- Email: [email protected]
A nonradioactive 96-well plate assay for screening of trans-sialidase activity
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 322 (2003) 139–147 www.elsevier.com/locate/yabio
A nonradioactive 96-well plate assay for screening of trans-sialidase activityq Silke Schrader,a Evelin Tiralongo,a Gast on Paris,b Teruo Yoshino,c and Roland Schauera,* a Biochemisches Institut, Christian-Albrechts-Universit€at zu Kiel, 24098 Kiel, Germany Instituto de Investigaciones Biotecnol ogicas, Universidad Nacional de Gral. San Martın, 1650 San Martın, Argentina c Department of Chemistry, Division of Natural Sciences, International Christian University, Tokyo 181-8585, Japan
b
Received 25 March 2003
Abstract Trans-sialidase (E.C. 3.2.1.18) catalyzes the transfer of preferably a2,3-linked sialic acid to another glycan or glycoconjugate, forming a new a2,3 linkage to galactose or N-acetylgalactosamine. Here, we describe a nonradioactive 96-well plate fluorescence test for monitoring trans-sialidase activity with high sensitivity, specificity, and reproducibility using sialyllactose and 4-methylumbelliferyl-b-D -galactoside as donor and acceptor substrates, respectively. The assay conditions were optimized using the trans-sialidase from Trypanosoma congolense and its general applicability was confirmed with recombinant trans-sialidase from Trypanosoma cruzi. Using this procedure, a large number of samples can be tested quickly and reliably, for instance in monitoring trans-sialidase during enzyme purification and the production of monoclonal antibodies, for enzyme characterization, and for identifying potential substrates and inhibitors. The trans-sialidase assay reported here was capable of detecting trans-sialidase activity in the low-mU range and may be a valuable tool in the search for further trans-sialidases in various biological systems. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Trans-sialidase; Nonradioactive screening assay; 96-well plates; Trypanosoma congolense; Trypanosoma cruzi
Trans-sialidase was first described in trypanosomes. These flagellated protozoa are the agents of several human diseases such as Chagas disease in South and Central America caused by Trypanosoma cruzi [1,2] and sleeping sickness in Africa caused by T. b. gambiense and T. b. rhodesiense [3]. They are also responsible for Nagana, a disease of cattle in central Africa, caused by T. b. brucei and Trypanosoma congolense [4,5]. Although these parasites are unable to synthesize sialic acids (N-acetylneuraminic acid, Neu5Ac, and derivatives thereof) [6], they possess an enzyme, trans-sialidase, which enables them to acquire this sugar from host glycoconjugates for sialylation of their plasma membrane glycoproteins [7]. Trans-sialidase is believed to q The financial support by Numico Research (Friedrichsdorf, Germany), Sialic Acids Society (Kiel, Germany), and Fonds der Chemischen Industrie (Frankfurt, Germany) is gratefully acknowledged. * Corresponding author. Fax: +49-431-880-2238. E-mail address: [email protected] (R. Schauer).
0003-2697/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2003.07.016
play an important role in the pathogenicity of trypanosomes [8,9]. This enzyme has also been detected in Endotrypanum species [10] and Corynebacterium diphtheriae [11]. Recently, the existence of trans-sialidase in human serum was also described [12]. Trans-sialidase catalyzes the transfer of preferably a2,3-linked sialic acid directly to terminal b-galactoseor b-N-acetylgalactosamine-containing acceptors, giving rise to a new a2,3 linkage [13–16]. In the absence of an appropriate acceptor, this enzyme acts as a sialidase, hydrolytically releasing glycosidically linked sialic acid. Interest in understanding the complete catalytic mechanism of trans-sialidase [17,18], its possible application in the regiospecific synthesis of sialylated carbohydrates and glycoconjugates [19,20], and the search for potent trans-sialidase inhibitors to reduce trypanosome pathogenicity has stimulated much research on this enzyme. Recently, Buschiazzo et al. [21] reported the crystal structure of T. cruzi trans-sialidase and could show that sialic acid binding triggers a conformational switch
140
S. Schrader et al. / Analytical Biochemistry 322 (2003) 139–147
activating the enzyme and modulating affinity for the acceptor substrate. Trans-sialidase activity is generally detected with a radioactive assay using sialyl-a2,3-lactose as a donor and radioactively labeled lactose or N-acetyllactosamine as an acceptor. Following the reaction, the radioactive, negatively charged sialylated products are separated from labeled uncharged acceptor molecules by anion exchange chromatography and quantified to give a measure of enzyme activity [2,13,15,16,22]. Another radioactive test was described by Tertov et al. [12] who used a variety of donors containing radioactively labeled Neu5Ac. Trans-sialidase activity was then monitored either by quantifying the amount of labeled sialic acid released from the donors in the presence of an appropriate acceptor or by directly quantifying radioactivity on the acceptor molecules. Additionally, nonradioactive trans-sialidase assays have been reported. Lee and Kim [23] described a spectrophotometric assay, in which the amount of Neu5Ac transferred from the sialyl donor to the o-nitrophenyl-a-D -galactopyranoside (ONPG)1 acceptor was determined by measuring the concentration of the remaining ONPG with b-galactosidase. Harrison et al. [24] investigated the transfer activity of trans-sialidase using Neu5Aca2,3Gal-b-O-p-nitrophenol as the donor together with an appropriate acceptor. The release of p-nitrophenol (PNP) from the desialylated donor substrate was measured using b-galactosidase. Furthermore, Engstler et al. [3] had introduced a fluorimetric trans-sialidase assay, in which sialyl-a2,3-lactose (30 -SL) and 4-methylumbelliferyl-a-D -galactoside (MUGal) were used as donor and acceptor, respectively. Despite this multitude of existing trans-sialidase assays, a highly sensitive and specific nonradioactive screening assay suitable for high-throughput applications has not yet been described. Such a test would be desirable, since interest in determining trans-sialidase activity, from both natural and recombinant sources, is increasing, due to its extensive pathophysiological, pharmacological, and biotechnological significance. To achieve this, we have modified and refined the fluorimetric assay described by Engstler et al. [3]. Moreover, we report on the adaptation of the assay to a 96-well plate format and show its advantages and effectiveness 1
Abbreviations used: 30 -SL, N-acetylneuraminic acid-a2,3-lactose; MUGal, 4-methyl-umbelliferyl-b-D -galactoside; 30 -SLN, N-acetylneuraminic acid-a2,3-N-acetyllactosamine; MULac, 4-methylumbelliferylb-D -lactoside; Pipes, piperazine-1,4-bis(2-ethanesulfonic acid); BSA, bovine serum albumin; BisTris, 2,2-bis-(hydroxymethyl)-2, 20 ,200 nitrilo-triethanol; MU, 4-methylumbelliferone; MUGalNeu5Ac, 20 (4-methylumbelliferyl)-N-acetylneuraminic acid-a2,3-galactose; MES, 2-morpholinoethanesulfonic acid; PNP-Neu5Ac, 2-O-(p-nitrophenyl)N-acetylneuraminic acid; MUNeu5Ac, 20 -(4-methylumbelliferyl)-Nacetylneuraminic acid; ONPG, o-nitrophenyl-a-D -galactopyranoside; DMSO, dimethyl sulfoxide.
using native trans-sialidase from T. congolense and recombinant trans-sialidase from T. cruzi.
Materials and methods Reagents Q-Sepharose Fast Flow was purchased from Pharmacia (Germany). All chemicals used were of analytical grade. MUGal was obtained from Sigma (Germany). Neu5Aca2,3-N-acetyllactosamine (30 -SLN) and fetuin were purchased from Dextra Laboratories (UK) and ICN (Germany), respectively. 4-Methylumbelliferyl-aD -lactoside (MULac) was synthesized according to Wang et al. [25]. 30 -SL was isolated from bovine milk as described by Veh et al. [26]. Cultivation of procyclic T. congolense Procyclic culture forms of T. congolense (STIB 249; kindly provided by Dr. Retro Brun from the Swiss Tropical Institute, Basel, Switzerland) were cultivated axenically in SM/SDM 79 medium [27], containing 10% fetal calf serum and 0.001% hemin. After 3 to 4 days of cultivation, the trypanosomes were transferred into new SM/SDM 79 medium lacking fetal calf serum and hemin. Following a further 3 days, the culture supernatant was harvested by centrifugation, filtered, concentrated in an Amicon filtration device (MWCO 20 kDa, Sartorius, Germany), and used for the trans-sialidase assay. Recombinant trans-sialidase from T. cruzi The recombinant trans-sialidase from T. cruzi was expressed in Escherichia coli by the clone h611/2, which does not have the SAPA repeat [17]. The enzyme protein was partially purified as described by Buschiazzo et al. [17] and precipitated in ammonium sulfate. An aliquot of the enzyme solution was dialyzed three times (two times for 1 h and then once overnight) against 20 mM Pipes/NaOH buffer, pH 7.0, at 4 °C. The enzyme solution was diluted 500- to 1000-fold in 100 mM Pipes/ NaOH, pH 7.0, containing 0.2% BSA, shortly before use. Radioactive trans-sialidase assay Trans-sialidase activity was determined using a modification of the method described by Schenkmann et al. [2]. Briefly, activity was measured in 20 mM BisTris buffer, pH 7.0, containing 1 mM 30 -SL as the sialic acid donor and 0.5 mM Galb1,4-[14C]GlcNAc (0.5 mCi/ lmol) as the acceptor in a final volume of 50 ll. After incubation at 37 °C for 30 min, the reaction was terminated by the addition of 1 ml ice-cold water and the
S. Schrader et al. / Analytical Biochemistry 322 (2003) 139–147
mixture was applied to a 0.3-ml column of Q-Sepharose FF (acetate form, generated by soaking the gel overnight in 1 M sodium acetate) which had been equilibrated with water (3 2 ml). Following washing (3 2 ml), the radiolabeled product was eluted with 2 ml 0.8 M ammonium acetate and quantified by b-scintillation counting. For the incubation time used, the formation of product was linear with respect to time. One unit of trans-sialidase activity is defined as 1 nmol of sialic acid transferred per minute. Nonradioactive trans-sialidase assay using minicolumns Trans-sialidase activity from T. congolense was monitored by incubating 25 ll of enzyme solution in 50 mM BisTris buffer, pH 7.0, containing 1 mM 30 -SL as the donor and 0.5 mM MUGal as the acceptor in a final volume of 50 ll at 37 °C for 2 h. To obtain a homogeneous dispersion, the 2 mM MUGal and, for the kinetic analysis of T. cruzi trans-sialidase, the 2 mM MULac stock suspensions were sonicated for 10 min prior to their use in the assay. T. cruzi trans-sialidase activity was measured by incubating 2 ll of diluted enzyme in 100 mM Pipes/NaOH, pH 7.0, containing 1 mM 30 -SL and 0.5 mM MUGal in a final volume of 50 ll at 20 °C for 45 min. In both cases, the reaction was terminated by the addition of 1 ml ice-cold water followed by application of the mixtures to minicolumns of 0.3 ml of QSepharose FF (acetate form) equilibrated with water (3 2 ml). The unbound MUGal was washed from the columns with 3 2 ml deionized water, 200 ll 1 M HCl was applied and the eluate discarded (dead volume). Subsequently, the sialylated product was eluted with 700 ll 1 M HCl into Eppendorf caps. Following acid hydrolysis of the eluted product at 95 °C for 45 min and cooling on ice, the samples were neutralized, adjusted to pH 10 by the addition of 250–290 ll 2 M NaOH and 300 ll 1 M glycine/NaOH, pH 10, and mixed thoroughly. After transferring 300 ll of the reaction mixture into black 96-well plates (Microfluor, Dynex, USA), fluorescence of the released 4-methyl-umbelliferone (MU) was measured with excitation and emission wavelengths of 365 and 450 nm, respectively, employing a 96-well plate fluorescence spectrophotometer (Fluorolite, Dynatech Lab., Inc. Chantilly, VA). Nonradioactive assay using 96-filter-well plates Trans-sialidase activity from T. congolense and T. cruzi was monitored essentially as described above, except that the assay was performed in polypropylene 96well plates (MicroWell plates; 0.5 ml, Nunc, Denmark) which were sealed with Nunc well caps. The enzyme reaction was stopped after the appropriate incubation time by the addition of 350 ll ice-cold water to each well. Using a multichannel pipette (CappAero, 25–200 ll,
141
Dunn Labortechnik GmbH, Germany) the mixture was transferred to a Unifilter 800-filter-well plate (GF/D glass fiber filter, Polyfiltronics, Whatman, UK) loaded with 300 ll of Q-Sepharose (acetate form) that had been washed with 6 500 ll of water. All wash and elution steps were performed using a vacuum manifold (QIAvac 96, Qiagen, Germany). To avoid drying out of the columns, the vacuum was applied slowly and only after the appropriate wash or elution buffers had been added to each well. Excess MUGal was removed from the columns by successive washing with water (6 500 ll). After discarding the first 100 ll of the eluent (1 M HCl), the sialylated product was eluted from the columns with 6 150 ll 1 M HCl. The eluate was collected in a 2-ml Nunc 96 DeepWell plate and sealed with polyolefin sealing tape (Nunc). Following hydrolysis of the eluted product in a water bath at 95 °C for 45 min, the sealing tape was immediately removed and the plate cooled on ice for 15 min. The pH was adjusted to pH 10 by addition of 120 ll of 6 M NaOH and 300 ll of 1 M glycine/NaOH, pH 10, and the plate was sealed using Nunc well caps. Following mixing by inversion of the plate two or three times, 300 ll of reaction mixture was transferred into black 96-well plates (Microfluor, Dynex); the released MU was quantified fluorimeterically as described above and taken as a measure for the amount of the product MUGalNeu5Ac. One unit of trans-sialidase was defined as 1 lmol of sialylated product formed per minute. Kinetic analyses of recombinant T. cruzi trans-sialidase were performed using either MUGal or MULac with concentrations from 0.1 to 1.0 mM at a constant 30 SL concentration of 1 mM. The donor specificity of recombinant T. cruzi trans-sialidase was studied with 1 mM 30 -SLN or 0.7% fetuin, containing 1 mM bound sialic acid, as measured by the microadaptation of the orcinol/Fe3þ /HCl method [28]. Direct determination of MUGalNeu5Ac, the trans-sialidase reaction product The amount of sialylated product, MUGalNeu5Ac, can also be determined without hydrolysis by measuring the fluorescence at excitation and emission wavelengths of 310 and 375 nm, respectively. In this case, the assay was run as described above under Nonradioactive assay using 96-filter-well plates, but elution from the Q-Sepharose minicolumns was performed with 1 M ammonium acetate instead of 1 M HCl and the fluorescence was measured with a Hitachi F-2500 fluorimeter (Hitachi, Ltd., Tokyo, Japan). Enzymatic determination and identification of MUGalNeu5Ac The assay was run as described above under Nonradioactive assay using 96-filter-well plates, but the
142
S. Schrader et al. / Analytical Biochemistry 322 (2003) 139–147
product MUGalNeu5Ac was eluted from the Q-Sepharose minicolumns with 1 M ammonium acetate instead of 1 M HCl and treated with 5 mU Vibrio cholerae sialidase (Behring Diagnostics GmbH, Marburg, Germany) and 1 U Aspergillus oryzae b-galactosidase (Sigma). The enzymes were added to 50 ll of eluate in 50 mM sodium acetate, pH 5.0, containing 1 mM CaCl2 in a total volume of 100 ll and incubated at 30 °C for 45 min. After stopping the reaction with 200 ll 0.5 M glycine/NaOH, pH 10, the amount of released MU was detected at excitation and emission wavelengths of 365 and 450 nm, respectively. Determination of N-acetylneuraminic acid Trans-sialidase from T. congolense and T. cruzi was incubated with 1 mM 30 -SL and 0.5 mM MUGal as described above under Nonradioactive trans-sialidase assay using minicolumns and the reaction was stopped with liquid nitrogen. The samples were lyophilized and the amount of free N-acetylneuraminic acid was determined as described by Hara et al. [29].
Results Optimization of the nonradioactive trans-sialidase test parameters Engstler et al. [3] introduced a nonradioactive transsialidase test using sialyllactose as a donor and MUGal as an acceptor, the sialylated product being separated from the substrates by anion exchange chromatography. The same principle was used here, though to increase sensitivity and handling, the procedure was further refined and some parameters were modified before adaptation to the 96-well plate format. Since MUGal has a low solubility in aqueous media, a stock suspension of 2 mM was prepared in water and dispersed by sonication for 10 min before use. With this procedure reproducible enzyme activities were obtained. Ethanol did not significantly improve the solubility of MUGal and decreased trans-sialidase activity slightly. Alternatively, MUGal was soluble in dimethyl sulfoxide (DMSO), which did not affect trans-sialidase activity following dilution to a final concentration of 0.6% DMSO in the assay. However, the dilution should be carried out shortly before use, otherwise, after storing on ice or freezing, crystallization of MUGal occurs and trans-sialidase activity was significantly reduced. Furthermore, MULac, which is more soluble in water than MUGal, can also be used as an acceptor (see also Fig. 7). As a further development of the original test, the separation of the sialylated product MUGalNeu5Ac from the acceptor MUGal was performed using minicolumns loaded with Q-Sepharose FF. In the radio-
active assay some authors use anion exchange chromatography media (Q-Sepharose FF, Dowex 2x8) in the acetate form [16,30]. In the nonradioactive test described here the acetate form of Q-Sepharose FF was employed. Equilibration of the media in sodium acetate overnight leads to about 25% higher recovery of activity than equilibration in water. Furthermore, the packed gel volume allowing the detection of both low and high trans-sialidase activities was determined. Fig. 1 shows the dependence of the amount of MU released from the product MUGalNeu5Ac on gel volume. At low enzyme activities, as shown for the T. congolense trans-sialidase, an increase in gel volume did not lead to higher amounts of eluted MUGalNeu5Ac. In contrast, larger gel volumes were necessary to detect increased enzyme activities, as shown for the recombinant T. cruzi trans-sialidase. The results in Fig. 1 suggest that 200–300 ll packed Q-Sepharose is sufficient to detect up to about 3,5 nmol of MUGalNeu5Ac. Investigating the elution of the product with 1 M HCl and acid hydrolysis revealed that the first 200 ll of eluate from the minicolumns can be discarded, since the total amount of product (MUGalNeu5Ac) eluted in the following 700 ll of 1 M HCl. Heating the column eluates containing MUGalNeu5Ac at 95 °C for 45 min gave an optimal recovery of MU. Neither longer periods of hydrolysis times nor higher HCl concentrations in the elution led to an increased yield of released MU (data not shown). Additionally, potential remaining sialidase activity of trans-sialidase was determined by measuring the amount of free N-acetylneuraminic acid after performing trans-sialidase reactions. No sialidase activity could be detected with either the T. congolense or the T. cruzi enzyme under the used assay conditions. The dependency of trans-sialidase activity on protein amount and incubation time was investigated under the described assay conditions. In the modified test, the rate of product formation was proportional to the amount of
Fig. 1. Dependence of the amount of released MU after hydrolysis of MUGalNeu5Ac (representing enzyme activity) on packed Q-Sepharose volume after reaction with T. congolense and recombinant T. cruzi trans-sialidase.
S. Schrader et al. / Analytical Biochemistry 322 (2003) 139–147
enzyme and the incubation time up to at least 3 h, as was tested with the T. congolense trans-sialidase from culture supernatant. Enzymatic analysis of the postulated product, MUGalNeu5Ac To investigate whether the postulated product is indeed MUGalNeu5Ac and whether MU can be measured after specific enzymatic cleavage of MUGalNeu5Ac, the column eluates were treated with either b-galactosidase or sialidase and with both enzymes together. The b-galactosidase from A. oryzae was chosen, since it can be used in a pH range also optimal for the sialidase (pH 5.0). The results (Table 1) show that MUGalNeu5Ac can be detected with high specificity using enzyme cleavage and measuring fluorescence of released MU. The fact that fluorescence is observed only after treatment with both enzymes is consistent with the postulated structure of this product. Development of a 96-well plate trans-sialidase assay To enhance throughput, the described minicolumnbased trans-sialidase assay was adapted to a 96-well plate format. Various 96-well plate types were tested. First, due to the low test volume of 50 ll it was necessary to incubate in sealable 96-well plates to prevent evaporation. Thus, for the trans-sialidase reaction polypropylene 96-well plates (MicroWell plates; 0.5 ml, Nunc) were chosen and sealed with Nunc well caps. Second, for the ion exchange chromatography 96-filter-well plates where each well is deep enough to hold 300 ll of packed gel and provide sufficient volume for the washing and elution media should be used. Furthermore, the 96-filter-well plates should contain a glass fiber membrane which does not bind the product and has a structure allowing a continuous and moderate flow under vacuum. Resistance to 1 M HCl and reusability are also important properties of the filter plates. Unifilter 800filter-well plates from Whatman were found to be ideal for this purpose. Deep 96-well plates (Nunc 96 DeepWell plates; 2 ml) were chosen to collect the eluate and perform the acid hydrolysis. These polypropylene plates
143
are heat stable, resistant to 1 M HCl, reusable, and deep enough for the volume of the eluate and neutralization after acid hydrolysis, and can be closed with Nunc well caps, to mix the samples after neutralization. Heat stability and resistance to HCl are also important properties of the sealing tape chosen. However, when adapted to the 96-well plate format, the conditions for the trans-sialidase test had to be slightly modified. The removal of unbound MUGal from the Q-Sepharose-loaded filter plates was monitored in test runs by collecting each wash fraction and measuring the fluorescence after acid hydrolysis. As shown in Fig. 2, five 0.5-ml washing steps were sufficient to remove unbound MUGal. Due to the increased flow rate during elution under vacuum, the product eluted earlier than found for the gravity run in minicolumns. Thus, after discarding the first 100 ll, the product was collected in six 150-ll elution steps using 1 M HCl (Fig. 2). To determine whether the MU fluorescence, representing enzyme activity, is proportional to the amount of product (MUGalNeu5Ac) formed, and since MUGalNeu5Ac is not commercially available, different volumes of enzyme reaction mixture, using T. congolense and T. cruzi trans-sialidase, were applied to the ion exchange medium in the wells and further treated in the usual manner. Fig. 3 shows clearly that if the assay is carried out in the described 96-well plate format, the amount of released MU is proportional to the amount of product applied to the ion exchanger. The dependence of MU release by acid hydrolysis on eluent concentration and reaction time was investigated in sealed polypropylene 96-deep-well plates in a water bath. As shown in Fig. 4, hydrolysis in the presence of 1 M HCl leads to a complete release of MU after 45–60 min. Therefore, when using trans-sialidase from T. congolense
Table 1 Enzymatic treatment of the trans-sialidase product MUGalNeu5Ac Treatment
+ TS
) TS (control)
Without enzymes + b-Galactosidase + Sialidase + Sialidase, + b-galactosidase
0 10 0 14.3 2.1
0 10 0 10
Fluorescence units (mean of three values standard deviation) after trans-sialidase (TS) reaction and different enzymatic treatments of the product MUGalNeu5Ac are given. As a control a sample without trans-sialidase added was treated in the same way.
Fig. 2. Elution profile of the acceptor MUGal and the product MUGalNeu5Ac on Q-Sepharose packed in wells, detected as nmol MU released after acid hydrolysis and neutralization, during separation with 96-filter-well plates. MUGal was washed from the columns with 500-ll steps of water. MUGalNeu5Ac was eluted with 100-ll steps of 1 M HCl (as indicated with arrows).
144
S. Schrader et al. / Analytical Biochemistry 322 (2003) 139–147
Fig. 3. Dependence of fluorescence units (representing enzyme activity) on the amount of MUGalNeu5Ac (expressed as reaction mixture volume) applied onto the minicolumns of 96-filter-well plates after reaction with T. cruzi and T. congolense trans-sialidase.
and T. cruzi activities, 1 M HCl was used to elute the sialylated product and hydrolysis was performed at 95 °C for 45 min. To use a calibration curve of MU for calculating enzyme activity, known amounts of MUGal were hydrolyzed in a water bath at 95 °C for 45 min. Fig. 5 shows that hydrolysis of MUGal performed under these conditions leads to the same fluorescence values as these observed for similarly treated MU. Detection of the product MUGalNeu5Ac without hydrolysis Based on the results presented in Fig. 6, the amount of the product MUGalNeu5Ac can alternatively be detected without acid hydrolysis by monitoring its fluorescence at excitation and emission wavelengths of 310– 315 and 375 nm, respectively. In this case, the product was eluted with 1 M ammonium acetate instead of 1 M HCl. Due to their identical excitation and emission wavelength maxima, the separation of MUGalNeu5Ac from MUGal with minicolumns or 96-filter-well plates is still necessary (data not shown). However, this method is not as sensitive as the detection of MU after acid hydrolysis (Table 2), but reduces assay time and could be a
Fig. 5. Dependence of fluorescence units on nmol MU and MUGal after incubation in 1 M HCl at 95 °C for 45 min in a water bath, followed by neutralization and adjustment to pH 10.
good alternative when relatively large amounts of product (due to high trans-sialidase activity) or a sufficiently sensitive fluorimeter are available. Applications The described 96-well plate assay was used to determine the pH and temperature optimum of the recombinant T. cruzi trans-sialidase. A neutral pH optimum in agreement with that of previous studies on the same enzyme performed with a radioactive assay [31] was detected (data not shown). The broad temperature optimum observed with the new 96-well plate assay was around 25 °C (data not shown). Buschiazzo et al. [31] found a similar temperature optimum between 15 and 25 °C for the same enzyme with the radioactive test. In addition, the dependence of the recombinant T. cruzi trans-sialidase on the concentration of both MUGal and MULac acceptors was investigated. The results in Fig. 7 show that T. cruzi trans-sialidase exhibits higher apparent Vmax and higher apparent Km values for MUGal than for MULac. Thus, apparent Km values of 0.2 and 0.1 mM and apparent Vmax values of 3.18 and 1.95 mU/lg protein for MUGal and MULac, respectively, were determined. The calculated apparent
Fig. 4. Dependence of nmol MU released from the product MUGalNeu5Ac on time of acid hydrolysis after elution with 1 M HCl (A) and on acid concentration of eluent (determined at 60 min) (B).
S. Schrader et al. / Analytical Biochemistry 322 (2003) 139–147
145
Fig. 6. Excitation (A) and emission (B) spectra of the product MUGalNeu5Ac after elution from the 96-filter-well plates with 1 M ammonium acetate.
Table 2 Detection of the trans-sialidase product MUGalNeu5Ac without hydrolysis
MUGalNeu5Ac MU
+TS
) TS (control)
231 12.1 1594 79.6
4.3 0.4 50.5 2.2
The values (fluorescence units) are the mean of three independent experiments standard deviation. In comparison, the values for the detection of released MU after acid hydrolysis of MUGalNeu5Ac from a corresponding sample are given. As controls, samples without trans-sialidase (TS) activity were treated in the same way.
Fig. 7. Dependence of recombinant T. cruzi trans-sialidase activity on different MUGal and MULac acceptor concentrations in the presence of 1 mM 30 -SL as donor. Inset shows the corresponding Lineweaver– Burk diagram. For kinetic data see text.
Vmax =Km -values were 15.9 and 19.5 for MUGal and MULac, respectively. In addition, the donor specificity of the recombinant T. cruzi trans-sialidase toward 30 SLN and fetuin was studied and compared with that toward 30 -SL (Table 3). Both substrates serve as donors with a higher trans-sialidase activity obtained with 30 SLN. Moreover, this nonradioactive test has been used to screen a large number of samples during the purification of T. congolense trans-sialidase, for substrate and inhibitor studies of this enzyme, and to monitor trans-sialidase activity during the production of monoclonal antibodies [32].
Table 3 Donor specificity of recombinant T. cruzi trans-sialidase Donor (1 mM sialic acid)
TS activity (mU/ml)
Neu5Aca2,3-lactose (30 -SL) Neu5Aca2,3-N-acetylactosamine (30 -SLN) Fetuin (0.7%)
5.76 6.31 4.21
Discussion In this paper, we report on the development of a new, nonradioactive 96-well plate assay to monitor trans-sialidase activity. Such an assay is of great interest, due to the increasing biotechnological and pharmacological interest in this enzyme [33,34]. The assay is specific, which was shown by enzymatic treatment of the product (Table 1). In addition, it is rapid and reproducible, allowing the screening of up to 96 samples in 3–4 h, depending on the enzyme activity and the corresponding incubation time. For the detection of higher enzyme activities, the processing time can be further reduced by quantifying the product MUGalNeu5Ac directly without prior hydrolysis to MU. In comparison to the more commonly employed radioactive test [16,22], the assay reported here uses the less expensive acceptor MUGal and is clearly more environmentally friendly, since no radioactive waste is produced and all of the 96-well plate equipment can be reused. To obtain a reliable test all assay parameters were studied carefully. The test is at least as accurate and sensitive as the radioactive assay with as little as about 0.1 nmol of product measurable depending on the fluorimeter used. Additionally, it exhibits a very low and constant background. The high sensitivity acquired is mainly due to a thorough and quantitative separation of substrates and products with the selective collection of the product peak only (Fig. 2) and the sensitive detection of MU after the application of a relatively long period of hydrolysis (Fig. 4). The applicability of this newly developed 96-well test was assessed successfully using this assay to monitor the purification and characterization of two trans-sialidase
146
S. Schrader et al. / Analytical Biochemistry 322 (2003) 139–147
forms from T. congolense and the screening of monoclonal antibodies [32]. Furthermore, the properties of recombinant T. cruzi trans-sialidase determined utilizing this assay were very similar to those reported in the literature, and only the temperature optimum was slightly higher in comparison to published values [23,31,35]. As was shown for MUGal and MULac, the assay is sufficiently quantitative and precise for determining kinetic constants (Fig. 7). Moreover, it can also be used for the investigation of potential trans-sialidase substrates and inhibitors, in particular for the testing of donor specificities. In common with the radioactive assay [13,15], the enzyme test described here monitors trans-sialidase activity by quantifying the formation of newly sialylated product and does not rely on measuring the released sialic acid or consumption of desialylated donor substrates in the presence of an appropriate acceptor. The latter approach can lead to an overestimation of transsialidase activity when sialidase activity is also present. Our test thus allows the accurate determination of transsialidase in extracts containing sialidase activity and can be used when analyzing crude extracts or serum samples or when investigating trans-sialidase in unknown biological systems. Sialidase activity can also influence the amount of sialylated product. In the assay described here, due to the relatively high concentration of the sialyllactose donor in comparison to the concentration of product formed, we assume that possible sialidase activities do not significantly reduce the amount of the sialylated trans-sialidase product. In addition, transsialidase itself acts as a sialidase and some authors reported that a part of this sialidase activity can be present at low acceptor concentrations or when using inefficient acceptors [22,14]. We investigated this possible sialidase activity with T. congolense and T. cruzi trans-sialidase, performing the assay described here and could not detect any significant amounts of free sialic acid. When employing this assay out of its linear range e.g. by using high trans-sialidase activity or incubating for long periods, the reverse direction becomes more significant and leads to an underestimation of trans-sialidase activity. The use of donors which do not act as good acceptors after desialylation by trans-sialidase might provide an alternative and is therefore of interest for preparative biotechnological applications. The synthetic a-sialosides PNP-Neu5Ac and MUNeu5Ac [22] both serve as such trans-sialidase donors. However, since some authors reported a much lower transfer rate using these donors in comparison to using sialyllactose [15,16,36], they are therefore unsuitable for a sensitive trans-sialidase test. The improved and specific assay described here will facilitate the development of other synthetic a-sialosides with higher donor specificities and the discovery and characterization of trans-sialidases in further biological systems. Moreover, it will assist in the screening of
potential trans-sialidase inhibitors, for instance using combinatorial substance libraries. Due to its specificity, sensitivity, and high-throughput potential, this assay may be also a valuable tool for diagnostic purposes [37,38].
Acknowledgments The technical assistance of Marzog El-Madani, Alice Schneider, and Renate Thun is gratefully acknowledged. We thank Dr. Alberto Carlos C. Frasch, Universidad Nacional de Gral. San Martin, Argentina and Dr. Joachim Thiem, University of Hamburg, Germany for collaboration and interest. Thanks are also due to Dr. Soerge Kelm, University of Bremen, Germany for useful advice. We are grateful to Dr. Ulf-Peter Hansen, Dr. Christoph Plieth, and Dr. Thisbe K. Lindhorst (all University of Kiel, Germany) for use of the laboratory and technical equipment during the end of this work. We also thank Dr. Guido Kohla, University of Kiel, Germany who carried out HPLC analyses for sialic acid determinations. Many thanks are due to Dr Joe Tiralongo, Medical University of Hannover, Germany and Dr. Lee Shaw, University of Kiel, Germany for useful suggestions and critical reading of the manuscript.
References [1] W. de Souza, Cell biology of Trypanosoma cruzi, Int. Rev. Cytol. 86 (1984) 197–283. [2] S. Schenkman, M.S. Jiang, G.W. Hart, V. Nussenzweig, A novel cell surface trans-sialidase of Trypanosoma cruzi generates a stagespecific epitope required for invasion of mammalian cells, Cell 65 (1991) 1117–1125. [3] M. Engstler, R. Schauer, R. Brun, Distribution of developmentally regulated trans-sialidases in the Kinetoplastida and characterization of a shed trans-sialidase activity from procyclic Trypanosoma congolense, Acta Trop. 59 (1995) 117–129. [4] S.J. Kemp, A.J. Teale, Genetic basis of trypanotolerance in cattle and mice, Parasitol. Today 14 (1998) 450–454. [5] L.C. Pontes de Carvalho, S. Tomlinson, F. Vandekerckhove, E.J. Bienen, A.B. Clarkson, M.S. Jiang, G.W. Hart, V. Nussenzweig, Characterization of a novel trans-sialidase of Trypanosoma brucei procyclic trypomastigotes and identification of procyclin as the main sialic acid acceptor, J. Exp. Med. 177 (1993) 465–474. [6] R. Schauer, G. Reuter, H. M€ uhlpfordt, A.F. Andrade, M.E. Pereira, The occurrence of N-acetyl- and N-glycoloylneuraminic acid in Trypanosoma cruzi, Hoppe-Seylers Z. Physiol. Chem. 364 (1983) 1053–1057. [7] S. Schenkman, M.A. Ferguson, N. Heise, M.L. de Almeida, R.A. Mortara, N. Yoshida, Mucin-like glycoproteins linked to the membrane by glycosylphosphatidylinositol anchor are the major acceptors of sialic acid in a reaction catalyzed by trans-sialidase in metacyclic forms of Trypanosoma cruzi, Mol. Biochem. Parasitol. 59 (1993) 293–304. [8] V.L. Pereira-Chioccola, S. Schenkman, Biological role of Trypanosoma cruzi trans-sialidase, Biochem. Soc. Trans. 27 (1999) 516–518.
S. Schrader et al. / Analytical Biochemistry 322 (2003) 139–147 [9] M. Engstler, R. Schauer, Trans-sialidases in the insect vector stages of African and American trypanosomes: Reply, Parasitol. Today 10 (1994) 180. [10] E. Medina-Acosta, S. Paul, S. Tomlinson, L.C. Pontes-de-Carvalho, Combined occurrence of trypanosomal sialidase/trans-sialidase activities and leishmanial metalloproteinase gene homologues in Endotrypanum sp., Mol. Biochem. Parasitol. 64 (1994) 273–282. [11] A.L. Mattos-Guaraldi, L.C. Formiga, A.F. Andrade, Transsialidase activity for sialic acid incorporation on Corynebacterium diphtheriae, FEMS Microbiol. Lett. 168 (1998) 167–172. [12] V.V. Tertov, V.V. Kaplun, I.A. Sobenin, E.Y. Boytsova, N.V. Bovin, A.N. Orekhov, Human plasma trans-sialidase causes atherogenic modification of low density lipoprotein, Atherosclerosis 159 (2001) 103–115. [13] F. Vandekerckhove, S. Schenkman, L. Pontes de Carvalho, S. Tomlinson, M. Kiso, M. Yoshida, A. Hasegawa, V. Nussenzweig, Substrate specificity of the Trypanosoma cruzi trans-sialidase, Glycobiology 2 (1992) 541–548. [14] A.J. Parodi, G.D. Pollevick, M. Mautner, A. Buschiazzo, D.O. Sanchez, A.C. Frasch, Identification of the gene(s) coding for the trans-sialidase of Trypanosoma cruzi, EMBO J. 11 (1992) 1705– 1710. [15] M. Engstler, G. Reuter, R. Schauer, The developmentally regulated trans-sialidase from Trypanosoma brucei sialylates the procyclic acidic repetitive protein, Mol. Biochem. Parasitol. 61 (1993) 1–14. [16] P. Scudder, J.P. Doom, M. Chuenkova, I.D. Manger, M.E. Pereira, Enzymatic characterisation of b-D -galactoside a2,3-transsialidase from Trypanosoma cruzi, J. Biol. Chem. 268 (1993) 9886– 9891. [17] A. Buschiazzo, G.A. Tavares, O. Campetella, S. Spinelli, M.L. Cremona, G. Paris, M.F. Amaya, A.C. Frasch, P.M. Alzari, Structural basis of sialyltransferase activity in trypanosomal sialidases, EMBO J. 19 (2000) 16–24. [18] G. Paris, M.L. Cremona, M.F. Amaya, A. Buschiazzo, S. Giambiagi, A.C. Frasch, P.M. Alzari, Probing molecular function of trypanosomal sialidases: single point mutations can change substrate specificity and increase hydrolytic activity, Glycobiology 11 (2001) 305–311. [19] K.B. Lee, Y.C. Lee, Transfer of modified sialic acids by Trypanosoma cruzi trans-sialidase for attachment of functional groups to oligosaccharide, Anal. Biochem. 216 (1994) 358–364. [20] N. Takahashi, K.B. Lee, H. Nakagawa, Y. Tsukamoto, Y. Kawamura, Y.T. Li, Y.L. Lee, Enzymatic sialylation of N-linked oligosaccharides using an a-(2,3)-specific trans-sialidase from Trypanosoma cruzi: structural identification using a three-dimensional elution mapping technique, Anal. Biochem. 230 (1995) 333– 342. [21] A. Buschiazzo, M.F. Amaya, M.L. Cremona, A.C. Frasch, P.M. Alzari, The crystal structure and mode of action of trans-sialidase, a key enzyme in Trypanosoma cruzi pathogenesis, Mol. Cell 10 (2002) 757–768. [22] S. Schenkmann, L. Pontes de Carvalho, V. Nussenzweig, Trypanosoma cruzi trans-sialidase and neuraminidase activities can be mediated by the same enzymes, J. Exp. Med. 175 (1992) 567–575. [23] S.G. Lee, B.G. Kim, Development of spectrophotometric method for monitoring trans-sialidase reaction and its application, Biotechnol. Lett. 22 (2000) 819–823.
147
[24] J.A. Harrison, K.P. Kartha, W.B. Turnbull, S.L. Scheuerl, J.H. Naismith, S. Schenkman, R.A. Field, Hydrolase and sialyltransferase activities of Trypanosoma cruzi trans-sialidase towards NeuAc-a-2,3-Gal-b-O-PNP, Bioorg. Med. Chem. Lett. 11 (2001) 141–144. [25] L.X. Wang, N.V. Pavlova, S.C. Li, Y.T. Li, Y.C. Lee, A fluorometric assay of ceramide glycanase with 4-methylumbelliferyl beta-D -lactoside derivatives, Glycoconj. J. 13 (1996) 359–365. [26] R.W. Veh, J.C. Michalski, A.P. Corfield, M. Sander-Wewer, D. Gies, R. Schauer, New chromatographic system for the rapid analysis and preparation of colostrum sialyloligosaccharides, J. Chromatogr. 212 (1981) 313–322. [27] R. Brun, M. Sch€ onberger, Cultivation and in vitro cloning of procyclic culture forms of Trypanosoma brucei in a semi-defined medium, Acta Trop. 36 (1979) 289–292. [28] R. Schauer, Analysis of sialic acids, Methods Enzymol. 138 (1987) 132–161. [29] S. Hara, M. Yamaguchi, Y. Takemori, K. Furuhata, H. Ogura, M. Nakamura, Determination of mono-O-acetylated N-acetylneuraminic acids in human and rat sera by fluorometric highperformance liquid chromatography, Anal. Biochem. 179 (1989) 162–166. [30] A.R. Todeschini, L. Mendonca-Previato, J.O. Previato, A. Varki, H. Halbeek, Trans-sialidase from Trypanosoma cruzi catalyzes sialoside hydrolysis with retention of configuration, Glycobiology 10 (2000) 213–221. [31] A. Buschiazzo, A.C. Frasch, O. Campetella, Medium scale production and purification to homogeneity of a recombinant trans-sialidase from Trypanosoma cruzi, Cell Mol. Biol. 42 (1996) 703–710. [32] E. Tiralongo, S. Schrader, H. Lange, H. Lemke, J. Tiralongo, R. Schauer, Two Trans-sialidase forms with different sialic acid transfer and sialidase activities from Trypanosoma congolense, J. Biol. Chem. 278 (2003) 23301–23310. [33] A. Vetere, S. Paoletti, Complete synthesis of 30 -sialyl-N-acetyllactosamine by regioselective transglycosylation, FEBS Lett. 399 (1996) 203–206. [34] A. Vetere, M. Miletich, M. Bosco, S. Paoletti, Regiospecific glycosidase-assisted synthesis of lacto-N-biose I (Galbeta1-3GlcNAc) and 30 -sialyl-lacto-N-biose I (NeuAcalpha2-3Galbeta13GlcNAc), Eur. J. Biochem. 267 (2000) 942–949. [35] M. Ribeirao, V.L. Pereira-Chioccola, D. Eichinger, M.M. Rodrigues, S. Schenkman, Temperature differences for trans-glycosylation and hydrolysis reaction reveal an acceptor binding site in the catalytic mechanism of Trypanosoma cruzi trans-sialidase, Glycobiology 7 (1997) 1237–1246. [36] S.G. Lee, D.H. Shin, B.G. Kim, Production of sialyloligosaccharides by trans-sialidase catalysed reaction using fetuin as a sialic acid donor, Enzyme Microb. Technol. 31 (2002) 742–746. [37] M.L. Leguizamon, G. Russomando, A. Luquetti, A. Rassi, M. Almiron, S. Gonzalez- Cappa, A.C. Frasch, O. Campetella, Longlasting antibodies detected by a trans-sialidase inhibition assay in parasite free/serologically cured chagasic patients, J. Infect. Dis. 175 (1997) 1272–1275. [38] M.S. Leguizamon, G. Russomando, A. Rojas de Arias, M. Saudio, M. Cabral, S.M. Gonzales-Cappa, A.C.C. Frasch, O. Campetella, Use of trans-sialidase inhibition assay in a population serologically negative for Trypanosoma cruzi but at a high risk of infection, Clin. Diagn. Lab. Immunol. 5 (1998) 254–255.
A nonradioactive 96-well plate assay for screening of trans-sialidase activityq Silke Schrader,a Evelin Tiralongo,a Gast on Paris,b Teruo Yoshino,c and Roland Schauera,* a Biochemisches Institut, Christian-Albrechts-Universit€at zu Kiel, 24098 Kiel, Germany Instituto de Investigaciones Biotecnol ogicas, Universidad Nacional de Gral. San Martın, 1650 San Martın, Argentina c Department of Chemistry, Division of Natural Sciences, International Christian University, Tokyo 181-8585, Japan
b
Received 25 March 2003
Abstract Trans-sialidase (E.C. 3.2.1.18) catalyzes the transfer of preferably a2,3-linked sialic acid to another glycan or glycoconjugate, forming a new a2,3 linkage to galactose or N-acetylgalactosamine. Here, we describe a nonradioactive 96-well plate fluorescence test for monitoring trans-sialidase activity with high sensitivity, specificity, and reproducibility using sialyllactose and 4-methylumbelliferyl-b-D -galactoside as donor and acceptor substrates, respectively. The assay conditions were optimized using the trans-sialidase from Trypanosoma congolense and its general applicability was confirmed with recombinant trans-sialidase from Trypanosoma cruzi. Using this procedure, a large number of samples can be tested quickly and reliably, for instance in monitoring trans-sialidase during enzyme purification and the production of monoclonal antibodies, for enzyme characterization, and for identifying potential substrates and inhibitors. The trans-sialidase assay reported here was capable of detecting trans-sialidase activity in the low-mU range and may be a valuable tool in the search for further trans-sialidases in various biological systems. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Trans-sialidase; Nonradioactive screening assay; 96-well plates; Trypanosoma congolense; Trypanosoma cruzi
Trans-sialidase was first described in trypanosomes. These flagellated protozoa are the agents of several human diseases such as Chagas disease in South and Central America caused by Trypanosoma cruzi [1,2] and sleeping sickness in Africa caused by T. b. gambiense and T. b. rhodesiense [3]. They are also responsible for Nagana, a disease of cattle in central Africa, caused by T. b. brucei and Trypanosoma congolense [4,5]. Although these parasites are unable to synthesize sialic acids (N-acetylneuraminic acid, Neu5Ac, and derivatives thereof) [6], they possess an enzyme, trans-sialidase, which enables them to acquire this sugar from host glycoconjugates for sialylation of their plasma membrane glycoproteins [7]. Trans-sialidase is believed to q The financial support by Numico Research (Friedrichsdorf, Germany), Sialic Acids Society (Kiel, Germany), and Fonds der Chemischen Industrie (Frankfurt, Germany) is gratefully acknowledged. * Corresponding author. Fax: +49-431-880-2238. E-mail address: [email protected] (R. Schauer).
0003-2697/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2003.07.016
play an important role in the pathogenicity of trypanosomes [8,9]. This enzyme has also been detected in Endotrypanum species [10] and Corynebacterium diphtheriae [11]. Recently, the existence of trans-sialidase in human serum was also described [12]. Trans-sialidase catalyzes the transfer of preferably a2,3-linked sialic acid directly to terminal b-galactoseor b-N-acetylgalactosamine-containing acceptors, giving rise to a new a2,3 linkage [13–16]. In the absence of an appropriate acceptor, this enzyme acts as a sialidase, hydrolytically releasing glycosidically linked sialic acid. Interest in understanding the complete catalytic mechanism of trans-sialidase [17,18], its possible application in the regiospecific synthesis of sialylated carbohydrates and glycoconjugates [19,20], and the search for potent trans-sialidase inhibitors to reduce trypanosome pathogenicity has stimulated much research on this enzyme. Recently, Buschiazzo et al. [21] reported the crystal structure of T. cruzi trans-sialidase and could show that sialic acid binding triggers a conformational switch
140
S. Schrader et al. / Analytical Biochemistry 322 (2003) 139–147
activating the enzyme and modulating affinity for the acceptor substrate. Trans-sialidase activity is generally detected with a radioactive assay using sialyl-a2,3-lactose as a donor and radioactively labeled lactose or N-acetyllactosamine as an acceptor. Following the reaction, the radioactive, negatively charged sialylated products are separated from labeled uncharged acceptor molecules by anion exchange chromatography and quantified to give a measure of enzyme activity [2,13,15,16,22]. Another radioactive test was described by Tertov et al. [12] who used a variety of donors containing radioactively labeled Neu5Ac. Trans-sialidase activity was then monitored either by quantifying the amount of labeled sialic acid released from the donors in the presence of an appropriate acceptor or by directly quantifying radioactivity on the acceptor molecules. Additionally, nonradioactive trans-sialidase assays have been reported. Lee and Kim [23] described a spectrophotometric assay, in which the amount of Neu5Ac transferred from the sialyl donor to the o-nitrophenyl-a-D -galactopyranoside (ONPG)1 acceptor was determined by measuring the concentration of the remaining ONPG with b-galactosidase. Harrison et al. [24] investigated the transfer activity of trans-sialidase using Neu5Aca2,3Gal-b-O-p-nitrophenol as the donor together with an appropriate acceptor. The release of p-nitrophenol (PNP) from the desialylated donor substrate was measured using b-galactosidase. Furthermore, Engstler et al. [3] had introduced a fluorimetric trans-sialidase assay, in which sialyl-a2,3-lactose (30 -SL) and 4-methylumbelliferyl-a-D -galactoside (MUGal) were used as donor and acceptor, respectively. Despite this multitude of existing trans-sialidase assays, a highly sensitive and specific nonradioactive screening assay suitable for high-throughput applications has not yet been described. Such a test would be desirable, since interest in determining trans-sialidase activity, from both natural and recombinant sources, is increasing, due to its extensive pathophysiological, pharmacological, and biotechnological significance. To achieve this, we have modified and refined the fluorimetric assay described by Engstler et al. [3]. Moreover, we report on the adaptation of the assay to a 96-well plate format and show its advantages and effectiveness 1
Abbreviations used: 30 -SL, N-acetylneuraminic acid-a2,3-lactose; MUGal, 4-methyl-umbelliferyl-b-D -galactoside; 30 -SLN, N-acetylneuraminic acid-a2,3-N-acetyllactosamine; MULac, 4-methylumbelliferylb-D -lactoside; Pipes, piperazine-1,4-bis(2-ethanesulfonic acid); BSA, bovine serum albumin; BisTris, 2,2-bis-(hydroxymethyl)-2, 20 ,200 nitrilo-triethanol; MU, 4-methylumbelliferone; MUGalNeu5Ac, 20 (4-methylumbelliferyl)-N-acetylneuraminic acid-a2,3-galactose; MES, 2-morpholinoethanesulfonic acid; PNP-Neu5Ac, 2-O-(p-nitrophenyl)N-acetylneuraminic acid; MUNeu5Ac, 20 -(4-methylumbelliferyl)-Nacetylneuraminic acid; ONPG, o-nitrophenyl-a-D -galactopyranoside; DMSO, dimethyl sulfoxide.
using native trans-sialidase from T. congolense and recombinant trans-sialidase from T. cruzi.
Materials and methods Reagents Q-Sepharose Fast Flow was purchased from Pharmacia (Germany). All chemicals used were of analytical grade. MUGal was obtained from Sigma (Germany). Neu5Aca2,3-N-acetyllactosamine (30 -SLN) and fetuin were purchased from Dextra Laboratories (UK) and ICN (Germany), respectively. 4-Methylumbelliferyl-aD -lactoside (MULac) was synthesized according to Wang et al. [25]. 30 -SL was isolated from bovine milk as described by Veh et al. [26]. Cultivation of procyclic T. congolense Procyclic culture forms of T. congolense (STIB 249; kindly provided by Dr. Retro Brun from the Swiss Tropical Institute, Basel, Switzerland) were cultivated axenically in SM/SDM 79 medium [27], containing 10% fetal calf serum and 0.001% hemin. After 3 to 4 days of cultivation, the trypanosomes were transferred into new SM/SDM 79 medium lacking fetal calf serum and hemin. Following a further 3 days, the culture supernatant was harvested by centrifugation, filtered, concentrated in an Amicon filtration device (MWCO 20 kDa, Sartorius, Germany), and used for the trans-sialidase assay. Recombinant trans-sialidase from T. cruzi The recombinant trans-sialidase from T. cruzi was expressed in Escherichia coli by the clone h611/2, which does not have the SAPA repeat [17]. The enzyme protein was partially purified as described by Buschiazzo et al. [17] and precipitated in ammonium sulfate. An aliquot of the enzyme solution was dialyzed three times (two times for 1 h and then once overnight) against 20 mM Pipes/NaOH buffer, pH 7.0, at 4 °C. The enzyme solution was diluted 500- to 1000-fold in 100 mM Pipes/ NaOH, pH 7.0, containing 0.2% BSA, shortly before use. Radioactive trans-sialidase assay Trans-sialidase activity was determined using a modification of the method described by Schenkmann et al. [2]. Briefly, activity was measured in 20 mM BisTris buffer, pH 7.0, containing 1 mM 30 -SL as the sialic acid donor and 0.5 mM Galb1,4-[14C]GlcNAc (0.5 mCi/ lmol) as the acceptor in a final volume of 50 ll. After incubation at 37 °C for 30 min, the reaction was terminated by the addition of 1 ml ice-cold water and the
S. Schrader et al. / Analytical Biochemistry 322 (2003) 139–147
mixture was applied to a 0.3-ml column of Q-Sepharose FF (acetate form, generated by soaking the gel overnight in 1 M sodium acetate) which had been equilibrated with water (3 2 ml). Following washing (3 2 ml), the radiolabeled product was eluted with 2 ml 0.8 M ammonium acetate and quantified by b-scintillation counting. For the incubation time used, the formation of product was linear with respect to time. One unit of trans-sialidase activity is defined as 1 nmol of sialic acid transferred per minute. Nonradioactive trans-sialidase assay using minicolumns Trans-sialidase activity from T. congolense was monitored by incubating 25 ll of enzyme solution in 50 mM BisTris buffer, pH 7.0, containing 1 mM 30 -SL as the donor and 0.5 mM MUGal as the acceptor in a final volume of 50 ll at 37 °C for 2 h. To obtain a homogeneous dispersion, the 2 mM MUGal and, for the kinetic analysis of T. cruzi trans-sialidase, the 2 mM MULac stock suspensions were sonicated for 10 min prior to their use in the assay. T. cruzi trans-sialidase activity was measured by incubating 2 ll of diluted enzyme in 100 mM Pipes/NaOH, pH 7.0, containing 1 mM 30 -SL and 0.5 mM MUGal in a final volume of 50 ll at 20 °C for 45 min. In both cases, the reaction was terminated by the addition of 1 ml ice-cold water followed by application of the mixtures to minicolumns of 0.3 ml of QSepharose FF (acetate form) equilibrated with water (3 2 ml). The unbound MUGal was washed from the columns with 3 2 ml deionized water, 200 ll 1 M HCl was applied and the eluate discarded (dead volume). Subsequently, the sialylated product was eluted with 700 ll 1 M HCl into Eppendorf caps. Following acid hydrolysis of the eluted product at 95 °C for 45 min and cooling on ice, the samples were neutralized, adjusted to pH 10 by the addition of 250–290 ll 2 M NaOH and 300 ll 1 M glycine/NaOH, pH 10, and mixed thoroughly. After transferring 300 ll of the reaction mixture into black 96-well plates (Microfluor, Dynex, USA), fluorescence of the released 4-methyl-umbelliferone (MU) was measured with excitation and emission wavelengths of 365 and 450 nm, respectively, employing a 96-well plate fluorescence spectrophotometer (Fluorolite, Dynatech Lab., Inc. Chantilly, VA). Nonradioactive assay using 96-filter-well plates Trans-sialidase activity from T. congolense and T. cruzi was monitored essentially as described above, except that the assay was performed in polypropylene 96well plates (MicroWell plates; 0.5 ml, Nunc, Denmark) which were sealed with Nunc well caps. The enzyme reaction was stopped after the appropriate incubation time by the addition of 350 ll ice-cold water to each well. Using a multichannel pipette (CappAero, 25–200 ll,
141
Dunn Labortechnik GmbH, Germany) the mixture was transferred to a Unifilter 800-filter-well plate (GF/D glass fiber filter, Polyfiltronics, Whatman, UK) loaded with 300 ll of Q-Sepharose (acetate form) that had been washed with 6 500 ll of water. All wash and elution steps were performed using a vacuum manifold (QIAvac 96, Qiagen, Germany). To avoid drying out of the columns, the vacuum was applied slowly and only after the appropriate wash or elution buffers had been added to each well. Excess MUGal was removed from the columns by successive washing with water (6 500 ll). After discarding the first 100 ll of the eluent (1 M HCl), the sialylated product was eluted from the columns with 6 150 ll 1 M HCl. The eluate was collected in a 2-ml Nunc 96 DeepWell plate and sealed with polyolefin sealing tape (Nunc). Following hydrolysis of the eluted product in a water bath at 95 °C for 45 min, the sealing tape was immediately removed and the plate cooled on ice for 15 min. The pH was adjusted to pH 10 by addition of 120 ll of 6 M NaOH and 300 ll of 1 M glycine/NaOH, pH 10, and the plate was sealed using Nunc well caps. Following mixing by inversion of the plate two or three times, 300 ll of reaction mixture was transferred into black 96-well plates (Microfluor, Dynex); the released MU was quantified fluorimeterically as described above and taken as a measure for the amount of the product MUGalNeu5Ac. One unit of trans-sialidase was defined as 1 lmol of sialylated product formed per minute. Kinetic analyses of recombinant T. cruzi trans-sialidase were performed using either MUGal or MULac with concentrations from 0.1 to 1.0 mM at a constant 30 SL concentration of 1 mM. The donor specificity of recombinant T. cruzi trans-sialidase was studied with 1 mM 30 -SLN or 0.7% fetuin, containing 1 mM bound sialic acid, as measured by the microadaptation of the orcinol/Fe3þ /HCl method [28]. Direct determination of MUGalNeu5Ac, the trans-sialidase reaction product The amount of sialylated product, MUGalNeu5Ac, can also be determined without hydrolysis by measuring the fluorescence at excitation and emission wavelengths of 310 and 375 nm, respectively. In this case, the assay was run as described above under Nonradioactive assay using 96-filter-well plates, but elution from the Q-Sepharose minicolumns was performed with 1 M ammonium acetate instead of 1 M HCl and the fluorescence was measured with a Hitachi F-2500 fluorimeter (Hitachi, Ltd., Tokyo, Japan). Enzymatic determination and identification of MUGalNeu5Ac The assay was run as described above under Nonradioactive assay using 96-filter-well plates, but the
142
S. Schrader et al. / Analytical Biochemistry 322 (2003) 139–147
product MUGalNeu5Ac was eluted from the Q-Sepharose minicolumns with 1 M ammonium acetate instead of 1 M HCl and treated with 5 mU Vibrio cholerae sialidase (Behring Diagnostics GmbH, Marburg, Germany) and 1 U Aspergillus oryzae b-galactosidase (Sigma). The enzymes were added to 50 ll of eluate in 50 mM sodium acetate, pH 5.0, containing 1 mM CaCl2 in a total volume of 100 ll and incubated at 30 °C for 45 min. After stopping the reaction with 200 ll 0.5 M glycine/NaOH, pH 10, the amount of released MU was detected at excitation and emission wavelengths of 365 and 450 nm, respectively. Determination of N-acetylneuraminic acid Trans-sialidase from T. congolense and T. cruzi was incubated with 1 mM 30 -SL and 0.5 mM MUGal as described above under Nonradioactive trans-sialidase assay using minicolumns and the reaction was stopped with liquid nitrogen. The samples were lyophilized and the amount of free N-acetylneuraminic acid was determined as described by Hara et al. [29].
Results Optimization of the nonradioactive trans-sialidase test parameters Engstler et al. [3] introduced a nonradioactive transsialidase test using sialyllactose as a donor and MUGal as an acceptor, the sialylated product being separated from the substrates by anion exchange chromatography. The same principle was used here, though to increase sensitivity and handling, the procedure was further refined and some parameters were modified before adaptation to the 96-well plate format. Since MUGal has a low solubility in aqueous media, a stock suspension of 2 mM was prepared in water and dispersed by sonication for 10 min before use. With this procedure reproducible enzyme activities were obtained. Ethanol did not significantly improve the solubility of MUGal and decreased trans-sialidase activity slightly. Alternatively, MUGal was soluble in dimethyl sulfoxide (DMSO), which did not affect trans-sialidase activity following dilution to a final concentration of 0.6% DMSO in the assay. However, the dilution should be carried out shortly before use, otherwise, after storing on ice or freezing, crystallization of MUGal occurs and trans-sialidase activity was significantly reduced. Furthermore, MULac, which is more soluble in water than MUGal, can also be used as an acceptor (see also Fig. 7). As a further development of the original test, the separation of the sialylated product MUGalNeu5Ac from the acceptor MUGal was performed using minicolumns loaded with Q-Sepharose FF. In the radio-
active assay some authors use anion exchange chromatography media (Q-Sepharose FF, Dowex 2x8) in the acetate form [16,30]. In the nonradioactive test described here the acetate form of Q-Sepharose FF was employed. Equilibration of the media in sodium acetate overnight leads to about 25% higher recovery of activity than equilibration in water. Furthermore, the packed gel volume allowing the detection of both low and high trans-sialidase activities was determined. Fig. 1 shows the dependence of the amount of MU released from the product MUGalNeu5Ac on gel volume. At low enzyme activities, as shown for the T. congolense trans-sialidase, an increase in gel volume did not lead to higher amounts of eluted MUGalNeu5Ac. In contrast, larger gel volumes were necessary to detect increased enzyme activities, as shown for the recombinant T. cruzi trans-sialidase. The results in Fig. 1 suggest that 200–300 ll packed Q-Sepharose is sufficient to detect up to about 3,5 nmol of MUGalNeu5Ac. Investigating the elution of the product with 1 M HCl and acid hydrolysis revealed that the first 200 ll of eluate from the minicolumns can be discarded, since the total amount of product (MUGalNeu5Ac) eluted in the following 700 ll of 1 M HCl. Heating the column eluates containing MUGalNeu5Ac at 95 °C for 45 min gave an optimal recovery of MU. Neither longer periods of hydrolysis times nor higher HCl concentrations in the elution led to an increased yield of released MU (data not shown). Additionally, potential remaining sialidase activity of trans-sialidase was determined by measuring the amount of free N-acetylneuraminic acid after performing trans-sialidase reactions. No sialidase activity could be detected with either the T. congolense or the T. cruzi enzyme under the used assay conditions. The dependency of trans-sialidase activity on protein amount and incubation time was investigated under the described assay conditions. In the modified test, the rate of product formation was proportional to the amount of
Fig. 1. Dependence of the amount of released MU after hydrolysis of MUGalNeu5Ac (representing enzyme activity) on packed Q-Sepharose volume after reaction with T. congolense and recombinant T. cruzi trans-sialidase.
S. Schrader et al. / Analytical Biochemistry 322 (2003) 139–147
enzyme and the incubation time up to at least 3 h, as was tested with the T. congolense trans-sialidase from culture supernatant. Enzymatic analysis of the postulated product, MUGalNeu5Ac To investigate whether the postulated product is indeed MUGalNeu5Ac and whether MU can be measured after specific enzymatic cleavage of MUGalNeu5Ac, the column eluates were treated with either b-galactosidase or sialidase and with both enzymes together. The b-galactosidase from A. oryzae was chosen, since it can be used in a pH range also optimal for the sialidase (pH 5.0). The results (Table 1) show that MUGalNeu5Ac can be detected with high specificity using enzyme cleavage and measuring fluorescence of released MU. The fact that fluorescence is observed only after treatment with both enzymes is consistent with the postulated structure of this product. Development of a 96-well plate trans-sialidase assay To enhance throughput, the described minicolumnbased trans-sialidase assay was adapted to a 96-well plate format. Various 96-well plate types were tested. First, due to the low test volume of 50 ll it was necessary to incubate in sealable 96-well plates to prevent evaporation. Thus, for the trans-sialidase reaction polypropylene 96-well plates (MicroWell plates; 0.5 ml, Nunc) were chosen and sealed with Nunc well caps. Second, for the ion exchange chromatography 96-filter-well plates where each well is deep enough to hold 300 ll of packed gel and provide sufficient volume for the washing and elution media should be used. Furthermore, the 96-filter-well plates should contain a glass fiber membrane which does not bind the product and has a structure allowing a continuous and moderate flow under vacuum. Resistance to 1 M HCl and reusability are also important properties of the filter plates. Unifilter 800filter-well plates from Whatman were found to be ideal for this purpose. Deep 96-well plates (Nunc 96 DeepWell plates; 2 ml) were chosen to collect the eluate and perform the acid hydrolysis. These polypropylene plates
143
are heat stable, resistant to 1 M HCl, reusable, and deep enough for the volume of the eluate and neutralization after acid hydrolysis, and can be closed with Nunc well caps, to mix the samples after neutralization. Heat stability and resistance to HCl are also important properties of the sealing tape chosen. However, when adapted to the 96-well plate format, the conditions for the trans-sialidase test had to be slightly modified. The removal of unbound MUGal from the Q-Sepharose-loaded filter plates was monitored in test runs by collecting each wash fraction and measuring the fluorescence after acid hydrolysis. As shown in Fig. 2, five 0.5-ml washing steps were sufficient to remove unbound MUGal. Due to the increased flow rate during elution under vacuum, the product eluted earlier than found for the gravity run in minicolumns. Thus, after discarding the first 100 ll, the product was collected in six 150-ll elution steps using 1 M HCl (Fig. 2). To determine whether the MU fluorescence, representing enzyme activity, is proportional to the amount of product (MUGalNeu5Ac) formed, and since MUGalNeu5Ac is not commercially available, different volumes of enzyme reaction mixture, using T. congolense and T. cruzi trans-sialidase, were applied to the ion exchange medium in the wells and further treated in the usual manner. Fig. 3 shows clearly that if the assay is carried out in the described 96-well plate format, the amount of released MU is proportional to the amount of product applied to the ion exchanger. The dependence of MU release by acid hydrolysis on eluent concentration and reaction time was investigated in sealed polypropylene 96-deep-well plates in a water bath. As shown in Fig. 4, hydrolysis in the presence of 1 M HCl leads to a complete release of MU after 45–60 min. Therefore, when using trans-sialidase from T. congolense
Table 1 Enzymatic treatment of the trans-sialidase product MUGalNeu5Ac Treatment
+ TS
) TS (control)
Without enzymes + b-Galactosidase + Sialidase + Sialidase, + b-galactosidase
0 10 0 14.3 2.1
0 10 0 10
Fluorescence units (mean of three values standard deviation) after trans-sialidase (TS) reaction and different enzymatic treatments of the product MUGalNeu5Ac are given. As a control a sample without trans-sialidase added was treated in the same way.
Fig. 2. Elution profile of the acceptor MUGal and the product MUGalNeu5Ac on Q-Sepharose packed in wells, detected as nmol MU released after acid hydrolysis and neutralization, during separation with 96-filter-well plates. MUGal was washed from the columns with 500-ll steps of water. MUGalNeu5Ac was eluted with 100-ll steps of 1 M HCl (as indicated with arrows).
144
S. Schrader et al. / Analytical Biochemistry 322 (2003) 139–147
Fig. 3. Dependence of fluorescence units (representing enzyme activity) on the amount of MUGalNeu5Ac (expressed as reaction mixture volume) applied onto the minicolumns of 96-filter-well plates after reaction with T. cruzi and T. congolense trans-sialidase.
and T. cruzi activities, 1 M HCl was used to elute the sialylated product and hydrolysis was performed at 95 °C for 45 min. To use a calibration curve of MU for calculating enzyme activity, known amounts of MUGal were hydrolyzed in a water bath at 95 °C for 45 min. Fig. 5 shows that hydrolysis of MUGal performed under these conditions leads to the same fluorescence values as these observed for similarly treated MU. Detection of the product MUGalNeu5Ac without hydrolysis Based on the results presented in Fig. 6, the amount of the product MUGalNeu5Ac can alternatively be detected without acid hydrolysis by monitoring its fluorescence at excitation and emission wavelengths of 310– 315 and 375 nm, respectively. In this case, the product was eluted with 1 M ammonium acetate instead of 1 M HCl. Due to their identical excitation and emission wavelength maxima, the separation of MUGalNeu5Ac from MUGal with minicolumns or 96-filter-well plates is still necessary (data not shown). However, this method is not as sensitive as the detection of MU after acid hydrolysis (Table 2), but reduces assay time and could be a
Fig. 5. Dependence of fluorescence units on nmol MU and MUGal after incubation in 1 M HCl at 95 °C for 45 min in a water bath, followed by neutralization and adjustment to pH 10.
good alternative when relatively large amounts of product (due to high trans-sialidase activity) or a sufficiently sensitive fluorimeter are available. Applications The described 96-well plate assay was used to determine the pH and temperature optimum of the recombinant T. cruzi trans-sialidase. A neutral pH optimum in agreement with that of previous studies on the same enzyme performed with a radioactive assay [31] was detected (data not shown). The broad temperature optimum observed with the new 96-well plate assay was around 25 °C (data not shown). Buschiazzo et al. [31] found a similar temperature optimum between 15 and 25 °C for the same enzyme with the radioactive test. In addition, the dependence of the recombinant T. cruzi trans-sialidase on the concentration of both MUGal and MULac acceptors was investigated. The results in Fig. 7 show that T. cruzi trans-sialidase exhibits higher apparent Vmax and higher apparent Km values for MUGal than for MULac. Thus, apparent Km values of 0.2 and 0.1 mM and apparent Vmax values of 3.18 and 1.95 mU/lg protein for MUGal and MULac, respectively, were determined. The calculated apparent
Fig. 4. Dependence of nmol MU released from the product MUGalNeu5Ac on time of acid hydrolysis after elution with 1 M HCl (A) and on acid concentration of eluent (determined at 60 min) (B).
S. Schrader et al. / Analytical Biochemistry 322 (2003) 139–147
145
Fig. 6. Excitation (A) and emission (B) spectra of the product MUGalNeu5Ac after elution from the 96-filter-well plates with 1 M ammonium acetate.
Table 2 Detection of the trans-sialidase product MUGalNeu5Ac without hydrolysis
MUGalNeu5Ac MU
+TS
) TS (control)
231 12.1 1594 79.6
4.3 0.4 50.5 2.2
The values (fluorescence units) are the mean of three independent experiments standard deviation. In comparison, the values for the detection of released MU after acid hydrolysis of MUGalNeu5Ac from a corresponding sample are given. As controls, samples without trans-sialidase (TS) activity were treated in the same way.
Fig. 7. Dependence of recombinant T. cruzi trans-sialidase activity on different MUGal and MULac acceptor concentrations in the presence of 1 mM 30 -SL as donor. Inset shows the corresponding Lineweaver– Burk diagram. For kinetic data see text.
Vmax =Km -values were 15.9 and 19.5 for MUGal and MULac, respectively. In addition, the donor specificity of the recombinant T. cruzi trans-sialidase toward 30 SLN and fetuin was studied and compared with that toward 30 -SL (Table 3). Both substrates serve as donors with a higher trans-sialidase activity obtained with 30 SLN. Moreover, this nonradioactive test has been used to screen a large number of samples during the purification of T. congolense trans-sialidase, for substrate and inhibitor studies of this enzyme, and to monitor trans-sialidase activity during the production of monoclonal antibodies [32].
Table 3 Donor specificity of recombinant T. cruzi trans-sialidase Donor (1 mM sialic acid)
TS activity (mU/ml)
Neu5Aca2,3-lactose (30 -SL) Neu5Aca2,3-N-acetylactosamine (30 -SLN) Fetuin (0.7%)
5.76 6.31 4.21
Discussion In this paper, we report on the development of a new, nonradioactive 96-well plate assay to monitor trans-sialidase activity. Such an assay is of great interest, due to the increasing biotechnological and pharmacological interest in this enzyme [33,34]. The assay is specific, which was shown by enzymatic treatment of the product (Table 1). In addition, it is rapid and reproducible, allowing the screening of up to 96 samples in 3–4 h, depending on the enzyme activity and the corresponding incubation time. For the detection of higher enzyme activities, the processing time can be further reduced by quantifying the product MUGalNeu5Ac directly without prior hydrolysis to MU. In comparison to the more commonly employed radioactive test [16,22], the assay reported here uses the less expensive acceptor MUGal and is clearly more environmentally friendly, since no radioactive waste is produced and all of the 96-well plate equipment can be reused. To obtain a reliable test all assay parameters were studied carefully. The test is at least as accurate and sensitive as the radioactive assay with as little as about 0.1 nmol of product measurable depending on the fluorimeter used. Additionally, it exhibits a very low and constant background. The high sensitivity acquired is mainly due to a thorough and quantitative separation of substrates and products with the selective collection of the product peak only (Fig. 2) and the sensitive detection of MU after the application of a relatively long period of hydrolysis (Fig. 4). The applicability of this newly developed 96-well test was assessed successfully using this assay to monitor the purification and characterization of two trans-sialidase
146
S. Schrader et al. / Analytical Biochemistry 322 (2003) 139–147
forms from T. congolense and the screening of monoclonal antibodies [32]. Furthermore, the properties of recombinant T. cruzi trans-sialidase determined utilizing this assay were very similar to those reported in the literature, and only the temperature optimum was slightly higher in comparison to published values [23,31,35]. As was shown for MUGal and MULac, the assay is sufficiently quantitative and precise for determining kinetic constants (Fig. 7). Moreover, it can also be used for the investigation of potential trans-sialidase substrates and inhibitors, in particular for the testing of donor specificities. In common with the radioactive assay [13,15], the enzyme test described here monitors trans-sialidase activity by quantifying the formation of newly sialylated product and does not rely on measuring the released sialic acid or consumption of desialylated donor substrates in the presence of an appropriate acceptor. The latter approach can lead to an overestimation of transsialidase activity when sialidase activity is also present. Our test thus allows the accurate determination of transsialidase in extracts containing sialidase activity and can be used when analyzing crude extracts or serum samples or when investigating trans-sialidase in unknown biological systems. Sialidase activity can also influence the amount of sialylated product. In the assay described here, due to the relatively high concentration of the sialyllactose donor in comparison to the concentration of product formed, we assume that possible sialidase activities do not significantly reduce the amount of the sialylated trans-sialidase product. In addition, transsialidase itself acts as a sialidase and some authors reported that a part of this sialidase activity can be present at low acceptor concentrations or when using inefficient acceptors [22,14]. We investigated this possible sialidase activity with T. congolense and T. cruzi trans-sialidase, performing the assay described here and could not detect any significant amounts of free sialic acid. When employing this assay out of its linear range e.g. by using high trans-sialidase activity or incubating for long periods, the reverse direction becomes more significant and leads to an underestimation of trans-sialidase activity. The use of donors which do not act as good acceptors after desialylation by trans-sialidase might provide an alternative and is therefore of interest for preparative biotechnological applications. The synthetic a-sialosides PNP-Neu5Ac and MUNeu5Ac [22] both serve as such trans-sialidase donors. However, since some authors reported a much lower transfer rate using these donors in comparison to using sialyllactose [15,16,36], they are therefore unsuitable for a sensitive trans-sialidase test. The improved and specific assay described here will facilitate the development of other synthetic a-sialosides with higher donor specificities and the discovery and characterization of trans-sialidases in further biological systems. Moreover, it will assist in the screening of
potential trans-sialidase inhibitors, for instance using combinatorial substance libraries. Due to its specificity, sensitivity, and high-throughput potential, this assay may be also a valuable tool for diagnostic purposes [37,38].
Acknowledgments The technical assistance of Marzog El-Madani, Alice Schneider, and Renate Thun is gratefully acknowledged. We thank Dr. Alberto Carlos C. Frasch, Universidad Nacional de Gral. San Martin, Argentina and Dr. Joachim Thiem, University of Hamburg, Germany for collaboration and interest. Thanks are also due to Dr. Soerge Kelm, University of Bremen, Germany for useful advice. We are grateful to Dr. Ulf-Peter Hansen, Dr. Christoph Plieth, and Dr. Thisbe K. Lindhorst (all University of Kiel, Germany) for use of the laboratory and technical equipment during the end of this work. We also thank Dr. Guido Kohla, University of Kiel, Germany who carried out HPLC analyses for sialic acid determinations. Many thanks are due to Dr Joe Tiralongo, Medical University of Hannover, Germany and Dr. Lee Shaw, University of Kiel, Germany for useful suggestions and critical reading of the manuscript.
References [1] W. de Souza, Cell biology of Trypanosoma cruzi, Int. Rev. Cytol. 86 (1984) 197–283. [2] S. Schenkman, M.S. Jiang, G.W. Hart, V. Nussenzweig, A novel cell surface trans-sialidase of Trypanosoma cruzi generates a stagespecific epitope required for invasion of mammalian cells, Cell 65 (1991) 1117–1125. [3] M. Engstler, R. Schauer, R. Brun, Distribution of developmentally regulated trans-sialidases in the Kinetoplastida and characterization of a shed trans-sialidase activity from procyclic Trypanosoma congolense, Acta Trop. 59 (1995) 117–129. [4] S.J. Kemp, A.J. Teale, Genetic basis of trypanotolerance in cattle and mice, Parasitol. Today 14 (1998) 450–454. [5] L.C. Pontes de Carvalho, S. Tomlinson, F. Vandekerckhove, E.J. Bienen, A.B. Clarkson, M.S. Jiang, G.W. Hart, V. Nussenzweig, Characterization of a novel trans-sialidase of Trypanosoma brucei procyclic trypomastigotes and identification of procyclin as the main sialic acid acceptor, J. Exp. Med. 177 (1993) 465–474. [6] R. Schauer, G. Reuter, H. M€ uhlpfordt, A.F. Andrade, M.E. Pereira, The occurrence of N-acetyl- and N-glycoloylneuraminic acid in Trypanosoma cruzi, Hoppe-Seylers Z. Physiol. Chem. 364 (1983) 1053–1057. [7] S. Schenkman, M.A. Ferguson, N. Heise, M.L. de Almeida, R.A. Mortara, N. Yoshida, Mucin-like glycoproteins linked to the membrane by glycosylphosphatidylinositol anchor are the major acceptors of sialic acid in a reaction catalyzed by trans-sialidase in metacyclic forms of Trypanosoma cruzi, Mol. Biochem. Parasitol. 59 (1993) 293–304. [8] V.L. Pereira-Chioccola, S. Schenkman, Biological role of Trypanosoma cruzi trans-sialidase, Biochem. Soc. Trans. 27 (1999) 516–518.
S. Schrader et al. / Analytical Biochemistry 322 (2003) 139–147 [9] M. Engstler, R. Schauer, Trans-sialidases in the insect vector stages of African and American trypanosomes: Reply, Parasitol. Today 10 (1994) 180. [10] E. Medina-Acosta, S. Paul, S. Tomlinson, L.C. Pontes-de-Carvalho, Combined occurrence of trypanosomal sialidase/trans-sialidase activities and leishmanial metalloproteinase gene homologues in Endotrypanum sp., Mol. Biochem. Parasitol. 64 (1994) 273–282. [11] A.L. Mattos-Guaraldi, L.C. Formiga, A.F. Andrade, Transsialidase activity for sialic acid incorporation on Corynebacterium diphtheriae, FEMS Microbiol. Lett. 168 (1998) 167–172. [12] V.V. Tertov, V.V. Kaplun, I.A. Sobenin, E.Y. Boytsova, N.V. Bovin, A.N. Orekhov, Human plasma trans-sialidase causes atherogenic modification of low density lipoprotein, Atherosclerosis 159 (2001) 103–115. [13] F. Vandekerckhove, S. Schenkman, L. Pontes de Carvalho, S. Tomlinson, M. Kiso, M. Yoshida, A. Hasegawa, V. Nussenzweig, Substrate specificity of the Trypanosoma cruzi trans-sialidase, Glycobiology 2 (1992) 541–548. [14] A.J. Parodi, G.D. Pollevick, M. Mautner, A. Buschiazzo, D.O. Sanchez, A.C. Frasch, Identification of the gene(s) coding for the trans-sialidase of Trypanosoma cruzi, EMBO J. 11 (1992) 1705– 1710. [15] M. Engstler, G. Reuter, R. Schauer, The developmentally regulated trans-sialidase from Trypanosoma brucei sialylates the procyclic acidic repetitive protein, Mol. Biochem. Parasitol. 61 (1993) 1–14. [16] P. Scudder, J.P. Doom, M. Chuenkova, I.D. Manger, M.E. Pereira, Enzymatic characterisation of b-D -galactoside a2,3-transsialidase from Trypanosoma cruzi, J. Biol. Chem. 268 (1993) 9886– 9891. [17] A. Buschiazzo, G.A. Tavares, O. Campetella, S. Spinelli, M.L. Cremona, G. Paris, M.F. Amaya, A.C. Frasch, P.M. Alzari, Structural basis of sialyltransferase activity in trypanosomal sialidases, EMBO J. 19 (2000) 16–24. [18] G. Paris, M.L. Cremona, M.F. Amaya, A. Buschiazzo, S. Giambiagi, A.C. Frasch, P.M. Alzari, Probing molecular function of trypanosomal sialidases: single point mutations can change substrate specificity and increase hydrolytic activity, Glycobiology 11 (2001) 305–311. [19] K.B. Lee, Y.C. Lee, Transfer of modified sialic acids by Trypanosoma cruzi trans-sialidase for attachment of functional groups to oligosaccharide, Anal. Biochem. 216 (1994) 358–364. [20] N. Takahashi, K.B. Lee, H. Nakagawa, Y. Tsukamoto, Y. Kawamura, Y.T. Li, Y.L. Lee, Enzymatic sialylation of N-linked oligosaccharides using an a-(2,3)-specific trans-sialidase from Trypanosoma cruzi: structural identification using a three-dimensional elution mapping technique, Anal. Biochem. 230 (1995) 333– 342. [21] A. Buschiazzo, M.F. Amaya, M.L. Cremona, A.C. Frasch, P.M. Alzari, The crystal structure and mode of action of trans-sialidase, a key enzyme in Trypanosoma cruzi pathogenesis, Mol. Cell 10 (2002) 757–768. [22] S. Schenkmann, L. Pontes de Carvalho, V. Nussenzweig, Trypanosoma cruzi trans-sialidase and neuraminidase activities can be mediated by the same enzymes, J. Exp. Med. 175 (1992) 567–575. [23] S.G. Lee, B.G. Kim, Development of spectrophotometric method for monitoring trans-sialidase reaction and its application, Biotechnol. Lett. 22 (2000) 819–823.
147
[24] J.A. Harrison, K.P. Kartha, W.B. Turnbull, S.L. Scheuerl, J.H. Naismith, S. Schenkman, R.A. Field, Hydrolase and sialyltransferase activities of Trypanosoma cruzi trans-sialidase towards NeuAc-a-2,3-Gal-b-O-PNP, Bioorg. Med. Chem. Lett. 11 (2001) 141–144. [25] L.X. Wang, N.V. Pavlova, S.C. Li, Y.T. Li, Y.C. Lee, A fluorometric assay of ceramide glycanase with 4-methylumbelliferyl beta-D -lactoside derivatives, Glycoconj. J. 13 (1996) 359–365. [26] R.W. Veh, J.C. Michalski, A.P. Corfield, M. Sander-Wewer, D. Gies, R. Schauer, New chromatographic system for the rapid analysis and preparation of colostrum sialyloligosaccharides, J. Chromatogr. 212 (1981) 313–322. [27] R. Brun, M. Sch€ onberger, Cultivation and in vitro cloning of procyclic culture forms of Trypanosoma brucei in a semi-defined medium, Acta Trop. 36 (1979) 289–292. [28] R. Schauer, Analysis of sialic acids, Methods Enzymol. 138 (1987) 132–161. [29] S. Hara, M. Yamaguchi, Y. Takemori, K. Furuhata, H. Ogura, M. Nakamura, Determination of mono-O-acetylated N-acetylneuraminic acids in human and rat sera by fluorometric highperformance liquid chromatography, Anal. Biochem. 179 (1989) 162–166. [30] A.R. Todeschini, L. Mendonca-Previato, J.O. Previato, A. Varki, H. Halbeek, Trans-sialidase from Trypanosoma cruzi catalyzes sialoside hydrolysis with retention of configuration, Glycobiology 10 (2000) 213–221. [31] A. Buschiazzo, A.C. Frasch, O. Campetella, Medium scale production and purification to homogeneity of a recombinant trans-sialidase from Trypanosoma cruzi, Cell Mol. Biol. 42 (1996) 703–710. [32] E. Tiralongo, S. Schrader, H. Lange, H. Lemke, J. Tiralongo, R. Schauer, Two Trans-sialidase forms with different sialic acid transfer and sialidase activities from Trypanosoma congolense, J. Biol. Chem. 278 (2003) 23301–23310. [33] A. Vetere, S. Paoletti, Complete synthesis of 30 -sialyl-N-acetyllactosamine by regioselective transglycosylation, FEBS Lett. 399 (1996) 203–206. [34] A. Vetere, M. Miletich, M. Bosco, S. Paoletti, Regiospecific glycosidase-assisted synthesis of lacto-N-biose I (Galbeta1-3GlcNAc) and 30 -sialyl-lacto-N-biose I (NeuAcalpha2-3Galbeta13GlcNAc), Eur. J. Biochem. 267 (2000) 942–949. [35] M. Ribeirao, V.L. Pereira-Chioccola, D. Eichinger, M.M. Rodrigues, S. Schenkman, Temperature differences for trans-glycosylation and hydrolysis reaction reveal an acceptor binding site in the catalytic mechanism of Trypanosoma cruzi trans-sialidase, Glycobiology 7 (1997) 1237–1246. [36] S.G. Lee, D.H. Shin, B.G. Kim, Production of sialyloligosaccharides by trans-sialidase catalysed reaction using fetuin as a sialic acid donor, Enzyme Microb. Technol. 31 (2002) 742–746. [37] M.L. Leguizamon, G. Russomando, A. Luquetti, A. Rassi, M. Almiron, S. Gonzalez- Cappa, A.C. Frasch, O. Campetella, Longlasting antibodies detected by a trans-sialidase inhibition assay in parasite free/serologically cured chagasic patients, J. Infect. Dis. 175 (1997) 1272–1275. [38] M.S. Leguizamon, G. Russomando, A. Rojas de Arias, M. Saudio, M. Cabral, S.M. Gonzales-Cappa, A.C.C. Frasch, O. Campetella, Use of trans-sialidase inhibition assay in a population serologically negative for Trypanosoma cruzi but at a high risk of infection, Clin. Diagn. Lab. Immunol. 5 (1998) 254–255.