Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, 21944970, Cidade Universitária, Rio de Janeiro, Brasil and 3Glycobiology Research and Training Center, School of Medicine, University of California San Diego, La Jolla, CA 920930687, USA
Received on June 9, 1999; revised on August 20, 1999; accepted on August 20, 1999.
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Abstract |
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Key words: enzyme mechanism/NMR spectroscopy/sialidase/trans-sialidase/Trypanosoma cruzi
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Introduction |
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In vitro studies have shown that T.cruzi TS preferentially catalyzes the transfer of sialic acid residues from Sia23Galß1-x containing donors and attaches them in
23 linkage to terminal ß-galactopyranosyl (ß-Galp) containing acceptors (Vandekerckhove et al., 1992
). Terminal
-Gal, Galß14(Fuc
13)GlcNAc and Galß13(Fuc
14)GlcNAc are not acceptors (Scudder et al., 1993
). Incorporation of one N-acetylneuraminic acid (Neu5Ac) residue onto an acceptor appears to hinder entry of a second residue when two potential acceptor sites are present on the same oligosaccharide (Previato et al., 1995
). In the absence of a suitable carbohydrate acceptor, T.cruzi TS irreversibly transfers sialic acid to a water molecule, thus functioning as a sialidase similar to viral, mammalian and bacterial sialidases (Scudder et al., 1993
). Sequencing of T.cruzi TS genes shows that, although the enzyme differs from those sialidases in acceptor specificity, it is a member of the sialidase superfamily (Roggentin et al., 1993
).
Members of the sialidase family display only a moderate degree of sequence homology. Firstly, the sequence motif S-X-D-X-G-X-T-W (the so called Asp-box) is found repeated three to five times in the sequences of bacterial, trypanosomatid, and mammalian sialidases, though it is barely recognizable in most viral sialidases (Roggentin et al., 1989, 1993). N-terminally from the Asp-box one finds the X-R-X-P (or FRIP) region, which includes one arginine residue out of the three arginines that are known to bind the carboxylate group of sialic acid (Garskell et al., 1995
). Crystallographic studies of bacterial and viral sialidases have shown that the overall fold of these molecules and the spatial arrangement of key amino acids at the active sites are similar (Crennell et al., 1993
). Furthermore, 1H nuclear magnetic resonance (NMR) spectroscopy has demonstrated that members of the sialidase family hydrolyze the sialyl glycosidic bond with retention of configuration at the anomeric center of sialic acid (Friebolin et al., 1981a
,b; Chong et al., 1992
; Wilson et al., 1995
, 1996; Kao et al., 1997
).
Inhibition by the transition-state analog 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en) appears to be a characteristic feature of most members of the sialidase family (Schauer and Kamerling, 1997). Furthermore, it has been shown by gas liquid chromatography/mass spectrometry (GC/MS) (Burmeister et al., 1993
) and by 1H NMR spectroscopy (Janakiraman et al., 1994
) that small amounts of Neu5Ac2en are formed during prolonged incubation of Neu5Ac or
(23)-sialyllactose (SL3) with influenza-B virus sialidase, but Neu5Ac2en has not been detected in reactions catalyzed by bacterial sialidases (Schauer and Kamerling, 1997
).
The catalytic site region in the N-terminal domain of T.cruzi TS contains all amino acid residues that are known to be involved in sialic acid binding for other sialidases studied (Pereira et al., 1991; Roggentin et al., 1993
; Crennell et al., 1993
, 1994; Garskell et al., 1995
). The T.cruzi TS N-terminal domain also contains the FRIP motif and five conserved Asp box sequences (Campetella et al., 1994
; Schenkman et al., 1994
). The C-terminal domain consists of a variable number of 12-amino-acid repeats (Cazzulo and Frasch, 1992
). It is not required for TS activity; rather, it enables oligomerization and binding of the enzyme to the parasite surface (Schenkman et al., 1994
).
In this study we use NMR spectroscopy to investigate the mechanism of the sialidase activity of a recombinant T.cruzi trans-sialidase (T.cruzi rTS). We show that the initial product of hydrolysis of a sialyl donor catalyzed by rTS is the -anomer of sialic acid, and that the reaction can be partially inhibited by Neu5Ac2en; we also investigate the effect of a nucleophile other than water on the hydrolysis reaction. On the basis of our results we suggest a mechanism for the sialidase activity of T.cruzi TS.
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Results |
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Hydrolysis of 4-methyl-umbelliferyl-N-acetylneuraminic acid by T.cruzi rTS
Figure 1 shows the progress of the hydrolysis of 4-methyl-umbelliferyl-N-acetylneuraminic acid (4-MU-Neu5Ac) catalyzed by T.cruzi rTS as monitored by 1H NMR spectroscopy. The time course of the reaction was monitored by observing the emergence of the H3 resonance signals of free sialic acid. The "time-zero" spectrum shows the spectral region from 1.5 to 3.1 p.p.m. of 4-MU-Neu5Ac (10 mM) at 37°C in deuterated phosphate-buffered saline (PBS), pH 5.8, before addition of enzyme. The H3eq and H3ax signals of 4-MU-Neu5Ac are observed at 2.87 and 1.98 p.p.m., respectively, not obscured by other resonances. The remaining spectra show the course of the hydrolysis reaction after addition of 1 unit (U) T.cruzi rTS. At t = 3 min, in the first spectrum recorded after the start of the incubation, the H3eq and H3ax signals corresponding to the -anomer of sialic acid are observed at 2.72 and 1.62 p.p.m., respectively. These signals increase in intensity as the reaction proceeds, with a concomitant decrease in the intensity of the H3 signals of the substrate. The H3 signals of the Neu5Ac
-anomer continually increase for about 28 min, after which they progressively decrease (Figure 1) as mutarotation takes place. The mutarotation starts immediately after liberation of
-Neu5Ac and at t = 3 min the H3eq and H3ax signals of ß-Neu5Ac are visible, albeit barely, at 2.21 and 1.84 p.p.m., respectively (Figure 1). Interestingly, the H3ax signal of
-Neu5Ac appeared as a multiplet rather than a well-defined triplet (Figure 1). This phenomenon is due to the chemical shifts of H4 and H5 being about 3.80 and 3.82 p.p.m., respectively, with J4,5 being 10.1 Hz (Rensch et al., 1983
). With H4 and H5 nearly coinciding,
between H4 and H5 is of the same magnitude as J4,5, causing H3ax to experience a virtual coupling to H5. That virtual coupling, in concert with the scalar couplings to H3eq and H4, leads to the multiplet pattern of the H3ax resonance.
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In an independent experiment the production of Neu5Ac2en during hydrolysis of SL3 by rTS was verified by GC/MS. The EI-mass spectrum of the per-O-trimethylsilylated (per-O-TMS) derivative of Neu5Ac2en is known to be characterized by a prominent fragment at m/z 285 (Burmeister et al., 1993; Reuter and Schauer, 1994
; Schauer and Kamerling, 1997
). Upon electron impact, per-O-TMS-Neu5Ac also produces a fragment ion at m/z 285, however less abundant than that of per-O-TMS-Neu5Ac2en. Moreover, the literature suggests that per-O-TMS-Neu5Ac has a shorter retention time on GC than per-O-TMS-Neu5Ac2en. Figure 6 shows the reconstructed ion chromatogram (i.e., the abundance of fragment m/z 285 as a function of time) of the TMS-derivatized mixture resulting from the incubation of SL3 (1 mM) with T.cruzi rTS (10 U) for 48 h at 37°C, pH 5.8. Three peaks were observed. The first two, at 24.52 and 24.69 min, correspond to the
and ß Neu5Ac anomers, respectively. The third peak, appearing at 25.75 min, corresponds to Neu5Ac2en. These assignments were verified by measuring the GC retention times of the per-O-TMS-derivatives of authentic Neu5Ac and Neu5Ac2en standards in separate experiments. In a nonenzymatic control experiment, no GC peaks producing a fragment ion at m/z 285 were observed.
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Discussion |
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The hydrolysis of SL3 by T.cruzi rTS was investigated by observing the resonance signal arising from the Gal H1 proton of the liberated lactose at 4.45 p.p.m., which is distinct from the Gal H1 signal of the substrate at 4.53 p.p.m. Since the aromatic proton signals of 4-MU-Neu5Ac are resolved from those of the product 4-MUone and are found in a region of the spectrum that is free of glycosyl proton signals, we were able to investigate the competition for rTS-catalyzed hydrolysis between 4-MU-Neu5Ac and SL3. Our experiment showed that 4-MU-Neu5Ac is a better substrate for T.cruzi rTS than SL3, a result in agreement with that reported by Ribeirao et al. (1997). Similar substrate specificities have been reported for T.rangeli sialidase (Reuter et al., 1987
), Arthrobacter sialophilus sialidase (Kessler et al., 1982
) and for a KDN sialidase from Sphingobacterium multivorum (Terada et al., 1997
). These results suggest that the cleavage rate is influenced by the nonsialic acid part of the molecules. The observed preferential cleavage of 4-MU-Neu5Ac over SL3 may be attributed to the fact that the 4-methyl-umbelliferyl aglycone is a better leaving group than the carbohydrates of natural substrates. Furthermore, the electronic characteristics of the 4-methyl-umbelliferyl moiety could permit a better stabilization of the positive charge possibly formed in the transition state (Tiralongo et al., 1995
; Terada et al., 1997
).
The hydrolysis of 4-MU-Neu5Ac in the presence of methanol has been documented for other sialidases (Kessler et al., 1982; Bouwstra et al., 1987
). Our data indicate that the amount of free Neu5Ac produced decreases with increasing methanol concentration, due to the simultaneous formation of
-Neu5Ac2Me. This means that nucleophiles other than water can attack the enzyme-substrate complex, which leads us to propose the following kinetic scheme for this reaction:
where E is the free enzyme, S is the substrate (4-MU-Neu5Ac), ES is the enzyme-substrate complex, ES" is the transition state, P1 is 4-MUone, P2 is Neu5Ac, and P3 is Neu5Ac2Me. Since methanol has no effect on the release of 4-MUone, k2 must be smaller than k3 and k4. Consequently, Kcat,P1 = k2, Ks = Km (app) [where Kcat is the catalytic rate constant, Km the Michaelis constant, and Ks the dissociation constant of the ES complex] and the release of 4-MUone is the rate-determining step in the solvolysis of 4-MU-Neu5Ac. These results suggest a reaction that involves an oxocarbonium ion as key intermediate formed in a slow step followed by a fast attack by the nucleophile (Carey and Sundberg, 1990).
X-Ray crystallographic and mechanistic studies of influenza virus sialidase suggest a reaction mechanism involving a sialosyl cation transition-state complex (Figure 8) (Chong et al., 1992; Burmeister et al., 1993
; Janakiraman et al., 1994
). During this reaction, planarization of the sialic acid around the ring oxygen, C1, C2, and C3, in the active center of the sialidase, induces the release of the ketoside partner of Neu5Ac along with the glycosidic oxygen at C2. This release generates a sialosyl cation intermediate, which is rapidly attacked by nucleophiles such as water or methanol. When the sialosyl cation is attacked from beneath the plane formed, the product is the
-anomer, which is consistent with our observations that free
-Neu5Ac is initially formed in the presence of water as the only acceptor, and
-Neu5Ac2Me is formed in the presence of methanol.
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Neu5Ac2en was found to be only a weak inhibitor for the sialoside hydrolysis reactions catalyzed by rTS. Its inhibiting activity was in the range of 102 M; this value differs from the inhibiting effect of Neu5Ac2en on bacterial sialidases by several orders of magnitude. As proposed in Figure 8, catalytic hydrolysis by T.cruzi rTS potentially leads to the formation of an oxocarbonium ion intermediate positively charged at C2. This implies that the Neu5Ac C2 geometry becomes planar during the course of the reaction. Therefore, in the putative transition state during sialoside hydrolysis catalyzed by T.cruzi rTS, the ketoside structure becomes similar to that of Neu5Ac2en. Furthermore, gene sequencing analysis has shown that T.cruzi TS and Salmonella typhimurium sialidase share the same conserved amino acid residues at the catalytic site that are implicated in the ring distortion of Neu5Ac from 2C5 to the boat conformation of the substrate during the sialidase catalyzed reaction (Cremona et al., 1995, Schauer and Kamerling, 1997
).
The apparent conservation of the sialidase mechanism in viral, bacterial, trypanosomal, and mammalian enzymes is consistent with a common origin and supports the hypothesis that the occurrence of related enzymes in phylogenetically distant organisms is the consequence of extensive horizontal gene transfer (Roggentin et al., 1993; Schauer et al., 1995
)
Our studies suggest that transition-state analogs may provide a strategy for the rational design of trans-sialidase inhibitors which could be highly effective agents for the chemotherapy of acute phase T.cruzi infections. Studies are currently in progress to further understand the catalytic mechanism of T.cruzi trans-sialidase.
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Materials and methods |
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Expression and purification of T.cruzi rTS
T.cruzi rTS was obtained from E.coli MC 1061 electro-transformed with a plasmid containing TS inserts (TSREP.C in pTrcHisA) (Buschiazzo et al., 1996). The T.cruzi trans-sialidase plasmid was a gift from Dr. A.C.C. Frasch, Instituto de Investigaciones Biotecnologicas, UNSAM, San Martin, Provincia de Buenos Aires, Argentina. Bacteria were grown in a medium (TB) (Tartof and Hobbs, 1987
), containing (per l) 12 g bacto-tryptone, 24 g bacto-yeast extract, and 4 ml glycerol supplemented with 100 µg/ml ampicillin at 28°C. When the culture reached an optical density of 1.5 at 600 nm, 30 mg/l of isopropyl-ß-D-thiogalactoside (IPTG) was added and incubation continued at 28°C with shaking (110 r.p.m.) overnight. Bacteria were lysed at 4°C in 20 mM TrisHCl containing 2 mg/ml lysozyme, 2% Triton X-100, 0.1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, and 0.1 µM iodoacetamide. Deoxyribonuclease I (0.1 mg/ml) was added in order to reduce the viscosity. The suspension was centrifuged at 21,000 x g for 30 min and the pellet was discarded. The T.cruzi rTS containing a six-tandem histidine tag was purified using Ni2+ chelating chromatography on a HiTrap column equilibrated with TrisHCl 20 mM (pH 7.2) and NaCl 0.5 M, and eluted with an imidazol gradient (01 M) (Buschiazzo et al., 1996
). Subsequently, the eluate was dialyzed against TrisHCl 20 mM and applied to Mono Q (HR 16/10) and Mono S (HR 5/5) columns, respectively. The enzyme was eluted with a linear gradient of NaCl (01 M). The homogeneity of the enzyme was evaluated by 10% SDSpolyacrylamide gel electrophoresis. The T.cruzi rTS was stored in 20 mM TrisHCl buffer, pH 7.4, at 4°C, until use.
T.cruzi rTS activity measurements
The T.cruzi rTS trans-sialidase activity was assayed by incubating the purified enzyme in 200 µl of cacodylate buffer (5 mM, pH 7.0) in the presence of 0.25 µmol SL3 and [D-glucose-114C]-lactose 0.25 µmol (40,000 c.p.m.) as described previously (Scudder et al., 1993). After incubation at 37°C for 30 min, the reaction mixture was diluted with 1 ml of water and applied to a column containing 1 ml of Dowex 2X8 (acetate form) equilibrated with water. The [D-glucose-114C]-lactose was eluted by washing the column with 3 ml of distilled water. The sialylated [D-glucose-114C]-lactose was eluted with 9 ml of 0.8 M ammonium acetate, and the radioactivity was determined by liquid scintillation counting on a Beckman LS 6500 instrument.
The T.cruzi rTS sialidase activity was determined by measuring the fluorescence of 4-MUone released by the hydrolysis of 0.1 mM 4-MU-Neu5Ac in a 200 µl volume containing 5 mM cacodylate buffer, pH 7.0 at 37°C for 30 min (Potier et al., 1979). The reaction was stopped with 1.8 ml of distilled water and the fluorescence of 4-MUone was measured in a Cyto Fluor II instrument.
Preparation of samples for NMR spectroscopy
Saccharides (4-MU-Neu5Ac, SL3, and Neu5Ac2en) were lyophilized from D2O (99.996% D) three times and dissolved in deuterated PBS, pH 5.8 or 7.6 (these values are not corrected for isotope effects). A T.cruzi rTS solution in 20 mM TrisHCl was exchanged several times with deuterated PBS in a concentrator with a cut-off range of 30 kDa. The enzyme concentration was adjusted based on the sialidase activity of T.cruzi rTS (1 U is defined as the amount of enzyme required to catalyze the hydrolysis of substrate at 1 µmol/min at 37°C).
NMR spectroscopy
500 MHz 1H NMR spectroscopy was conducted on a Varian Unity Inova 500 spectrometer, at 37°C in sample volumes of 0.6 or 0.7 ml in 5 mm tubes (Wilmad 528-PP or better). A Varian 5-mm PFG-ID probe was used for all experiments; data acquisition was controlled by a SUN Sparc5 computer running Varians VNMR software (version 5.3B). Acetate was used as internal standard for chemical shift calibration; its chemical shift was set to 1.908 p.p.m. Prior to an enzyme-catalyzed reaction, a spectrum of the substrate was acquired as a "zero time" spectrum at 37°C in a total sample volume of 0.6 ml. Next, the sample was removed from the probe and the reaction was initiated by mixing 0.1 ml of T.cruzi rTS solution with the substrate at 37°C, and then the tube was swiftly placed back into the magnet. Hydrolysis reactions were monitored as a function of time. The first spectrum was taken within 23 min after the reaction had begun, and from then on spectra were acquired every 12 min over 35 h. The HDO signal was suppressed with a low-power transmitter pulse of 1.0 s during the relaxation delay.
The hydrolysis reactions of 4-MU-Neu5Ac (10 mM) and SL3 (10 mM) were performed in 10 mM deuterated PBS, pH 5.8, in the presence of T.cruzi rTS (1.01.2 U). The competition hydrolysis experiment between 4-MU-Neu5Ac (5 mM) and SL3 (5 mM) was performed at pH 7.6. Hydrolysis reactions in the presence of methanol (CD3OD) and Neu5Ac2en (10 mM) were carried out with 4-MU-Neu5Ac (1 mM) and 0.2 U of T.cruzi rTS.
For each spectrum, 16 transients were acquired in 16K data points. No line broadening and no zero filling were applied before Fourier transformation. Proton peaks were integrated relative to the amount of free acetate in the sample and the values were plotted as a function of time of reaction using Cricket Graph (version 1.53) software on a Macintosh 5400/120 computer.
Mutarotation rate constants were determined using the peak integrals of the H3 signals of the and ß anomers of Neu5Ac after 60 min when the 4-MU-Neu5Ac signals had dropped below the detection limit. The points were fitted for a two-state kinetic-model (Noggle, 1988
): A = A0 {(kf x A0 kb x B0)/(kf + kb)} x {1 exp[(kf + kb)t]} and B = B0 + {(kf x A0 kb x B0)/(kf + kb)} x {1 exp[(kf + kb)t]}, where A0 and B0 represent the initial H3 peak integrals of
and ß anomers at t = 60 min (the time at which substrate disappears, i.e., time-zero for the study of the mutarotation process), and kf (
ß) and kb (ß
) are the mutarotation rate constants (f, forward; b, back). The fitting was done using Jandels Sigma Plot Scientific Graph System software (version 4.17) on a Macintosh 5400/120 computer, with kf and kb as adjustable parameters.
GC/MS analysis of the reaction product of incubation of (23)-sialyllactose with T.cruzi rTS
SL3 (1 mM) was incubated with T.cruzi rTS (10 U) in PBS at pH 5.8 in a total volume of 0.5 ml for 48 h at 37°C. After incubation, the reaction mixture was dried under a stream of nitrogen gas and the resulting products were derivatized with 100 µl of Tri-Sil (Pierce, Rockford, IL) for 1 h at room temperature. The precipitate was removed by centrifugation, and 2 µl of the supernatant was immediately subjected to GC/MS analysis. GC of the TMS-derivatives was carried out with a Hewlett-Packard 5890 gas chromatograph, using a DB-5 fused silica column (30 m x 2.25 mm internal diameter) with helium as carrier gas. The column temperature was programmed from 50200°C at 20°/min and 200300°C at 2°/min. Electron impact (EI) mass spectra were recorded with a Hewlett-Packard 591 mass selective detector at an ionization energy of 70 eV. In order to optimize detection of Neu5Ac2en, the fragment ion at m/z 285 was selectively monitored throughout the GC elution.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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2 Present address: CMM-East Building, Suite 1056, 9500 Gilman Drive, M/C 0687, University of California at San Diego, La Jolla, CA 920930687
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References |
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