A rapid, semi-automated method for detection of Galß1-4GlcNAc {alpha}2,6-sialyltransferase (EC 2.4.99.1) activity using the lectin Sambucus nigra agglutinin

Judy A.W. Halliday1,2, Alison H. Franks2, Tracie E. Ramsdale3, Rodney Martin2 and Elka Palant2

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
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Abbreviations
 References
 
Sialyltransferase activity has traditionally been studied by determining the rate at which the enzyme transfers a labeled donor sugar to an acceptor substrate. These types of assays can be difficult to quantitate, and the separation of untransfered donor sugar from the sialylated acceptor is time-consuming. The biosensor-based method described here is both rapid and semi-automated. The NeuAc-{alpha}2-6Gal-R-specific lectin Sambucus nigra agglutinin (SNA) immobilized to the carboxymethyl dextran surface of a BIAcore sensor chip was used to detect and measure the formation of the NeuAc-{alpha}2-6Gal-R moieties. The sialyltransferase assays were carried out using modified protocols based on the method described in Rearick, J.I., Sadler, J.E., Paulson, J.C., and Hill, R.L. (1979) Enzymatic characterization of ßD-galactoside {alpha}2-3 sialyltransferase from porcine submaxillary gland. J. Biol. Chem., 254, 4444–4451. The complete assay mixture was simply diluted before injection into the instrument. All injections were performed automatically using the robotics of the BIAcore instrument. Using this technique it is possible to detect product from 0.4 µU of commercial Galß1-4GlcNAc {alpha}2,6-sialyltransferase (EC 2.4.99.1) (ST6Gal I). One unit of sialyltransferase is defined as the quantity that will transfer 1 µmol of N-acetylneuraminic acid from cytidine monophosphate (CMP)-N-acetylneuraminic acid to asialofetuin per min at pH 6.5 and 37°C. The method described here requires as little as 10 µl total assay volume, thus reducing the consumption of reagents. In addition, the sample is completely recoverable from the sensor chip surface, which allows for downstream analysis of the reaction product if desired. This method eliminates the need for labeled donor and acceptor molecules and does not require the separation of the substrates from the product before analysis. Although some kinetic properties of the enzyme can be estimated using this method, further development and validation is required. The method is most useful in determining qualitative estimates of ST6Gal I activity in tissue extracts and in characterizing the production of enzymes in cultured cell systems. The use of a microtiter plate assay format enables the rapid screening of multiple fractions for sialyltransferase activity.

Key words: biosensor/glycosyltransferase activity/lectin/sialyltransferase


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Abbreviations
 References
 
Glycosylation is one of the most frequently occurring posttranslational modifications made to proteins and lipids in eukaryotic organisms. The resultant carbohydrate side chains often have very complex oligosaccharide sequences and concomitant structural diversity. The functional roles of these carbohydrate side chains cover a wide spectrum, from relatively trivial to crucial for the growth, development, and survival of an organism. Of particular importance are the sialic acid residues, which often terminate carbohydrate chains. Sialic acid residues act as receptors for a number of bacterial and viral pathogens; they determine the half-lives of many circulating glycoproteins; and they play critical roles in cellular communication and differentiation (Varki, 1993Go, 1997; Brockhausen et al., 1998Go).

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., 1979Go; Beyer et al., 1981Go; Weinstein et al., 1982Go). 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)Go, 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)Go 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., 1992Go; Yeh and Cummings, 1996Go). 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, 1992Go).

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 mm–2 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 lectin–carbohydrate binding specificities and kinetics has recently been reported by Haseley et al. (1999)Go. They reported that SNA immobilized to the carboxymethyl dextran sensor chip surface binds both oligosaccharides and glycoproteins containing the NeuAc-{alpha}2-6Gal epitope moiety. They found that the association constants (KA) for various epitope ranged between 3.4 x 107 M–1 for a multibranched oligosaccharide and 7.80 x 105 M–1 for human transferrin. Hutchinson (1994)Go 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 {alpha}2,6-sialyltransferase (EC 2.4.99.1) (ST6Gal I).


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Abbreviations
 References
 
Immobilization
Using the immobilization protocol described in materials and methods, between 7000 and 10,000 RUs (approx. 10 ng on the chip surface) of SNA was immobilized on the carboxymethyl dextran hydrogel of the CM5 sensor chip. A typical immobilization protocol consisted of three phases: (1) activation, (2) ligand immobilization, and (3) residual surface ester inactivation. In preparation for use, the surface was washed with several pulses of 0.02 M HCl to remove nonspecifically bound protein. The stability of the surface was tested by repeated injection and surface regeneration cycles using an 0.01 mU ST6Gal I assay mixture that contained 100 µg ASF, 150 µM CMP-Neu5Ac, 10 mM MnCl2, 35 mM MES pH 6.5, and 0.1% Triton X-100 in a total volume of 40 µl; the mixture was incubated at 37°C for 5.5 h. The assay mixture was then diluted 1:50 with 10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% surfactant P20 (HBS) before 30 injection/regeneration cycles were done. An average signal of 476.9 RU bound (range 454.9–517.5 RU) and a starting baseline, which lost 22.7 RUs across the injection set, was obtained. Each time a new SNA surface was prepared these tests were done. The variations in RU bound values obtained from different immobilizations can be accounted for by the variations in the levels of SNA bound to the surface during the amine coupling steps.

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, 1992Go). 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 {alpha}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, 1981Go; Gross et al., 1989Go; Gross and Brossmer, 1995Go) 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 ({Delta}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 {Delta}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 10–9 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)Go used concentrations of fetuin in the range of 0.31–10 µM to determine binding kinetics for the SNA–fetuin 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.



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Fig. 1. Calibration plot of glycoprotein bovine fetuin binding to the SNA surface of a sensor chip. Assay conditions and dilutions are described in Materials and methods. Each diluted assay sample was analyzed by injecting the sample over the flow cell surface for 90 s. {Delta}RU represents the change in response measured 60 s after the end of the sample injection ({Delta}RU = 60-s RU value – RU value prior to injection). After each sample injection the bound sample was removed by a pulse of 10 µl 0.02 M HCl at 10 µl min–1. Data points are the mean ± SEM of duplicate determinations. The figure inset shows the product concentration range (0.06–0.75 µg) over which there is a linear relationship between reaction product produced and {Delta}RU. The equation for the linear regression of these data points is y = 147.9 ± 9.4x – 6.7 ± 3.4.

 
It is important to note that each time a new sensor chip surface is coated with immobilized lectin it must be calibrated. It is also important that the calibration uses the same acceptor substrate as used in the enzyme assays to account for the batch to batch variation in ASF. It is also desirable to check the surface calibration at intervals to ensure that the characteristics of the lectin–acceptor interaction are stable.

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.



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Fig. 2. Reaction progress curves for commercial rat liver ST6Gal I and recombinant human ST6Gal I. Assay mixtures containing 0.3 mU of either rat liver ST6Gal I or recombinant human ST6Gal I were carried out as described in Materials and methods. All assays were done in triplicate. The figure inset shows the first 80 min of the reactions for both enzymes where the reaction curve approached linearity. The equations for the lines as determined by GraphPad Prism are y = 0.025 ± 0.0007x + 23.09 ± 2.1 for rat liver ST6Gal I and y = 0.025 ± 0.001x + 30.3 ± 4.8 for rhST6Gal I.

 
Comparison of the radioactive transfer assay with the bisensor-based assay.
For comparison of the radioactive transfer method (Rearick et al., 1979Go) with the biosensor-based method, a comparatively large-scale radioactively labeled 14C-CMP-NeuAc transfer assay was carried out as described in Materials and methods. Half of the assay mixture was separated using standard gel filtration techniques to separate the unused donor from the labeled product. Fractions were collected from the gel filtration column and the amount of radioactivity incorporated into the product quantitiated by liquid scintillation counting and expressed as percent transfer. The other half of the same assay was used to determine the level of NeuAc{alpha}2-6Gal-R product formation using immobilized SNA as described above. A qualitative comparison of the rate of product formation by the two methods over 2 h is shown in Figure 3. These experiments were performed in duplicate.



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Fig. 3. Qualitative comparisons of percent transfer of 14C-CMP-Neu5Ac to ASF as measured by the radioactive transfer method (left panel) and {Delta}RU as measured by product binding to SNA (right panel). As detailed in Materials and methods, a comparatively large volume radioactive transfer reaction was carried out and aliquots removed for analysis at the times shown. All data points are the mean ± SEM of duplicate determinations.

 
Determination of apparent Km values
A preliminary series of experiments was performed to determine whether the biosensor method could be used to derive values for apparent Km for the CMP-Neu5Ac donor substrate (Figures 4 and 5). It should be noted that in all experiments the acceptor substrate used was ASF. Although a simple defined acceptor substrate like Galß1-4GlcNAcß1-6Man would be most suitable for these types of assays the moleculer weight of the oligosaccharide is too low for reliable detection in the BIAcore 1000 instrument. Details of the calculations are included in the figure legends. In all experiments shown rat liver ST6Gal I was used. Analysis of the plot of 1/{Delta}RU min–1 versus 1/[CMP-Neu5Ac] (Figure 5, {Delta}RU min–1 values for each concentration of CMP-Neu5Ac are derived from the linear regression of the data shown in Figure 4) gives an apparent Km for CMP-Neu5Ac of 35 µM. This value falls between the values of 16 and 47 µM reported in the literature (Scudder and Chantler, 1981Go; Weinstein et al., 1982Go). Analysis of the plot of {Delta}RU min–1 versus [CMP-Neu5Ac] (Figure 5, inset) gives an apparent Km of 30 µM from the x intercept = –1/Km. This preliminary array of data indicate that this method could be extended to develop a rigorous protocol for the determination of apparent Kmand Vmax values. Future studies will include the use of various glycoprotein, oligosaccharide, and synthetic substrates. The BIAcore 1000 instrument used in these experiments has a limited dynamic range and cannot reliably be used to detect product formation if the product has a relatively low molecular weight (< 3000 Da). Haseley et al. (1999)Go have reported the use of the BIAcore 2000 in characterizing the binding specificity and kinetics of SNA for a number of oligosaccharides. Their studies indicate that the types of sialyltransferase kinetics studies described here could be extended to include a variety of oligosaccharides and other glycoconjugates using either a BIAcore 2000 or 3000 instrument.



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Fig. 4. Effect of variations in the concentration of CMP-Neu5Ac on the reaction rate. Assay reaction mixes contained 2.5 µg µl–1 ASF, 10 mM MnCl2, and 0.1% Tx-100 in 35 mM MES pH 6.5, the concentration of CMP-Neu5Ac used were 12.5 µM (square), 25 µM (triangle, base down), 50 µM (triangle, base up), 100 µM (diamonds), and 200 µM (circles). In all experiment 0.15 mU of rat liver ST6Gal I was added to the reaction mixture to give a final reaction mixture volume of 100 µl. Addition of the enzyme was designated 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 and injected across the SNA sensor chip surface as described in Materials and methods. All reactions were done in duplicate, data points are mean ± SEM.

 


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Fig. 5. Experimental derivation of an apparent Km for CMP-Neu5Ac. The slopes of the lines derived from the data in Figure 4 were plotted as shown. The larger figure shows a Lineweaver-Burk type plot of 1/slope, calculated from data in Figure 4, versus 1/[CMP-Neu5Ac] where the x intercept value of –0.029 predicts a Km value of 29 µM. The inset shows the plot of {Delta}RU min–1 versus [CMP-Neu5Ac]. This plot predicts a Km value of 35 µM. All data analyses and plots were done using GraphPad Prism.

 
Biosensor sialyltransferase assays used to monitor the expression and purification of recombinant human ST6Gal I (rhST6Gal I)
In our hands the method described here has been most useful in allowing the rapid monitoring of the expression of rhST6Gal I in a variety of expression systems (Halliday and Franks, unpublished data). In cases where low concentrations of high specific activity rhST6Gal I are produced this method is used to track the purification of the enzyme. Sensorgrams showing the production of active rhST6Gal I by Sf9 insect cells are shown in Figure 6a. Calibration of the SNA surface and inclusion of the appropriate controls allows the estimation of the yields of active enzyme from this system (Figure 6b). In experiments where the expression of rhST6Gal I was directed to the media by a secretion signal, the yield was estimated to be approximately 12 U of rhST6Gal I activity per L of culture medium (Halliday and Franks, unpublished data).




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Fig. 6. Biosensor monitoring of the production and purification of rhST6Gal I. Sensorgrams illustrating the binding of reaction product to immobilized SNA are shown in the top panel. The fetuin was produced by the activity of recombinant human ST6Gal I containing culture media, in an assay mixture containing the acceptor ASF, and the donor CMP-Neu5Ac in microtiter format, as detailed in Materials and methods. The diluted assay mixture was injected across the SNA surface for 300 s at a flow rate of 5 µl min–1 (solid line). Sensorgrams of control assays are also shown. The positive control was 0.01 mU of rat liver ST6Gal I (dotted line), negative controls included H2O (dashed line), and culture media from uninfected control cells (dashed-and-dotted line) are also shown. The bottom panel is a bar graph showing the data collected from the sensorgrams in the top panel.

 
The sensitivity of the method described here allows small-volume assays to be carried out in a microtiter plate format as described in Materials and methods. This enables the rapid screening of purification protocols by tracing sialyltransferase activity through various extraction and chromatography conditions whilst consuming small quantities of sample.


    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Abbreviations
 References
 
To detect the activity of sialyltransferases, or other glycosyltransferases for that matter, that exist in very low concentrations, it has become necessary to develop sensitive activity assays. The use of radioactive CMP-NeuAc, though sensitive, can become prohibitively expensive when numerous expression and purification conditions are being screened. The need to separate the unused radioactive donor from the reaction product includes several steps and typically is quite labor-intensive. Enhancing the signal of assay products using light and fluorescence approaches have been effective (Mattox et al., 1992Go; Yeh and Cummings, 1996Go). The interpretation of the data from these methods, which use a modified donor substrate containing a bulky fluorescent reporter, was complicated by the resultant alteration of the enzyme affinity and kinetics with respect to the donor molecule. The method described here, using the enhanced sensitivity and carbohydrate linkage discrimination afforded by lectins in combination with the BIAcore’s advantages of detection and robotic automation, results in a reliable tool for the study of sialyltransferases with a minimum of expense and handling per reaction. It is possible to use the microtiter assay protocol to screen over 150 assay mixtures overnight, which is very useful in the development and optimization of chromatographic purification protocols.

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., 1982Go) and 120 µM (Gross et al., 1989Go) 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 {alpha}2,3 or {alpha}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)Go 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., 1982Go) 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, 1981Go; Gross et al., 1989Go; Gross and Brossmer, 1995Go). 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-{alpha}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., 1990Go). 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, 1988Go). 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
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Abbreviations
 References
 
Instrumentation and reagents
The Upgraded BIAcore 1000 instrument, CM5 sensor chip (research grade), N-hydroxy succinimide (NHS) 1-ethyl-3- (3-dimethylamino-propyl) carbodiimide (EDC) amine coupling kit and P20 Surfactant were all from BIAcore AB. Other reagents, including buffers, SNA, ASF, CMP-NeuAc, and cytidine 5' triphosphate (CTP) were purchased from Sigma. Rat liver Galß1-4GlcNAc {alpha}2,6-sialyltransferase (EC 2.4.99.1) was purchased from Calbiochem. CMP- [4,5,6,7,8,9-14C] NeuAc (specific activity 286 mCi/mmol) was obtained from Amersham (Buckinghamshire, England). The liquid scintillant used was OptiPhase HiSafe 3 from Wallac (Finland).

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 min–1. The protein was dissolved in 10 mM sodium acetate pH 4.0 at a concentration of 200 µg ml–1 and 30 µl was injected across the activated surface at 1 µl min–1. Injecting 35 µl of 1.0 M ethanolamine in water at 5 µl min–1 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 min–1 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 min–1. 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 ({Delta}RU). The SNA surface was regenerated using 10-µl pulses of 0.02 M HCl injected at 10 µl min–1; this disrupts the binding between the NeuAc{alpha}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 {alpha}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, 1981Go; Gross et al., 1989Go; Gross and Brossmer, 1995Go). 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 ml–1 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 min–1 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 min–1. 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 ({Delta}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 µl–1 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 {alpha}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 ml–1 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
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Abbreviations
 References
 
ASF, asialofetuin; CMP, cytidine monophosphate; EDC, 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide; Gal, galactose; GlcNAc, N-acetylglucosamine; HBS, HEPES buffered saline; NeuAc, N-acetylneuraminic acid; NHS, N-hydroxy succinimide; RU, response unit; SNA, Sambucus nigra agglutinin; SPR, surface plasmon resonance.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Abbreviations
 References
 
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