Trans-sialidase from Trypanosoma cruzi catalyzes sialoside hydrolysis with retention of configuration

Adriane R. Todeschini, Lucia Mendonça-Previato1, José O. Previato, Ajit Varki3 and Herman van Halbeek2,3

Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, 21944–970, Cidade Universitária, Rio de Janeiro, Brasil and 3Glycobiology Research and Training Center, School of Medicine, University of California San Diego, La Jolla, CA 92093–0687, USA

Received on June 9, 1999; revised on August 20, 1999; accepted on August 20, 1999.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The trans-sialidase from Trypanosoma cruzi is a member of the sialidase superfamily that functions as a sialidase in the absence of a carbohydrate acceptor. We have used 1H nuclear magnetic resonance (NMR) spectroscopy to investigate the stereospecificity of the hydrolysis of two substrates, namely, 4-methyl-umbelliferyl-N-acetylneur­aminic acid and {alpha}(2–3)-sialyllactose, catalyzed by a recombinant T.cruzi trans-sialidase. We demonstrate that, in aqueous solution, the thermodynamically less stable {alpha}-form of N-acetylneuraminic acid is the initial product of the hydrolysis; subsequent mutarotation leads eventually to an equilibrium mixture of the {alpha} and ß forms, in molar ratio 8:92. In a mixed water/methanol solution, the hydrolysis reaction produces also the {alpha}-methyl sialoside but not its ß-methyl counterpart. We also show that 4-methyl-umbelliferyl-N-acetylneuraminic acid is a significantly better substrate for the sialidase than {alpha}(2–3)-sialyllactose. Prolonged incubation of {alpha}(2–3)-sialyllactose with an excess of trans-sialidase produced a trace of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, as identified by NMR spectroscopy and by gas liquid chromatography/mass spectro­metry. In conclusion, this study shows that the stereo­selectivity of the sialidase activity of T.cruzi trans-sialidase is identical to that of bacterial, viral, and mammalian sialidases, suggesting a similar active-site architecture.

Key words: enzyme mechanism/NMR spectroscopy/sialidase/trans-sialidase/Trypanosoma cruzi


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The protozoan parasite Trypanosoma cruzi, the causative agent of Chagas’ disease, is unable to synthesize sialic acid, instead scavenging it from exogenous glycoconjugates and transferring it to mucin-like acceptor molecules on the parasite surface by means of a trans-sialidase (TS) (Previato et al., 1985Go). The T.cruzi TS differs from mammalian sialyltransferases that use exclusively CMP-N-acetylneuraminic acid (CMP-Neu5Ac) as the sialic acid (Sia) donor. T.cruzi TS-mediated surface sialyl­ation is thought to be necessary for trypomastigote forms of the parasite to attach to and enter host cells (Burleigh and Andrews, 1995Go).

In vitro studies have shown that T.cruzi TS preferentially catalyzes the transfer of sialic acid residues from Sia{alpha}2–3Galß1-x containing donors and attaches them in {alpha}2–3 linkage to terminal ß-galactopyranosyl (ß-Galp) containing acceptors (Vandekerckhove et al., 1992Go). Terminal {alpha}-Gal, Galß1–4(Fuc{alpha}1–3)GlcNAc and Galß1–3(Fuc{alpha}1–4)GlcNAc are not acceptors (Scudder et al., 1993Go). 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., 1995Go). 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., 1993Go). 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., 1993Go).

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., 1989Go, 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., 1995Go). 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., 1993Go). 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., 1981aGo,b; Chong et al., 1992Go; Wilson et al., 1995Go, 1996; Kao et al., 1997Go).

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, 1997Go). Furthermore, it has been shown by gas liquid chromatography/mass spectrometry (GC/MS) (Burmeister et al., 1993Go) and by 1H NMR spectroscopy (Janakiraman et al., 1994Go) that small amounts of Neu5Ac2en are formed during prolonged incubation of Neu5Ac or {alpha}(2–3)-sialyllactose (SL3) with influenza-B virus sialidase, but Neu5Ac2en has not been detected in reactions catalyzed by bacterial sialidases (Schauer and Kamerling, 1997Go).

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., 1991Go; Roggentin et al., 1993Go; Crennell et al., 1993Go, 1994; Garskell et al., 1995Go). The T.cruzi TS N-terminal domain also contains the FRIP motif and five conserved Asp box sequences (Campetella et al., 1994Go; Schenkman et al., 1994Go). The C-terminal domain consists of a variable number of 12-amino-acid repeats (Cazzulo and Frasch, 1992Go). It is not required for TS activity; rather, it enables oligomerization and binding of the enzyme to the parasite surface (Schenkman et al., 1994Go).

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 {alpha}-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.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Purification of T.cruzi rTS
A recombinant analog of T.cruzi TS that has a six-tandem histidine N-terminal tail was purified from E.coli lysates using iminodiacetic metal affinity chromatography as described by Buschiazzo et al. (1996)Go, followed by sequential ion exchange chromatography on Mono Q and Mono S. The final yield of recombinant enzyme was 1 mg of protein per liter of bacterial culture. The purified T.cruzi rTS migrated on a 10% polyacrylamide denaturing gel as a diffuse band of 90 kDa, exhibited kinetic properties similar to those of the native enzyme (Scudder et al., 1993Go; Ribeirao et al., 1997Go) (results not shown) and was stable for extended periods of time at 4°C without loss of activity.

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 {alpha}-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 {alpha}-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 {alpha}-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 {alpha}-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., 1983Go). With H4 and H5 nearly coinciding, {Delta}{delta} 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.



View larger version (48K):
[in this window]
[in a new window]
 
Fig. 1. Reaction scheme and time course of hydrolysis of 4-MU-Neu5Ac (10 mM) by T.cruzi rTS (1 U) in deuterated PBS, pH 5.8 at 37ºC, monitored by 500 MHz 1H NMR spectroscopy. The times at which spectral data were acquired refer to the addition of T.cruzi rTS as t = 0.

 
The hydrolysis of the substrate was found to be complete after 58 min. As shown in Figure 2, mutarotation of {alpha}-Neu5Ac continues for an additional 40 min until final equilibrium values of 92% ß and 8% {alpha} sialic acid are reached, as determined by integration of the H3 signals. The rate constants for the Neu5Ac mutarotation were calculated. Under the conditions employed (pH 5.8 and 37°C) the kf ({alpha}->ß) and kb->{alpha}) were found to be 6.27 x 10–4 s–1 and 6.43 x 10–5 s–1, respectively.



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 2. Kinetics of the hydrolysis of 4-MU-Neu5Ac (10 mM) by T.cruzi rTS (1 U) in deuterated PBS, pH 5.8 at 37ºC. The reaction was monitored by 500 MHz 1H NMR spectroscopy. Shown are the normalized peak area integration values for H3eq of 4-MU-Neu5Ac (open squares), {alpha}-Neu5Ac (solid diamonds), and ß-Neu5Ac (open circles).

 
Hydrolysis of {alpha}(2–3)-sialyllactose by T.cruzi rTS
The time course of the rTS-catalyzed hydrolysis of SL3 was monitored by observing the emergence of the resonance signal (at 4.45 p.p.m.) of the Gal H1 proton of the released lactose and the concomitant decrease of the substrate Gal H1 resonance (at 4.53 p.p.m.). Hydrolysis of SL3 by T.cruzi rTS followed a course similar to that observed for 4-MU-Neu5Ac except that the reaction was significantly slower. Figure 3 shows the progress of the hydrolysis of SL3 (10 mM) in the presence of T.cruzi rTS (1 U) at 37°C in deuterated PBS, pH 5.8. The signals for H3eq and H3ax of {alpha}-Neu5Ac were visible after the first 3 min of the reaction, at 2.72 and 1.62 p.p.m., respectively. The signal for H3eq of the ß-anomer was first detected after about 10 min of incubation. The product signals increase in intensity as the reaction proceeds, with a concomitant decrease in the signal intensity of protons from the substrate. Since SL3 acts as a donor in the TS reaction, some of the released lactose is resialylated in a trans-sialidase catalyzed side reaction. An equilibrium between acceptor and donors was established after about 5 h of incubation.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 3. Kinetics of the hydrolysis of SL3 (10 mM) by T.cruzi rTS (1 U) in deuterated PBS, pH 5.8 at 37°C, monitored by 500 MHz 1H NMR spectroscopy. Shown are the normalized peak area integration values for the signals arising from Gal H1 in SL3 (squares); H3eq of ß-Neu5Ac (diamonds) and H3ax of {alpha}-Neu5Ac (circles).

 
Competition of 4-MU-Neu5Ac and {alpha}(2–3)-sialyllactose hydrolysis by T.cruzi rTS
4-MU-Neu5Ac (5 mM) and SL3 (5 mM) together were incubated with 1.2 U T.cruzi rTS in deuterated PBS, pH 7.6 at 37°C and 1H NMR spectra were recorded every 2 min. Monitoring the 4-methyl-umbelliferone (4-MUone) Hb signal (for definition, see inset in Figure 1) at 6.09 p.p.m. revealed that the enzymatic hydrolysis of 4-MU-Neu5Ac goes to completion in about 60 min. In contrast, the doublet corresponding to Gal H1 of SL3 at 4.53 p.p.m. only disappears after 180 min. Figure 4 illustrates that in a competition reaction 4-MU-Neu5Ac is a better substrate for the T.cruzi rTS-catalyzed hydrolysis reaction than SL3. The rate of hydrolysis of 4-MU-Neu5Ac was found to be 90 nmol·min–1 while the rate of hydrolysis of SL3 was 25 nmol·min–1.



View larger version (39K):
[in this window]
[in a new window]
 
Fig. 4. Competition between the hydrolysis of SL3 (5 mM) and 4-MU-Neu5Ac (5 mM) by T.cruzi rTS (1.2 U) in deuterated PBS, pH 7.6 at 37°C, monitored by 500 MHz 1H NMR spectroscopy. Spectral data were acquired at the times indicated on the spectra.

 
Effect of Neu5Ac2en on the sialidase activity of T.cruzi rTS
The hydrolytic activity of T.cruzi rTS toward 4-MU-Neu5Ac (1 mM) was determined in the absence and the presence of Neu5Ac2en (10 mM). The same experiment was carried out with SL3 (1 mM) as substrate. As shown in Figure 5, Neu5Ac2en partially inhibited the sialidase activity of rTS under the conditions described. The hydrolysis reactions of 4-MU-Neu5Ac and SL3 by rTS were followed for 120 min (Figure 5A,B). The relative intensities of the 4-MUone Hb signal at 6.09 p.p.m. and the SL3 Gal H1 signal at 4.53 p.p.m. each were compared in the spectra recorded in the absence and in the presence of Neu5Ac2en. It was found that Neu5Ac2en reduced the rTS sialidase activity by 25% and 23%, respectively (Figure 5A,B).



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 5. Effect of Neu5Ac2en on the kinetics of the hydrolysis of (A) 4-MU-Neu5Ac (1 mM) and (B) SL3 (1 mM), by T.cruzi rTS (0.2 U) in deuterated PBS, pH 7.6 at 37°C. The reactions were monitored by 500 MHz 1H NMR spectroscopy in the absence (squares) and presence (diamonds) of Neu5Ac2en (10 mM). Shown are the normalized peak area integration values for Gal H1 of SL3 and Hb of 4-MUone (see inset in Figure 1).

 
Production of Neu5Ac2en during T.cruzi rTS-catalyzed hydrolysis of {alpha}(2–3)-sialyllactose
We investigated the possibility of production of Neu5Ac2en during T.cruzi rTS-catalyzed hydrolysis of SL3 first by NMR spectroscopy. SL3 (10 mM) was incubated with T.cruzi rTS (1 U) in deuterated PBS, pH 5.8 at 37°C. After 48 h, a trace of Neu5Ac2en was detected, as indicated by the appearance of the characteristic H3 signal at 5.805 p.p.m. and by the presence of the signal at 2.063 p.p.m. attributed to the 5Ac protons of Neu5Ac2en (result not shown) (Vliegenthart et al., 1982Go; Reuter and Schauer, 1994Go).

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., 1993Go; Reuter and Schauer, 1994Go; Schauer and Kamerling, 1997Go). 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 {alpha} 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.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 6. (A) Reconstructed m/z 285 fragment ion gas chromatogram of the per-O-trimethylsilyl derivatives of the products of incubation of SL3 (1 mM) with T.cruzi rTS (10 U), pH 5.8 at 37°C for 48 h. The peaks are attributed to {alpha}-Neu5Ac (I), ß-Neu5Ac (II), and Neu5Ac2en (III). (B) EI-mass spectrum of per-O-TMS I and II; (C) EI-mass spectrum of per-O-TMS III.

 
Effect of methanol on the hydrolysis of 4-MU-Neu5Ac by T.cruzi rTS
In order to study the T.cruzi rTS-catalyzed hydrolysis in the presence of a nucleophile other than water, the effect of addition of methanol to an aqueous solution of 4-MU-Neu5Ac on its enzymatic hydrolysis was examined by 1H NMR spectro­scopy. In the presence of methanol, the products of 4-MU-Neu5Ac hydrolysis were identified as 4-MUone, {alpha}-Neu5Ac, and ß-Neu5Ac, and {alpha}-methyl sialoside ({alpha}-Neu5Ac2Me). An increase in methanol concentration (from 0 to 20%) did not affect the relative rate of release of 4-MUone but increased the formation of {alpha}-Neu5Ac2Me with concomitant decrease of the amount of free ß-Neu5Ac produced. Figure 7 shows the 1H NMR spectra of the products of hydrolysis of 4-MU-Neu5Ac in the absence and in the presence of methanol (20%, v/v). The H3ax signal of {alpha}-Neu5Ac2Me was observed at 1.62 p.p.m. as a well-defined triplet, and the H3eq signal of the glycoside was detected at 2.73 p.p.m., in accordance with Vliegenthart et al. (1982)Go. No ß-Neu5Ac2Me was produced, by virtue of the absence of H3ax and H3eq resonances at 1.65 and 2.34 p.p.m., respectively (Vliegenthart et al., 1982Go).



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 7. (A) 500 MHz 1H NMR spectrum of the products of hydrolysis of 4-MU-Neu5Ac (1 mM) by T.cruzi rTS (0.2 U) in 10 mM deuterated PBS, pH 7.6 at 37°C in aqueous (D2O) solution; (B) as in (A), but in a D2O:CD3OD solution (4:1, v/v). Please note that the H3ax and H3eq signals of {alpha}-Neu5Ac virtually coincide with their counterparts of {alpha}-Neu5Ac2Me.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
NMR spectroscopy is a powerful tool for studying the kinetics of enzymatic reactions, because levels of both substrates and products can be simultaneously measured without perturbing the system. Moreover, the stereoselectivity of the action of sialidases can be monitored by 1H NMR spectroscopy, as the technique is eminently suited for determination of the anomeric configuration of the released sialic acid (Friebolin et al., 1981aGo), provided that the H3ax and H3eq resonances characteristic of the Neu5Ac {alpha} and ß anomers are resolved from substrate resonances. Those conditions were satisfied with 4-MU-Neu5Ac and SL3 as substrates. However, at equilibrium in aqueous solution the ß-anomer of free sialic acid is known to be the predominant isomer. Thus, {alpha}->ß mutarotation could hamper observation of hydrolytic release of {alpha}-Neu5Ac as initial product from the substrate. However, the rate of {alpha}->ß mutarotation is known to be pH-dependent; it was established to be at a minimum around pH 5.4 (Friebolin et al., 1981bGo). Therefore, we chose a pH value of 5.8 for most of the rTS-catalyzed hydrolysis experiments in this study, a value close to the pH that gives the best opportunity to observe initial release of the {alpha}-anomer of Neu5Ac. Furthermore, the amount of enzyme added was chosen such that substrate hydrolysis was complete before equilibrium between the anomers was established. Under these conditions we have shown that the {alpha}-anomer of sialic acid is released from 4-MU-Neu5Ac and SL3 by T.cruzi rTS. Thus the stereoselectivity of the sialidase activity of T.cruzi TS is identical to that of bacterial, viral, and mammalian sialidases (Friebolin et al., 1981aGo; Kao et al., 1997Go).

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)Go. Similar substrate specificities have been reported for T.rangeli sialidase (Reuter et al., 1987Go), Arthrobacter sialophilus sialidase (Kessler et al., 1982Go) and for a KDN sialidase from Sphingobacterium multivorum (Terada et al., 1997Go). 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., 1995Go; Terada et al., 1997Go).

The hydrolysis of 4-MU-Neu5Ac in the presence of methanol has been documented for other sialidases (Kessler et al., 1982Go; Bouwstra et al., 1987Go). Our data indicate that the amount of free Neu5Ac produced decreases with increasing methanol concentration, due to the simultaneous formation of {alpha}-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, 1990Go).

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., 1992Go; Burmeister et al., 1993Go; Janakiraman et al., 1994Go). 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 {alpha}-anomer, which is consistent with our observations that free {alpha}-Neu5Ac is initially formed in the presence of water as the only acceptor, and {alpha}-Neu5Ac2Me is formed in the presence of methanol.



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 8. Proposed reaction mechanism for the sialidase activity of T.cruzi rTS.

 
While the exact catalytic mechanism of the sialidase activity of T.cruzi trans-sialidase cannot be deduced from the data obtained in this study, our results with respect to the role of Neu5Ac2en may provide additional insight in the matter. Similar to influenza virus sialidase, but different from bacterial sialidases, T.cruzi trans-sialidase catalyzes hydrolysis of SL3 with the production of both {alpha}-Neu5Ac and a tiny amount of Neu5Ac2en. The finding that Neu5Ac2en is a by-product of the sialidase activity of T.cruzi recombinant trans-sialidase is remarkable. The latter compound is a transition-state analog of the oxocarbonium ion intermediate proposed to be formed during sialoside hydrolysis by viral sialidases. It has been speculated (Burmeister et al., 1993Go) that Neu5Ac2en is produced by proton elimination at C3 of the oxocarbonium ion intermediate.

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 10–2 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., 1995Go, Schauer and Kamerling, 1997Go).

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., 1993Go; Schauer et al., 1995Go)

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Materials
SL3 was kindly supplied by Dr. G.Strecker, Laboratoire de Chimie Biologique, Université des Sciences et Technologies de Lille, France. 4-MU-Neu5Ac was purchased from Sigma (St. Louis, MO), Neu5Ac was from Kantoishi Pharmaceuticals (Tokyo, Japan), and Neu5Ac2en was from Calbiochem (La Jolla, CA). D2O (99.996% D) and CD3OD (99.9% D) were purchased from Cambridge Isotope Laboratories (Andover, MA). Chromatographic media (Mono S, Mono Q and a HiTrap chelating column) were from Pharmacia Biotech (Uppsala, Sweden). All other reagents were of the highest available quality.

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., 1996Go). 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, 1987Go), 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 Tris–HCl 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 Tris–HCl 20 mM (pH 7.2) and NaCl 0.5 M, and eluted with an imidazol gradient (0–1 M) (Buschiazzo et al., 1996Go). Subsequently, the eluate was dialyzed against Tris–HCl 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 (0–1 M). The homogeneity of the enzyme was evaluated by 10% SDS–polyacrylamide gel electrophoresis. The T.cruzi rTS was stored in 20 mM Tris–HCl 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-1–14C]-lactose 0.25 µmol (40,000 c.p.m.) as described previously (Scudder et al., 1993Go). 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-1–14C]-lactose was eluted by washing the column with 3 ml of distilled water. The sialylated [D-glucose-1–14C]-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., 1979Go). 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 Tris–HCl 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 Varian’s 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 2–3 min after the reaction had begun, and from then on spectra were acquired every 1–2 min over 3–5 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 (1.0–1.2 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 {alpha} 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, 1988Go): A = A0 – {(kf x A0 kb x B0)/(kf + kb)} x {1 – exp[–(kf + kb)t]} and B = B0 + {(kf x A0kb x B0)/(kf + kb)} x {1 – exp[–(kf + kb)t]}, where A0 and B0 represent the initial H3 peak integrals of {alpha} 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 ({alpha}->ß) and kb ->{alpha}) are the mutarotation rate constants (f, forward; b, back). The fitting was done using Jandel’s 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 {alpha}(2–3)-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 50–200°C at 20°/min and 200–300°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.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Dr. A.C.C.Frasch from the Instituto de Investigaciones Biotecnologicas, Universidad Nacional de General San Martin (UNSAM), San Martin, Provincia de Buenos Aires, Argentina, for the T.cruzi trans-sialidase plasmid. A.R.T. is a recipient of a Ph.D. student fellowship from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brasil. L.M-P. is a Howard Hughes International Research Scholar. This work was supported by grants from the Programa Núcleo de Excelência, Finaciadora de Estudos e Projetos, CNPq, Conselho de Ensino para Graduados de UFRJ, Fundação Universitária José Bonifácio, Centro Nacional de Ressonância Magnética Nuclear na UFRJ, and the G. Harold and Leila Y. Mathers Foundation. A preliminary communication of this investigation was presented at the third Annual Conference of the Society for Glycobiology, Baltimore, MD, November 1998. This work was performed during A.R.T.’s tenure as a visiting scientist at UCSD.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
ax, axial; D, deuterium; EI, electron impact; eq, equatorial; Fuc, fucose; Gal, galactose; Galp, galactopyranose; GC, gas liquid chromatography; GlcNAc, N-acetylglucosamine; IPTG, isopropyl-ß-D-thiogalactoside; J, scalar coupling constant; Lac, lactose; Me, methyl; MS, mass spectrometry; 4-MU-Neu5Ac, 4-methyl-umbelliferyl-N-acetylneuraminic acid; 4-MUone, 4-methyl-umbelliferone; Neu5Ac, N-acetylneuraminic acid; Neu5Ac2en, 2-deoxy-2,3-didehydro-N-acetylneuraminic acid; Neu5Ac2Me, N-acetylneuraminic acid methylglycoside; NMR, nuclear magnetic resonance; PBS, phosphate buffered saline; rTS, recombinant trans-sialidase; SDS, sodium dodecyl sulfate; Sia, sialic acid; SL3, {alpha}(2–3)-sialyllactose; TB, terrific broth; TMS, trimethylsilyl; TS, trans-sialidase; UCSD, University of California San Diego; UFRJ, Universidade Federal do Rio de Janeiro.


    Footnotes
 
1 To whom correspondence should be addressed Back

2 Present address: CMM-East Building, Suite 1056, 9500 Gilman Drive, M/C 0687, University of California at San Diego, La Jolla, CA 92093–0687 Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Bouwstra,J.B., Deyl,C.M. and Vliegenthart,J.F.G. (1987) Purification and kinetic properties of sialidase from Clostridium perfringens. Biol. Chem. Hoppe Seyler, 368, 269–275.

Burleigh,B.A. and Andrews,N.W. (1995) The mechanisms of Trypanosoma cruzi invasion of mammalian cells. Annu. Rev. Microbiol., 49, 175–200.[ISI][Medline]

Burmeister,W.P., Henrissat,B., Bosso,C., Cusack,S. and Ruigrok,R.W. (1993) Influenza B virus neuraminidase can synthesize its own inhibitor. Structure, 1, 19–26.[ISI][Medline]

Buschiazzo,A., Frasch,A.C.C. and Campetella,O. (1996) Medium scale production and purification to homogeneity of a recombinant trans-sialidase from Trypanosoma cruzi. Cell. Mol. Biol., 42, 703–710.[ISI]

Campetella,O.E., Uttaro,A.D., Parodi,A.J. and Frasch,A.C.C. (1994) A recombinant Trypanosoma cruzi trans-sialidase lacking the amino acid repeats retains the enzymatic activity. Mol. Biochem. Parasitol., 64, 337–340.[ISI][Medline]

Carey,F. and Sundberg,R.J. (1990) Advanced Organic Chemistry (3rd ed.). Plenum, New York, pp. 257–340.

Cazzulo,J.J. and Frasch,A.C.C. (1992) SAPA/trans-sialidase and cruzipain: two antigens from Trypanosoma cruzi contain immunodominant but enzymatically inactive domains. FASEB J., 6, 3259–3264.[Abstract/Free Full Text]

Chong,A.K.J., Pegg,M.S., Taylor,N.R. and Von Itzstein,M. (1992) Evidence for a sialosyl cation transition-state complex in the reaction of sialidase from influenza virus. Eur. J. Biochem., 207, 335–343.[Abstract]

Cremona,M.L., Sanchez,D.O., Frasch,A.C. and Campetella,O. (1995) A single tyrosine differentiates active and inactive Trypanosoma cruzi trans-sialidases. Gene, 160, 123–128.[ISI][Medline]

Crennell,S.J., Garman,E.F., Laver,W.G., Vimr,E.R. and Taylor,G.L. (1993) Crystal structure of a bacterial sialidase (from Salmonella typhimurium LT2) shows the same fold as an influenza neuraminidase. Proc. Natl. Acad. Sci. USA, 90, 9852–9856.[Abstract]

Crennell,S.J., Garman,E.F., Laver,W.G., Vimr,E.R. and Taylor,G.L. (1994) Crystal structure of a Vibrio cholerae neuraminidase reveals a dual lectin-like domain in addition to the catalytic domain. Structure, 2, 535–544.[ISI][Medline]

Friebolin,H., Baumann,W., Keilich,G., Ziegler,D., Brossmer,R. and Von Nicolai,H. (1981a) 1H-NMR spectroscopy: a potent method for the determination of the substrate specificity of sialidases. Hoppe Seyler’s Z. Physiol. Chem., 362, 1455–1463.[ISI][Medline]

Friebolin,H., Kunzelmann,P., Supp,M., Brossmer,R., Keilich,G. and Ziegler,D. (1981b) 1H NMR Spektroskopische Untersuchungen zur Mutarotation der N-Acetyl-D-neuraminsaüre; pH-Abhängigkeit der Mutarotationsgeschwindigkeit. Tetrahedron Lett., 22, 1383–1386.[ISI]

Garskell,A., Crennell,S.J. and Taylor,G. (1995) The three domains of a bacterial sialidase: a ß-propeller, an immunoglobulin module and a galactose-binding jelly-roll. Structure, 15, 1197–1205.

Janakiraman,M.N., White,C.L., Laver,W.G., Air,G.M. and Luo,M. (1994) Structure of influenza virus neuraminidase B/Lee/40 complexed with sialic acid and a dehydro analog at 1.8-Å resolution: implications for the catalytic mechanism. Biochemistry, 33, 8172–8179.[ISI][Medline]

Kao,Y.H., Lerner,L. and Warner,T.G. (1997) Stereoselectivity of the Chinese hamster ovary cell sialidase: sialoside hydrolysis with overall retention of configuration. Glycobiology, 7, 559–563.[Abstract]

Kessler,J., Heck,J., Tannenbaum,S.W. and Flashner,M. (1982) Substrate and product specificity of Arthrobacter sialophilus neuraminidase. J. Biol. Chem., 257, 5056–5060.[Abstract/Free Full Text]

Noggle,J.H. (1988) Physical Chemistry. Scott, Foresman, Glenview, IL, pp. 546–560.

Pereira,M.E.A., Mejia,J.S., Ortega-Barria,E., Matzilevich,D. and Prioli,R.P. (1991) The Trypanosoma cruzi neuraminidase contains sequences similar to bacterial neuraminidases, YWTD repeats of low density lipoprotein receptor and type III modules of fibronectin. J. Exp. Med., 174, 179–191.[Abstract]

Potier,M., Mameli,L., Bélisle,M., Dellaire,L. and Melançon,S.B. (1979) Fluorometric assay of neuraminidase with a sodium (4-methyl-umbelliferyl-{alpha}-D-N-acetylneuraminate) substrate. Anal. Biochem., 94, 287–296.[ISI][Medline]

Previato,J.O., Andrade,A.F.B., Pessolani,M.C.V and Mendonça-Previato,L. (1985) Incorporation of sialic acid into Trypanosoma cruzi macromolecules. A proposal for a new metabolic route. Mol. Biochem. Parasitol., 16, 85–96.[ISI][Medline]

Previato,J.O., Jones,C., Xavier,M.T., Wait,R., Travassos,L.R., Parodi,A.J. and Mendonça-Previato,L. (1995) Structural characterization of the major glycosylphosphatidylinositol membrane anchored glycoprotein of epimastigote forms of Trypanosoma cruzi Y-strain. J. Biol. Chem., 270, 7241–7250.[Abstract/Free Full Text]

Rensch,R., Baumann,W. and Friebolin,H. (1983) 1H-NMR Untersuchungen an Sialinsaüren. V. N-Acetyl-{alpha}-D-neuraminsaüre: Chemische Verschiebungen und Kopplungskonstanten; Konfiguration und Konformation in Lösung. J. Carbohydr. Chem., 2, 415–425.[ISI]

Reuter,G. and Schauer,R. (1994) Determination of sialic acids. Methods Enzymol., 230, 168–199.[ISI][Medline]

Reuter,G., Schauer,R., Prioli,R.P. and Pereira,M.E.A. (1987) Isolation and properties of a sialidase from Trypanosoma rangeli. Glycoconj. J., 4, 339–348.[ISI]

Ribeirao,M., Pereira-Chioccola,V.L., Eichinger,D., Rodrigues,M.M. and Schenkman,S. (1997) Temperature differences for trans-glycosylation and hydrolysis reaction reveal an acceptor binding site in the catalytic mechanism of Trypanosoma cruzi trans-sialidase. Glycobiology, 7, 1237–1246.[Abstract]

Roggentin,P., Rothe,B., Kaper,J.B., Galen,J., Lawrisuk,L., Vimr,E.R. and Schauer,R. (1989) Conserved sequences in bacterial and viral sialidases. Glycoconj. J., 6, 349–353.[ISI][Medline]

Roggentin,P., Schauer,R., Hoyer,L.L. and Vimr,E.R. (1993) The sialidase superfamily and its spread by horizontal gene transfer. Mol. Microbiol., 9, 915–921.[ISI][Medline]

Schauer,R. and Kamerling,J.P. (1997) Chemistry, biochemistry and biology of sialic acid. In Montreuil,J., Vliegenthart,J.F.G. and Schachter,H. (eds.), Glycoproteins II. Elsevier, Amsterdam, pp. 243–402.

Schauer,R., Kelm,S., Reuter,G., Roggentin,P. and Shaw,L. (1995) Biochemistry and role of sialic acids. In Rosenberg,A. (ed.), Biology of the Sialic Acids. Plenum, New York, pp. 7–67.

Schenkman,S., Chaves,L.B., Pontes de Carvalho,L.C. and Eichinger,D. (1994) A proteolytic fragment of Trypanosoma cruzi trans-sialidase lacking the C-terminal domain is active, monomeric and generates antibodies that inhibit enzymatic activity. J. Biol. Chem., 269, 7970–7975.[Abstract/Free Full Text]

Scudder,P., Doom,J.P., Chuenkova,M., Manger,I.D. and Pereira,M.E.A. (1993) Enzymatic characterization of ß-D-galactoside {alpha}2,3-trans-sialidase from Trypanosoma cruzi. J. Biol. Chem., 268, 9886–9891.[Abstract/Free Full Text]

Tartof,K.D. and Hobbs,C.A. (1987) Improved media for growing plasmid and cosmid clones. Bethesda Res. Lab. Focus, 9, 12.

Terada,T., Kitajima,K., Inoue,S., Wilson,J.C., Norton,A.K., Kong,D.C.M., Thomson,R.J., Von Itzstein,M. and Inoue,Y. (1997) Catalysis by a new sialidase, deaminoneuraminic acid residue-cleaving enzyme (KDNase Sm), initially forms the less stable {alpha}-anomer of 3-deoxy-D-glycero-D-galacto-nonulosonic acid and is strongly inhibited by the transition state analogue, 2-deoxy-2,3-didehydro-D-glycero-D-galacto-2-nonulopyranosonic acid, but not by 2-deoxy-2,3-didehydro-N-acetylneuraminic acid. J. Biol. Chem., 272, 5452–5456.[Abstract/Free Full Text]

Tiralongo,J., Pegg,M.S. and Von Itzstein,M. (1995) Effect of substrate aglycone on enzyme mechanism in the reaction of sialidase from influenza virus. FEBS Lett., 372, 148–150.[ISI][Medline]

Vandekerckhove,F., Schenkman,S., Pontes de Carvalho,L., Tomlinson,S., Kiso,M., Yoshida,M., Hasegawa,A. and Nussenzweig,V. (1992) Substrate specificity of the Trypanosoma cruzi trans-sialidase. Glycobiology, 2, 541–548.[Abstract]

Vliegenthart,J.F.G., Dorland,L., Van Halbeek,H. and Haverkamp,J. (1982) NMR spectroscopy of sialic acids. In Schauer,R. (ed), Sialic Acids—Chemistry, Metabolism and Function. Springer, Vienna, pp. 128–172.

Wilson,J.C., Kong,D.C., Angus,D.L. and Von Itzstein,M. (1995) 1H NMR evidence that Salmonella typhimurium sialidase hydrolyses sialosides with overall retention of configuration. J. Am. Chem. Soc., 117, 4214–4217.[ISI]

Wilson,J.C., Kong,D.C., Li,Y.-T. and Von Itzstein,M. (1996) A 1H NMR investigation of the hydrolysis of a synthetic substrate by KDN-sialidase from Crassostrea virginica. Glycoconj. J., 13, 927–931.[ISI][Medline]