochem., 220, 303307.[ISI][Medline]
Jonsson, U. (1992) Biosensors: Fundamentals, Technologies and Applications. VCH, New York.
Laroy, W., Maras, M., Fiers, W., and Contreras, R. (1997) A radioactive assay for sialyltransferase activity using 96-Well Multiscreen filtration plates. Anal. Biochem., 249, 108111.[ISI][Medline]
Manzi, A.E., and Varki, A. (1992) Compositional analysis of Glycoproteins. In Fukuda, M. and Kobata, A., eds., Glycobiology: A Practical Approach. Oxford University Press, Oxford, pp. 2777.
Mattox, S., Walrath, K., Ceiler, D., Smith, D.F., and Cummings, R.D. (1992) A solid phase assay for the activity of CMPNeuAc:Gal-2,6-Sialyltransferase. Anal. Biochem., 206, 430436.[ISI][Medline]
Rearick, J.I., Sadler, J.E., Paulson, J.C., and Hill, R.L. (1979) Enzymatic characterization of D-galactoside 2-3 Sialyltransferase from porcine submaxillary gland. J. Biol. Chem., 254, 44444451.[Abstract]
Scudder, P.R., and Chantler, E.N. (1981) Glycosyltransferases of the human cervical epithelium. II. Characterization of a CMP-N-acetylneuraminate: galactosyl-glycoprotein sialyltransferase. Biochim. Biophys. Acta, 660, 136141.[ISI][Medline]
Varki, A. (1993) Biological roles of oligosaccharides: all of the theories are correct. Glycobiology, 3, 97130.[Abstract]
Varki, A. (1997) Sialic acids as ligands in recognition phenomena. FASEB J., 11, 248255.
Weinstein, J., de Souza-e-Silva, U., and Paulson, J.C. (1982) Sialylation of glycoprotein oligosaccharides N-linked to asparagine. Enzymatic characterization of a Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialyltransferase and a Gal beta 1 to 4GlcNAc alpha 2 to 6 sialyltransferase from rat liver. J. Biol. Chem., 257, 1384513853.
Yeh, J.-C., and Cummings, R.D. (1996) Absorbance- and light-based solid-phase assays for CMPNeuAc:Galbeta1-4GlcNAc-R alpha-2,3-sialyltransferase. Anal. Biochem., 236, 126133.[Medline]