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.
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Abstract |
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Key words: trypanosomatids/sialidase/trans-sialidase/substrate specificity/catalytic site
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Introduction |
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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 Chagass disease in North America and South America (Frasch, 2000). 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
-(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., 1991
; Ferrero-García et al., 1993
).
TS has been extensively studied in T. cruzi. It shares about 30% sequence identitity and several characteristic motifs with bacterial sialidases (Cremona et al., 1995). 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., 1997
). 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., 1998
) or using unusually high concentrations of the acceptor substrate (Watt et al., 1998
).
Recently, the 3D structure of TrSA was obtained and compared with the modeled structure of TcTS (Buschiazzo et al., 2000). Trypanosomal sialidases fold into two globular domains tightly associated with each other. The catalytic domain (residues 1372) displays a six-bladed ß-propeller topology similar to that of bacterial and viral sialidases, connected by a long
-helical segment to a C-terminal domain (residues 398614) 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., 2000
). 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., 1993
; Ribeirao et al., 1997
).
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.
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Results |
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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 Pro283TrSA Gln284 (Smith and Eichinger, 1997) and TcTS Tyr119TrSA Ser120 (Buschiazzo et al., 2000
). 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., 2000
), 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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Discussion |
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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., 1983; Chong et al., 1992
). 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., 2000
). 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 aciddonor 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., 1992
; Vandekerckhove et al., 1992
).
Substrate binding and specificity
Trypanosomal sialidases (for example, TrSA) and TSs (for example, TcTS) (Vandekerckhove et al., 1992; Crennell et al., 1994
; Buschiazzo et al., 1997
) as well as STNA (Hoyer et al., 1991
) and the sialidase from Macrobdella decora (Chou et al., 1996
) display a high specificity for
-(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
-(2,3),
-(2,6), and even
-(2,8)-linked sialic acid conjugates (Colman et al., 1983
; Taylor et al., 1992
; Crennell et al., 1994
). We have demonstrated here that the conserved Trp 312/313 in TrSA and TcTS is directly involved in the binding of the sialic aciddonor 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 TrpAla mutants of TrSA and TcTS retained the wild-type capability of hydrolyzing
-(2,3)-linked substrates but were also able to cleave
-(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 Trp312Ala mutation, independently of the sialic aciddonor 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 -(2,3)- and
-(2,6)-linked substrates, although the Trp312Ala 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 aciddonor and the ß-galactose-containing acceptor, bind simultaneously to the catalytic cleft (Buschiazzo et al., 2000
). In this case, the loss of TS activity for the TcTS Trp312Ala 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 Tyr119Ser 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., 1999). This enzyme is strictly regiospecific for
-(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., 1996
). 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 acceptors 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., 1993
; Ribeirao et al., 1997
).
A single GlnPro 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 Gln284Pro 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 Pro283Gln 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 Pro283Gln 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). In these experiments, the exchange of sequence segments from TrSA and TcTS allowed the identification of the single amino acid substitution Gln283Pro, as able to partially reconstitute transferase activity onto a hybrid TrSATcTS protein that originally expressed only hydrolytic activity.
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Materials and methods |
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Recombinant protein expression and enzyme activity assays
The mutagenized clones were used to transform Escherichia coli XL1Blue (Stratagene) or DH5F'-RT (Gibco BRL). After growth in LB broth up to Abs600 0.60.8, 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 1216 h. Cells were harvested and frozen (80°C) until needed. After thawing, lysis was achieved in the presence of 20 mM TrisCl, pH 8.0, 0.5 M NaCl, 0.5% NP 40, 1 mg ml1 lisozyme, 100 µg ml1 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 TrisCl, pH 8, 0.5 M NaCl. The activity peak was pooled, dialyzed against 20 mM TrisHCl (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 0.50.7 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 SDSpolyacrylamide 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., 1997). 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 TrisCl, 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 TrisCl, pH 7.6, using 1 mM 3'SL, 6'SL, or MUNANA as donor and 12 µM [D-glucose-1-14C]-lactose (55 mCi mmol1) (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, 1986; Romero et al., 1997
). Wild-type or mutated proteins were incubated with 5 mM of either 3'SL or 6'SL and 50 mM TrisHCl, 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.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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2 To whom correspondence should be addressed
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References |
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