Probing molecular function of trypanosomal sialidases: single point mutations can change substrate specificity and increase hydrolytic activity

Gaston Paris1,3, Maria Laura Cremona1,3, Maria Fernanda Amaya4, Alejandro Buschiazzo4, Susana Giambiagi4, Alberto C.C. Frasch3 and Pedro M. Alzari2,4

3Instituto de Investigaciones Biotecnológicas, Universidad Nacional de San Martín, CC30, 1650 San Martín, Argentina, and 4Unité de Biochimie Structurale, CNRS URA 2185, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris, France

Received on September 20, 2000; revised on November 24, 2000; accepted on December 12, 2000.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Sialidases are present on the surface of several trypanosomatid protozoan parasites. They are highly specific for sialic acid linked in {alpha}-(2,3) to a terminal ß-galactose and include the strictly hydrolytic enzymes and trans-sialidases (sialyl-transferases). Based on the structural comparison of the sialidase from Trypanosoma rangeli and the trans-sialidase from T. cruzi (the agent of Chagas’ disease in humans), we have explored the role of specific amino acid residues sought to be important for substrate specificity. The substitution of a conserved tryptophanyl residue in the two enzymes, Trp312/313-Ala, changed substrate specificity, rendering the point mutants capable to hydrolyze both {alpha}-(2,3)- and {alpha}-(2,6)-linked sialoconjugates. The same mutation abolished sialyl-transferase activity, indicating that transfer (but not hydrolysis) requires a precise orientation of the bound substrate. The exchange substitution of another residue that modulates oligosaccharide binding, Gln284-Pro, was found to significantly increase the hydrolytic activity of sialidase, and residue Tyr119 was confirmed to be part of a second binding site for the acceptor substrate in trans-sialidase. Together with the structural information, these results provide a consistent framework to account for the unique enzymatic properties of trypanosome trans-sialidases.

Key words: trypanosomatids/sialidase/trans-sialidase/substrate specificity/catalytic site


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Protozoan parasites cause widespread diseases in humans as well as in domestic animals. To infect the vertebrate and insect vectors, parasites have developed a number of different strategies that require parasite-specific proteins. Some of the best-studied examples are the variable surface glycoproteins of African trypanosomes (Borst and Fairlamb, 1998Go; Rudenko et al., 1998Go) and a number of surface molecules present in Plasmodium spp. (Holder, 1994Go) and Leishmania spp. (Ferguson, 1997Go; Turco and Descoteaux, 1992Go). Some of these parasite-specific molecules display novel enzymatic activities, like trypanothione reductase, NAD-linked aromatic {alpha}-hydroxy acid dehydrogenase, and trans-sialidase (TS) from trypanosomes. The origin of these enzymes can usually be traced to molecules present in other eukaryotic cells in terms of their primary structure or activity. Trypanothione reductase is related to the mammalian enzyme glutathione reductase, and both are involved in keeping a stable cellular redox balance (Stoll et al., 1997Go). However, both enzymes are highly specific for their respective substrates, which can be explained by specific amino acid changes within the active site cleft (Bradley et al., 1991Go; Bond et al., 1999Go). NAD-linked {alpha}-hydroxy acid dehydrogenase is responsible, together with tyrosine aminotransferase, for the excretion of aromatic lactate derivatives in Trypanosoma cruzi. The enzyme clearly derives from a cytosolic malate dehydrogenase, but it is unable to use malate as substrate (Cazzulo Franke et al., 1999Go). These results suggest that protozoan parasites are likely to be a rich source of novel enzymatic activities.

TS is an enzyme present in a few trypanosomatids, like Endotrypanum spp.; Trypanosoma brucei, the agent of spleeping sickness in Africa; and T. cruzi, the agent of Chagas’s disease in North America and South America (Frasch, 2000Go). TS belongs to the family of sialidases (EC 3.2.1.18), but, instead of hydrolyzing sialic acid, it preferentially transfers the monosaccharide to an acceptor sugar molecule. Suitable substrates are molecules having a sialic acid linked in {alpha}-(2-3) to a terminal ß-galactose residue. Because terminal ß-galactoses are also the acceptors of the transferred sialic acid, the reaction is freely reversible. Although TS is by far more efficient in transferring than in hydrolyzing sialic acid, its activity is different from mammalian sialyltransferases (EC 2.4.99.X) because TS cannot use the sugar nucleotide CMP-sialic acid as the monosaccharide donor (Schenkman et al., 1991Go; Ferrero-García et al., 1993Go).

TS has been extensively studied in T. cruzi. It shares about 30% sequence identitity and several characteristic motifs with bacterial sialidases (Cremona et al., 1995Go). Another trypanosome, T. rangeli, was found to express a sialidase, TrSA, that, although highly similar to TcTS (70% identity), is completely devoid of TS activity (Buschiazzo et al., 1997Go). Therefore, the comparison of TcTS and TrSA might unravel why TS is such an efficient transferase. Few glycosidases are able to efficiently add a monosaccharide to an acceptor molecule in an overall inverse direction of the hydrolysis reaction. However, because hydrolysis is greatly favored, special assay conditions have to be used in these cases to detect transfer activity, such as performing the reaction in organic solvents (Bousquet et al., 1998Go) or using unusually high concentrations of the acceptor substrate (Watt et al., 1998Go).

Recently, the 3D structure of TrSA was obtained and compared with the modeled structure of TcTS (Buschiazzo et al., 2000Go). Trypanosomal sialidases fold into two globular domains tightly associated with each other. The catalytic domain (residues 1–372) displays a six-bladed ß-propeller topology similar to that of bacterial and viral sialidases, connected by a long {alpha}-helical segment to a C-terminal domain (residues 398–614) showing the characteristic ß-barrel topology of plant lectins. The two domains interact through an extended contact surface area (2600 Å2 of occluded molecular surface), which is significantly greater than that observed for other microbial sialidases that also have one or more lectin-like domains flanking the catalytic domain. The comparison of TrSA and TcTS reveals that a few amino acid changes close to the substrate-binding cleft might modulate sialyltransferase activity. These structural studies, together with a preliminary analysis of appropriate exchange mutants, suggested the existence of distinct donor and acceptor binding sites that might account for the sialyltransferase activity of TcTS (Buschiazzo et al., 2000Go). This observation was consistent with previous kinetic studies of TcTS that indicated a sequential mechanism, rather than the ping-pong mechanism usually expected for transglycosidases (Scudder et al., 1993Go; Ribeirao et al., 1997Go).

Taking advantage of the differences in amino acid residues between TcTS and TrSA at key positions, we constructed and analyzed a number of mutants. Their characterization allowed the identification of the amino acid residues conferring substrate specificity and involved in the formation of the binding site for the terminal galactose accepting the sialic acid in the trans-sialylation reaction. The results confirm and extend the main hypotheses derived from the crystallographic study and provide a molecular framework to account for the unusual enzymatic activity of TcTS.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Amino acid positions to be mutated were selected for their possible role in catalysis, as deduced from the comparison of sialidase and TS 3D structures (Buschiazzo et al., 2000Go) (Figure 1a and b). In one of the mutants generated, Trp312/313-Ala of TcTS and TrSA, respectively, the tryptophanyl residue is conserved in both molecules but was selected for mutagenesis due to its possible implication on determining substrate specificity. Enzymatic activities of purified mutant proteins were first assayed under standard conditions. When modifications in enzymatic activities were evident, kinetic constants were determined.




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Fig. 1. Amino acid positions that were subjected to site-directed mutagenesis in recombinant TrSA and TcTS. Equivalent positions differ by one amino acid because of a one-residue deletion present in TcTS close to the N-terminus (Buschiazzo et al., 1997Go). (A) The positions differing in the catalytic domain of both enzymes are indicated on the bars (TrSA, dark rectangle; TcTS, light gray rectangle). (B) View of the polypeptide backbone of the TrSA active site complexed with DANA, showing the location of the four amino acid residues mutated in this work.

 
Previous structural studies of STNA (Crennell et al., 1993Go, 1996) and TrSA (Buschiazzo et al., 2000Go) in complex with the inhibitor DANA suggested that an exposed aromatic side chain at a position close to the active site cleft (Tyr307 in STNA, Trp313 in TrSA) is favorably located to interact with substrate (Figure 1b). This amino acid residue is part of a loop insertion that is missing in other microbial sialidases having a broader specificity. The high specificity of TrSA, TcTS, and STNA for sialyl-{alpha}-(2,3) substrates could thus be explained by unfavorable interactions of this Trp residue with sialyl-{alpha}-(2,6)-linked oligosaccharides (Figure 2). To test this prediction, the recombinant mutants TrSA Trp313-Ala and TcTS Trp312-Ala were obtained and assayed for activity using three different substrates: sialyl-{alpha}-(2,3)-lactose (3'SL), sialyl-{alpha}-(2,6)-lactose (6'SL), and 4-metylumbelliferyl-N-acetylneuraminic acid (MUNANA) (Table I). The mutated enzymes were now capable to hydrolyze 3'SL and 6'SL substrates, hence losing the strict specificity of the wild-type enzymes for the {alpha}-(2,3) regioisomer.



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Fig. 2. Close-up view of the active site cleft of TcTS/TrSA, indicating the relative location of the amino acid residues at positions 312/313 (Trp/Ala) and 283/284 (Pro/Gln). The arrow indicates the position of the glycosidic oxygen of the bound sialic acid molecule. The top left view corresponds to wild-type TrSA, the top right view to wild-type TcTS, the bottom left to TrSA Trp313-Ala, and the bottom right to TcTS Trp312-Ala.

 

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Table I. Effect of the amino acid substitution Trp312/313-Ala on the sialidase (SA) and trans-sialidase (TS) activities of TcTS and TrSA using different substrates
 
Interestingly, the Trp312/313-Ala mutants of TrSA and TcTS essentially lost the capability to hydrolyze MUNANA (Table I). These results indicate that this fluorogenic substrate, widely used in sialidase assays, does not behave as the natural oligosaccharidic substrate because MUNANA, but not sialyl-ß-galactosyl substrates, requires a bulky hydrophobic residue at position 312/313 for hydrolysis to occur. Also, the mutation Trp312-Ala completely abolished the trans-sialylation activity of TcTS, independently of which regioisomer of sialyl-lactose was used (Table I).

Two other amino acid residues, which are close to the active site cleft and differ between TcTS and TrSA, have been previously suggested to be important for catalysis: TcTS Pro283–TrSA Gln284 (Smith and Eichinger, 1997Go) and TcTS Tyr119–TrSA Ser120 (Buschiazzo et al., 2000Go). The single substitution of Gln284 by a proline residue in TrSA was found to increase sevenfold the sialidase activity against MUNANA and threefold against 3'SL (Table II). According to the crystal structure of TrSA (Buschiazzo et al., 2000Go), the side chain of Gln284, adjacent to Trp313, is well positioned to interact directly with substrate (Figure 2), thus accounting at least in part for the observed increase in hydrolytic activity. In the case of MUNANA, a significantly lower Km for hydrolysis (0.03 mM for the mutant and 0.2 mM for the wild type) confirmed that the smaller proline side chain at position 284 facilitates binding of the aromatic methylumbelliferyl moiety of the substrate.


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Table II. Effect of the amino acid substitution Gln284–Pro in TrSA on the sialidase activity using MUNANA and {alpha}-(2,3)-sialyl-lactose as substrates.
 
Tyr119 was suggested to play a role in TcTS catalysis (Buschiazzo et al., 2000Go). Results in this work lend further support to the hypothesis that this residue is part of a second carbohydrate-binding site in the catalytic cleft. This site should be involved in binding the sialic acid-acceptor molecule, because the TcTS substitution Tyr119-Ser (see Figure 1) practically abolishes the sialyltransferase activity while preserving most sialidase activity (Buschiazzo et al., 2000Go). If this is true, TrSA mutants including both amino acid positions 120 (Tyr) and 284 (Pro) that are essential for trans-sialylation in TcTS are expected to gain TS activity. Instead, the mutant protein bearing these two mutations has no TS activity (Table III). These results strongly suggest that the donor substrate binds differently to the active site clefts of TrSA and TcTS and other amino acid residues must be implicated in the modulation of TS activity. All the point mutants of TrSA that include the exchange substitution, Gln284-Pro showed a significant increase of the sialidase activity (Table II): They strongly differ in their Vmax values (27,238 nmol sialic acid min–1 mg–1 for the mutant and 3750 nmol sialic acid min–1 mg–1 for the wild-type enzyme). Conversely, the reverse mutations in TcTS led to inactive recombinant proteins lacking not only TS but also sialidase activity. The single Pro283-Gln, double Tyr248–Gly/Pro283–Gln and triple Tyr119–Ser/Tyr248–Gly/Pro283–Gln TcTS mutants show neither TS nor sialidase activity. These results suggest that the Pro–Gln substitution in TcTs could destabilize the active conformation of the catalytic center.


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Table III. Specific activity of purified mutant proteins in 0.5 mM MUNANA
 
Although Tyr119 in TcTS seems to be important for sialic acid transfer activity its precise role in the general transferase reaction remains unclear. First, several amino acid residues are necessarily involved in the definition of the transferase-competent active site architecture. Thus, the inverse substitution Ser120–Tyr in TrSA proved insufficient by itself to confer transferase activity to TrSA (Table III). Second, although kinetic measurements for 3'SL and lactose as sialic acid donor and acceptor substrates, respectively, showed a six times higher apparent Km for lactose, compatible with a lactose-binding, the apparent Vmax for both substrates were significantly reduced and a small increase of the apparent Km for sialyl-lactose was also detected (Table IV). The possibility remains that these apparent Km values are far from true association/dissociation constants and a definitive model of substrate-enzyme interaction must await further structural studies of TcTS-ligand complexes.


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Table IV. Kinetic constants of recombinant mutant protein TcTS Tyr119–Ser
 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Extensive work has been accomplished to elucidate the general mechanism(s) by which glycosidases catalyze the hydrolysis of glycosidic bonds (see Ly and Withers, 1999Go for a recent review). These studies confirmed, to a large extent, the catalytic mechanisms originally proposed by Koshland (1953)Go for inverting and retaining glycosidases. The two classes of enzymes hydrolyze the glycosidic linkage using similar mechanisms that proceed through oxocarbenium ion–like transition states and that usually involve a pair of carboxylic acids in the active site acting as the general acid (proton donor) and the nucleophile or general base in the reaction.

In the case of sialidases, the detailed mechanisms are less clear. Nuclear magnetic resonance (NMR) and polarimetry studies established that these enzymes are retaining glycosidases, and a general model of sialidase action was proposed from crystallographic, NMR, and kinetic isotope effect studies of influenza virus sialidase (Varghese et al., 1983Go; Chong et al., 1992Go). Recent NMR experiments on TcTS have confirmed that sialidase activity of this enzyme follow the same mechanism, with retention of anomeric carbon configuration and the oxocarbenium ion intermediate (Todeschini et al., 2000Go). In addition, there are at least two examples of sialidases (TcTS and the homologous enzyme from T. brucei) that preferentially catalyze the transglycosylation of the sialic acid moiety from a ß-galactose-containing sialic acid–donor substrate to a chemically equivalent glycosidic acceptor. The transfer reaction of TcTS has a Kcat at least 10 times higher than that corresponding to the hydrolytic reaction catalyzed by the same enzyme, leading in fact to negligible amounts of hydrolyzed free sialic acid if millimolar quantities of a suitable sugar acceptor molecule are present (Schenkman et al., 1992Go; Vandekerckhove et al., 1992Go).

Substrate binding and specificity
Trypanosomal sialidases (for example, TrSA) and TSs (for example, TcTS) (Vandekerckhove et al., 1992Go; Crennell et al., 1994Go; Buschiazzo et al., 1997Go) as well as STNA (Hoyer et al., 1991Go) and the sialidase from Macrobdella decora (Chou et al., 1996Go) display a high specificity for {alpha}-(2,3)-linked sialic acid conjugates. In general, many other microbial sialidases, such as the enzymes from Vibrio cholerae, Micromonospora viridifaciens, and influenza virus, for which the 3D structures have been determined, can cleave {alpha}-(2,3), {alpha}-(2,6), and even {alpha}-(2,8)-linked sialic acid conjugates (Colman et al., 1983Go; Taylor et al., 1992Go; Crennell et al., 1994Go). We have demonstrated here that the conserved Trp 312/313 in TrSA and TcTS is directly involved in the binding of the sialic acid–donor substrates, as suggested by previous crystallographic studies. The single point mutants in which the bulky tryptophanyl residue (structurally homologous to STNA Tyr307 and equivalent to M. decora sialidase Trp734) was changed to alanine allow a looser accommodation of the donor substrate. As a consequence of this, the Trp–Ala mutants of TrSA and TcTS retained the wild-type capability of hydrolyzing {alpha}-(2,3)-linked substrates but were also able to cleave {alpha}-(2,6) regioisomers, thus broadening their substrate specificity. On the other hand, the mutants showed a significant decrease of their hydrolytic activity against the fluorogenic substrate MUNANA (hydrolysis was undetectable for TcTS and reduced 100-fold for TrSA), further stressing the critical role of Trp312/313 in the binding of the sialylated substrate.

Ping-pong versus sequential mechanism for trans-sialylation
Interestingly, the TS activity of TcTS was completely abolished by the Trp312–Ala mutation, independently of the sialic acid–donor molecule used as substrate (MUNANA, 3'SL, and 6'SL). For MUNANA, the hydrolysis reaction is also impaired, suggesting that the amino acid substitution could simply hinder substrate binding. However, sialyl-transferase activity is also undetectable for {alpha}-(2,3)- and {alpha}-(2,6)-linked substrates, although the Trp312–Ala mutant can still excise sialic acid from these substrates. If we assume that the transfer reaction takes place through a ping-pong mechanism with a long-lived sialosyl-cation intermediate (with both donor and acceptor substrates binding to the same site), the loss of activity would imply that Trp312 is critical for binding the acceptor but not the donor molecule because 3'SL and 6'SL are good substrates for hydrolysis. Although we cannot strictly rule out this possibility, a simpler explanation for these results is provided by an alternative model in which the two substrates, the sialic acid–donor and the ß-galactose-containing acceptor, bind simultaneously to the catalytic cleft (Buschiazzo et al., 2000Go). In this case, the loss of TS activity for the TcTS Trp312–Ala mutant would indicate that a precise orientation of the donor substrate within the catalytic center is required for the transfer reaction (but not for hydrolysis) to take place.

Additional experimental evidence sustain the two-sites model. From the 3D model of TcTS, we had identified another aromatic residue (Tyr119) that is missing in TrSA (Ser120). The Tyr side chain is located on a lateral wall of the catalytic cleft, apparently too far from Trp312 to be part of the same sugar-binding site. Modeling the substrate 3'SL with the lactose moiety stacked against the side chain of Trp312 leaves enough place for an acceptor (a second lactose) molecule that could interact with Tyr119. Furthermore, we have now confirmed that the exchange mutant TcTS Tyr119–Ser retained almost the same level of hydrolytic activity (78%) as the wild-type enzyme, whereas its TS activity dropped significantly (2.5%). This result is consistent with Tyr119 being a critical residue of the acceptor substrate-binding site.

Another important precedent for a TS-like activity was reported for the leech M. decora sialidase (Luo et al., 1999Go). This enzyme is strictly regiospecific for {alpha}-(2,3) sialoglycoconjugates and does not release free sialic acid as product of hydrolysis but an intramolecular cyclic sialic acid derivative: 2,7-anhydro-neuraminic acid (Chou et al., 1996Go). Based on the crystal structure of the enzyme complexed with DANA, the authors proposed that an oxygen atom from the axially located glycerol group of the same sialic acid molecule performs the nucleophilic attack on the C-2 carbonium of the reaction intermediate, leading to intramolecular transglycosylation. Along similar lines, the TcTS active site cleft could display a modified sialidase architecture with the aromatic side chains of Tyr119 and Trp312 on either side of the scissile glycosidic bond, allowing the simultaneous binding of donor and acceptor substrates in such a way that the acceptor’s galactosyl group could perform the nucleophilic attack on the sialosyl anomeric carbon C-2. This double-substrate model maintains a ping-pong (double displacement) mechanism common to retaining glycosidases but can also account for kinetic reports on TcTS consistent with a sequential mechanism that requires the concurrent presence of both substrates at the reaction center (Scudder et al., 1993Go; Ribeirao et al., 1997Go).

A single Gln–Pro substitution increases the sialidase activity of TrSA
We also obtained important modifications on enzymatic activities performing exchange mutations at position 283/284 (Gln in TrSA, Pro in TcTS). This residue is adjacent to Trp312/313 in the active site cleft (Figure 2) and could therefore be directly involved in substrate binding. In TrSA, all mutants containing the Gln284–Pro substitution consistently showed a significant increase of their hydrolytic activity (Tables IIIII). Interestingly, the activity increase with respect to the wild-type enzyme was greater when using MUNANA as substrate (Table III), suggesting that the bulkier Gln side chain could partially impair binding of the rigid methylumbelliferyl moiety of this substrate in wild-type TrSA.

In contrast with TrSA, the inverse Pro283–Gln substitution in TcTS abolished both the sialidase and TS activities. These results suggest that the proline residue in TcTS is required to stabilize the functional conformation of the loop. Indeed, a glycine residue (conferring flexibility to the polypeptide backbone) is found at position 284 of TcTS, whereas an aspartic acid residue occupies the equivalent position in TrSA. However, the lack of transferase activity in the TcTS Pro283–Gln mutant could be also related to steric hindrance, with Gln283 providing a steric obstacle for the correct orientation of the sialyl donor substrate. This would also explain the transferase activity reconstitution on hybrid constructs reported by Smith and Eichinger (1997)Go. In these experiments, the exchange of sequence segments from TrSA and TcTS allowed the identification of the single amino acid substitution Gln283–Pro, as able to partially reconstitute transferase activity onto a hybrid TrSA–TcTS protein that originally expressed only hydrolytic activity.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Site-directed mutagenesis
Site-directed point mutagenesis was performed using the Chameleon Double-Stranded site-directed mutagenesis kit (Stratagene, La Jolla, CA), according to the manufacturer’s instructions. Double-stranded recombinant pTrcHisA (Invitrogen, San Diego, CA) plasmids were used as templates in the mutagenesis reactions. The clone pTrcTS611/2 (Buschiazzo et al., 1997Go) contains an insert encoding the globular nonrepetitive core of T. cruzi TS, and the clone pTrcTrSA4_Nhe (which derives from clone pTrSA4 [Buschiazzo et al., 1997Go] by PCR-subcloning into the restriction site NheI of plasmid pTrcHisA) contains an insert encoding the sialidase of T. rangeli. Both clones encode fusion recombinant proteins with the vector-encoded histidine tag at the N-terminus. A second procedure to obtain mutated clones was performed by the megaprimer method (Sarkar and Sommer, 1990Go). All clones were entirely sequenced confirming mutation of the target sites only.

Recombinant protein expression and enzyme activity assays
The mutagenized clones were used to transform Escherichia coli XL1Blue (Stratagene) or DH5{alpha}F'-RT (Gibco BRL). After growth in LB broth up to Abs600 0.6–0.8, with constant agitation 250 RPM at 37°C, bacteria were induced to overexpress recombinant protein by adding 0.5 mM isopropylthiogalactoside (Sigma, St. Louis, MO). Induction was maintained with normal agitation at 28°C for 12–16 h. Cells were harvested and frozen (–80°C) until needed. After thawing, lysis was achieved in the presence of 20 mM Tris–Cl, pH 8.0, 0.5 M NaCl, 0.5% NP 40, 1 mg ml–1 lisozyme, 100 µg ml–1 DNase I, and 1 mM phenylmethylsulphonyl fluoride. Supernatants were centrifuged at 21,000 x g for 30 min and subjected to iminodiacetic acid metal affinity chromatography (IMAC) Ni2+-charged after adding NaCl to 0.5 M. Elution was achieved using 100 mM imidazole in 20 mM Tris–Cl, pH 8, 0.5 M NaCl. The activity peak was pooled, dialyzed against 20 mM Tris–HCl (pH 8) and further purified by FPLC anionic exchange (MonoQ) applying a linear NaCl elution gradient. With TrSA and TrSA-derived proteins we initially had problems at high pH and in the presence of high concentrations of chloride ions. Following a typical TcTS purification protocol, sialidase was irreversibly inactivated after IMAC purification. Lysis buffer was changed to 50 mM sodium phosphate, pH 7.4, and NaCl was replaced by 0.5–0.7 M sodium acetate in the IMAC equilibration and elution buffers. The activity peak was pooled and dialyzed against 50 mM sodium acetate, pH 5.5, and resolved by FPLC applying a linear NaCl elution gradient to a cationic exchange column (against, for example, MonoS). When needed, the production of recombinant proteins was scaled up using 4lt-fermentors without changing the general protocols.

Purified proteins were analyzed by SDS–polyacrylamide gel electrophoresis under reducing conditions, stained with Coomassie Blue R250, and quantitated with Image Quantifier 3.22 (Molecular Dynamics) using purified BSA as standard. After the ionic exchange columns, the proteins were > 95% pure by these criteria. Enzyme activity assays were carried out using this material as previously described (Buschiazzo et al., 1997Go). Briefly, neuraminidase activity was determined by measuring the fluorescence of 4-methylumbelliferone released by the hydrolysis of 0.2 mM MUNANA (Sigma). The assay was performed in 50 µl Tris–Cl, pH 7.6, or 50 mM sodium acetate, pH 5.5, when required. After incubation at 25°C, the reaction was stopped by dilution in 0.2 M carbonate, pH 10, and fluorescence was measured with a DYNA QuantTM 200 fluorometer (Hoefer Pharmacia Biotech Inc., Uppsala). TS was measured in 30 mM NaCl, 20 mM Tris–Cl, pH 7.6, using 1 mM 3'SL, 6'SL, or MUNANA as donor and 12 µM [D-glucose-1-14C]-lactose (55 mCi mmol–1) (Amersham, Buckinghamshire) as acceptor in 30 µl final volume at 25°C. The reaction was stopped by dilution, and sialyl-14C-lactose was quantitated with a ß-scintillation counter after adding 0.1 ml of a dense slurry of QAE-Sephadex (Pharmacia Biotech, Uppsala). Suitable modifications were made to the standard reaction to obtain the kinetic constants.

Measurements of 3'SL and 6'SL hydrolysis were done by the thiobarbituric method (Powell and Hart, 1986Go; Romero et al., 1997Go). Wild-type or mutated proteins were incubated with 5 mM of either 3'SL or 6'SL and 50 mM Tris–HCl, pH 7.5, in a final volume of 10 µl for 30 min at room temperature. The enzymatic reactions were stopped by adding 7.5 µl of a 25 mM NaIO4 solution prepared in 125 mM sulfuric solution. The mixtures were vortexed and allowed to react in a water bath at 37°C for 30 min (sialic acid oxidation period). Samples were then neutralized with 6.5 µl of sodium arsenite 2% w/v in HCl (0.5 N) by slow addition of the reactive. Tubes were gently vortexed to complete the reduction reaction. After the total disappearance of yellow color (~5 min) 76 µl of thiobarbituric acid (36 mM, pH 9.0) were added and then incubated in a boiling water bath for 15 min (chromophore formation). Samples were then cooled in an ice-water bath for 5 min, followed by room-temperature color stabilization. The samples were centrifuged, and 20 µl were separated by high-performance liquid chromatography through a C18 reverse phase column (Pharmacia Biotech) using 2:3:5 water:methanol:buffer (buffer: 0.2% phosphoric acid; 0.23 M sodium perchlorate). Absorbance was measured at 549 nm. A sialic acid calibration curve was previously set, and absorbance values were always read in the linear range. The sensitivity of the assay is 1 pmol sialic acid.


    Acknowledgments
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Michel Diaquin and Jean Paul Latgé (Département de Bactériologie et Mycologie, Institut Pasteur, Paris) for the use of the fermentors. This work was supported by grants from the World Bank/UNDP/WHO Special Program for Research and Training in Tropical Diseases (TDR), ECOS-SeCyT (France-Argentina), the Institut Pasteur and the CNRS (France), the CONICET, the Agencia Nacional de Promoción Científica y Tecnológica and the Fundación Antorchas (Argentina). The research from ACCF was supported in part by an International Research Scholars Grant from the Howard Hughes Medical Institute.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
3'SL, sialyl-{alpha}-(2,3)-lactose; 6'SL, sialyl-{alpha}-(2,6)-lactose; DANA, 2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid; IMAC, iminodiacetic acid metal affinity chromatography; MUNANA, 4-methylumbelliferyl-N-acetylneuraminic acid; NMR, nuclear magnetic resonance; STNA, Salmonella typhimurium sialidase; TS, trans-sialidase; TcTS, Trypanosoma cruzi trans-sialidase; TrSA, T. rangeli sialidase.


    Footnotes
 
1 These authors contributed equally to this work. Back

2 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Bond, C.S., Zhang, Y., Berriman, M., Cunningam, M.L., Fairlamb, A.H., and Hunter, W.N. (1999) Crystal structure of Trypanosoma cruzi trypanothione reductase in complex with trypanothione, and the structure-based discovery of new natural product inhibitors. Structure Fold Des., 7, 81–89.[ISI][Medline]

Borst, P., and Fairlamb, A.H. (1998) Surface receptors and transporters of Trypanosoma brucei. Annu. Rev. Microbiol., 52, 745–778.[ISI][Medline]

Bousquet, M.P., Willemot, R.M., Monsan, P., and Boures, E. (1998) Enzymatic synthesis of alkyl-alpha-glucoside catalyzed by a thermostable alpha-transglucosidase in solvent-free organic medium. Appl. Microbiol. Biotechnol., 50, 167–173.[ISI]

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