The cestodes constitute important but understudied human and veterinary parasites. Their surfaces are rich in carbohydrates, on which very little structural information is available. The tissue-dwelling larva (hydatid cyst) of the cestode Echinococcus granulosus is outwardly protected by a massive layer of carbohydrate-rich extracellular matrix, termed the laminated layer. The monosaccharide composition of this layer suggests that its major carbohydrate components are exclusively mucin-type O-glycans. We have purified these glycans after their release from the crude laminated layer and obtained by MS and NMR the complete structure of 10 of the most abundant components. The structures, between two and six residues in length, encompass a limited number of biosynthetic motifs. The mucin cores 1 and 2 are either nondecorated or elongated by a chain of Galpbeta1-3 residues. This chain can be capped by a single Galpalpha1-4 residue, such capping becoming more dominant with increasing chain size. In addition, the core 2 N-acetylglucosamine residue is in cases substituted with the disaccharide Galpalpha1-4Galpbeta1-4, giving rise to the blood P(1)-antigen motif. Larger, also related, glycans exist, reaching at least 18 residues in size. The glycans described are related but larger than those previously described from an Echinococcus multilocularis mucin [Hulsmeier, A. J., et al. (2002) J. Biol. Chem. 277, 5742-5748]. Our results reveal that the E. granulosus cyst exposes to the host only a few different major carbohydrate motifs. These motifs are composed essentially of galactose units and include the elongation by (Galpbeta1-3)(n) and the capping by Galpalpha1-4, novel in animal mucin-type O-glycans.
Host/parasite interaction mediated by carbohydrate/lectin recognition results in the attachment to and invasion of host cells and immunoregulation, enabling parasite replication and establishment of infection. Trypanosoma cruzi, the protozoan responsible for Chagas disease, expresses on its surface a family of enzymatically active and inactive trans-sialidases. The parasite uses the active trans-sialidase for glycoprotein sialylation in an unusual trans-glycosylation reaction. Inactive trans-sialidase is a sialic acid-binding lectin that costimulates host T cells through leucosialin (CD43) engagement. The co-mitogenic effect of trans-sialidase can be selectively abrogated by N-acetyllactosamine, suggesting the presence of an additional carbohydrate binding domain for galactosides, in addition to that for sialic acid. Here we investigated the interaction of inactive trans-sialidase in the presence of β-galactosides. By using NMR spectroscopy, we demonstrate that inactive trans-sialidase has a β-galactoside recognition site formed following a conformational switch induced by sialoside binding. Thus prior positioning of a sialyl residue is required for the β-galactoside interaction. When an appropriate sialic acid-containing molecule is available, both sialoside and β-galactoside are simultaneously accommodated in the inactive trans-sialidase binding pocket. This is the first report of a lectin recognizing two distinct ligands by a sequential ordered mechanism. This uncommon binding behavior may play an important role in several biological aspects of T. cruzi/host cell interaction and could shed more light into the catalytic mechanism of the sialic acid transfer reaction of enzymatically active trans-sialidase. Host/parasite interaction mediated by carbohydrate/lectin recognition results in the attachment to and invasion of host cells and immunoregulation, enabling parasite replication and establishment of infection. Trypanosoma cruzi, the protozoan responsible for Chagas disease, expresses on its surface a family of enzymatically active and inactive trans-sialidases. The parasite uses the active trans-sialidase for glycoprotein sialylation in an unusual trans-glycosylation reaction. Inactive trans-sialidase is a sialic acid-binding lectin that costimulates host T cells through leucosialin (CD43) engagement. The co-mitogenic effect of trans-sialidase can be selectively abrogated by N-acetyllactosamine, suggesting the presence of an additional carbohydrate binding domain for galactosides, in addition to that for sialic acid. Here we investigated the interaction of inactive trans-sialidase in the presence of β-galactosides. By using NMR spectroscopy, we demonstrate that inactive trans-sialidase has a β-galactoside recognition site formed following a conformational switch induced by sialoside binding. Thus prior positioning of a sialyl residue is required for the β-galactoside interaction. When an appropriate sialic acid-containing molecule is available, both sialoside and β-galactoside are simultaneously accommodated in the inactive trans-sialidase binding pocket. This is the first report of a lectin recognizing two distinct ligands by a sequential ordered mechanism. This uncommon binding behavior may play an important role in several biological aspects of T. cruzi/host cell interaction and could shed more light into the catalytic mechanism of the sialic acid transfer reaction of enzymatically active trans-sialidase. Trypanosoma cruzi is the etiologic agent of Chagas disease or American trypanosomiasis that affects ∼18 million people in Central and South America. Another 100 million people are at risk of infection (1Moncayo A. Mem. Inst. Oswaldo Cruz. 2003; 98: 577-591Crossref PubMed Scopus (295) Google Scholar). Mammalian cell invasion is crucial for T. cruzi survival (2Brener Z. Annu. Rev. Microbiol. 1973; 27: 347-382Crossref PubMed Scopus (492) Google Scholar). Elucidation of molecular components regulating the initiation of the parasitic infection is critical for understanding the pathogenesis of Chagas disease and will enable the development of novel, effective, and selective treatments. Initial communication between T. cruzi trypomastigotes and mammalian cells requires contact of soluble or membrane-bound parasite molecules with host ligands. T. cruzi expresses on its surface a family of glycosylphosphatidylinositol-anchored active and inactive trans-sialidase (TS) 1The abbreviations used are: TStrans-sialidaseβGalpβ-galactopyranoseNeu5AcN-acetylneuraminic acidSTDsaturation transfer differenceTOCSYtotal correlation spectroscopyPBSphosphate-buffered saline. proteins (3Uemura H. Schenkman S. Nussenzweig V. Eichinger D. EMBO J. 1992; 11: 3837-3844Crossref PubMed Scopus (76) Google Scholar, 4Cremona M.L. Sanchez D.O. Frasch A.C.C. Campetella O. Gene (Amst.). 1995; 160: 123-128Crossref PubMed Scopus (88) Google Scholar, 5Frasch A.C. Parasitol. Today. 2000; 16: 282-286Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar), which contain a Tyr or a His residue, respectively, at position 342 (4Cremona M.L. Sanchez D.O. Frasch A.C.C. Campetella O. Gene (Amst.). 1995; 160: 123-128Crossref PubMed Scopus (88) Google Scholar). The parasite uses active TS to sialylate its surface glycoproteins by a trans-sialidase reaction (6Previato J.O. Andrade A.F.B. Pessolani M.C.V. Mendonça-Previato L. Mol. Biochem. Parasitol. 1985; 16: 85-96Crossref PubMed Scopus (176) Google Scholar). Thus, TS activity is capable of extensively remodeling the T. cruzi cell surface by using host glycoconjugates as sialyl donor. Alternatively, the enzyme may sialylate host cell glycomolecules to generate receptors used by the trypanosome for adherence to and penetration of target cells. Results with sialic acid-deficient mutants of Chinese hamster ovary cells support this hypothesis (7Ming M. Chuenkova M. Ortega-Barria E. Pereira M.E.A. Mol. Biochem. Parasitol. 1993; 59: 243-252Crossref PubMed Scopus (100) Google Scholar, 8Ciavaglia M.C. De Carvalho T.U. De Souza W. Biochem. Biophys. Res. Commun. 1993; 193: 718-721Crossref PubMed Scopus (42) Google Scholar, 9Schenkman R.P.F. Vandekerckhove F. Schenkman S. Infect. Immun. 1993; 61: 898-902Crossref PubMed Google Scholar). Sialic acid-deficient cells are less infected than wild type cells, suggesting that recognition of sialyl residues on Chinese hamster ovary cells is necessary during T. cruzi invasion. trans-sialidase β-galactopyranose N-acetylneuraminic acid saturation transfer difference total correlation spectroscopy phosphate-buffered saline. Recently, we demonstrated that TS binds to α2,3-linked sialic acid from CD43 on murine T cells and initiates CD43-dependent co-stimulatory responses that increase mitogenesis, cytokine secretion, and promote rescue from apoptosis (10Todeschini A.R. Nunes M.P. Pires R.S. Lopes M.F. Previato J.O. Mendonça-Previato L. DosReis G.A. J. Immunol. 2002; 168: 5192-5198Crossref PubMed Scopus (58) Google Scholar). The co-mitogenic effects of inactive TS on T cells were selectively abrogated by addition of N-acetyllactosamine, suggesting the inactive form of this glycosyltransferase is a sialic acid-binding lectin with an additional binding site for lactosides. In this work, we demonstrate by using NMR spectroscopy that inactive TS possesses two sugar-binding sites, one for α2,3-sialic acid-containing molecules and a second for β-galactosides. We also show that inactive TS recognizes its ligands in a sequential ordered mode. Sialoside binding is necessary to trigger a conformational modification of the inactive TS, allowing it to interact with β-galactosides. Knowledge of binding properties of TS could clarify its role in the molecular mechanism of T. cruzi/host cell interaction. Materials—Most of the chemical products used were from Sigma or Fisher. The following materials were obtained from other sources: pre-packed Ni2+-chelating HP HiTrap, Mono Q HR 10/10, and Mono S HR 5/5 columns. α-2,3-Sialyllacto-N-tetraose was kindly supplied by Dr. G. Strecker, Laboratoire de Chimie Biologique, Université des Sciences et Technologies de Lille, France. Recombinant Inactive trans-Sialidase—Recombinant inactive TS, containing the C-terminal repeats, was obtained from Escherichia coli MC1061 electrotransformed with plasmids containing the inactive mutant TS insert bearing a Tyr342 → His substitution, pTrcHisA (4Cremona M.L. Sanchez D.O. Frasch A.C.C. Campetella O. Gene (Amst.). 1995; 160: 123-128Crossref PubMed Scopus (88) Google Scholar). Bacteria were grown in supplemented TB medium in the presence of 100 μg/ml ampicillin. When the culture reached an A600 nm of 1.5, 30 mg/liter isopropyl-β-d-thiogalactoside was added, and incubation was continued overnight. Bacteria were lysed at 4 °C in 20 mm Tris-HCl containing 2.0 mg/ml lysozyme, 2% Triton X-100, 0.1 mm phenylmethylsulfonyl fluoride, 5.0 μg/ml leupeptin, 1.0 μg/ml trypsin inhibitor, and 0.1 μm iodoacetamide. Inactive TS containing a poly(His) tag was purified as described (11Todeschini A.R. Mendonça-Previato L. Previato J.O. Varki A. Van Halbeek H. Glycobiology. 2000; 10: 213-221Crossref PubMed Scopus (47) Google Scholar), using Ni2+-chelating chromatography on a HiTrap column, eluted with imidazole gradient. The eluates were applied to Mono Q and Mono S columns and eluted with NaCl gradient. The homogeneity of the protein was evaluated by 10% SDS-PAGE, and inactive TS was stored in 20 mm Tris-HCl buffer, pH 7.4, at 4 °C until used. Frontal Affinity Chromatography Using Lactose-Sepharose Column—Frontal affinity chromatography was carried out using a lactose-Sepharose column (7.4 × 0.8 cm) equilibrated with PBS buffer in the absence or presence of α-2,3-sialyllacto-N-tetraose (2 mm). BSA and inactive TS were run at a flow rate of 0.5 ml/min at 4 °C. Fractions of 1 ml were collected, lyophilized, and suspended in 200 μl, and 20 μl were analyzed by SDS-PAGE (10%) under reducing conditions. NMR Experiments—α2,3-Sialillactose, α2,6-sialillactose, methyl β-lactoside, methyl β-melibioside, methyl α-mannoside, or Galβ-1,3-GlcNAcβ-1,3-Galβ-1,4-Glc (lacto-N-tetraose) were dissolved in deuterated PBS, pH 7.6 (not corrected for isotope effects). Inactive TS solution in 20 mm Tris-HCl was exchanged with deuterated PBS by gel filtration on a G-25 column. Twenty μl of a stock solution containing 10 mg/ml of inactive TS was added to a solution of sialylglycoside (2 mm final concentration), and the total volume was adjusted to 500 μl. NMR spectra were obtained at a probe temperature of 20 °C on a Bruker DMX 600 equipped with a 5-mm self-shielded gradient triple resonance probe or on a Bruker DRX 600 with a 5-mm triple resonance probe. One-dimensional Saturation Transfer Difference (STD)—One-dimensional STD experiments were performed by low power presaturation of the methyl region of the protein during the 2-s relaxation delay. The pulse scheme was as follow: relaxation delay with or without presaturation of the protein resonances, 90° pulse and acquisition. Two hundred and fifty six scans of 16,000 points over a 10 ppm spectral width were collect. Data were obtained with an interspersed acquisition of on-resonance and control spectra in order to minimize the effects of temperature and magnet instability. Saturation Transfer Difference-Total Correlation Spectroscopy (STD-TOCSY)—STD-TOCSY spectra were recorded with a mixing time of 66 ms, 32 scans per t1 increment. 200 t1 increments were collected in an interlaced mode with presaturation on or off for 2 s. Prior subtraction both spectra were processed and phased identically. The acquisition times for the two-dimensional experiments were typically 16 h. The spectra were multiplied with a square cosine bell function in both dimensions and zero-filled two times. All spectra were referenced relative to trimethylsilyl-2,2′,3,3′-d4-propionic acid-sodium salt (δ = 0.0 ppm). Recently, we applied STD-NMR techniques to demonstrate that enzymatically inactive T. cruzi TS is a sialic acid-binding lectin, allowing atomic resolution of the epitope involved in the interaction (12Todeschini A.R. Girard M.F. DosReis G.A. Mendonça-Previato L. Previato J.O. J. Biol. Chem. 2002; 277: 45962-45968Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). By using the same methodology, the interaction of inactive TS with lacto-N-tetraose, methyl-β-lactose, methyl β-melibiose, and methyl α-mannoside was studied. Fig. 1 shows the one-dimensional STD-NMR spectra of inactive TS in the presence of lacto-N-tetraose. From an inspection in the spectra, it is immediately obvious that no proton from lacto-N-tetraose or methyl β-lactoside (results not shown) receives magnetization from the protein. The STD-TOCSY acquired under the same conditions confirmed these results (Fig. 2). Neither saccharide studied makes contact with the inactive TS, because no cross-peaks were observed (Fig. 2B).Fig. 2STD-TOCSY of lacto-N-tetraose in presence of inactive TS.A, reference TOCSY spectrum of inactive TS in the presence of lacto-N-tetraose in PBS/D2O, pH 7.6, 20 °C. B, STD-TOCSY spectrum. Spectra were recorded with a mixing time of 66 ms, 32 scans per t1 increment. 200 t1 increments were collected in an interlaced mode for on or off presaturation, as described under "Experimental Procedures."View Large Image Figure ViewerDownload Hi-res image Download (PPT) Schudder et al. (13Scudder P. Doom J.P. Chuenkova M. Manger I.D. Pereira M.E.A. J. Biol. Chem. 1993; 268: 9886-9891Abstract Full Text PDF PubMed Google Scholar) and Ribeirão et al. (14Ribeirão M. Pereira-Chioccola V.L. Eichinger D. Rodrigues M.M. Schenkman S. Glycobiology. 1997; 7: 1237-1246Crossref PubMed Scopus (67) Google Scholar) demonstrated by kinetic studies that active TS catalyzes the transfer of α-2,3-sialyllactose to terminal β-Galp-containing molecules through a bisubstrate sequential mechanism involving an enzyme-substrate ternary complex, indicating that the β-galactoside must bind to the enzyme at some stage in the catalytic cycle. If inactive TS exhibits the same binding properties, it must interact with lacto-N-tetraose or methyl β-lactoside when a sialoside is present. To test this hypothesis, we used STD experiments to probe the interaction of inactive TS with lacto-N-tetraose in the presence of α-2,3-sialyllactose. Fig. 3 shows the reference TOCSY (Fig. 3A) and the STD-TOCSY (Fig. 3, B and C) spectra of the lacto-N-tetraose incubated with inactive TS in the presence of α-2,3-sialyllactose. The STD-TOCSY spectrum indicates that the NAc and H3ax protons of Neu5Ac residue are in close contact with the inactive enzyme and that H3eq, H4, and H5 from the Neu5Ac residue and H1, H3, and H4 from the β-Galp ring receive saturation from the protein (Fig. 4). The contacts and intensities observed for α-2,3-sialyllactose interaction with inactive TS are in complete agreement with those found previously (12Todeschini A.R. Girard M.F. DosReis G.A. Mendonça-Previato L. Previato J.O. J. Biol. Chem. 2002; 277: 45962-45968Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). In addition, Fig. 3C clearly shows additional cross-peaks at 4.47 and 3.68 ppm arising from H1 and H3 of β-Galp of the lacto-N-tetraose, respectively. Similarly, contact between the β-Galp residue and inactive enzyme was observed when methyl β-lactoside was incubated at the same conditions (results not shown).Fig. 4Relative intensities of STD-TOCSY cross-peaks. The intensity is normalized to the intensity of the corresponding cross-peak in the reference TOCSY spectrum.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Binding of inactive TS with β-lactoside in the presence of α-2,3-linked sialic acid was also verified by frontal affinity chromatography. When the inactive TS was loaded on the lactose-Sepharose column pre-equilibrated in PBS buffer with α-2,3-sialyllacto-N-tetraose, the enzyme was eluted in a higher elution volume in relation to the front found when the column was equilibrated in absence of the sialoside (Fig. 5). These results demonstrate that binding of α-2,3-sialyllactose to inactive TS elicits a conformational alteration in the protein framework that allows the β-galactoside to bind. The α-2,3-sialyllactose and β-galactoside simultaneously accommodate in the inactive TS-binding pocket of inactive TS, suggesting that the inactive members of the TS family interact with their ligands by a sequential ordered mechanism. Previously, we demonstrated that inactive TS binds only weakly to α-2,6-sialyllactose, because in contrast to α-2,3-sialyllactose, no contact between the protein and the β-Galp ring was detected (12Todeschini A.R. Girard M.F. DosReis G.A. Mendonça-Previato L. Previato J.O. J. Biol. Chem. 2002; 277: 45962-45968Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). To investigate whether the conformational switch of inactive TS depends on the correct positioning of the sialoside in the enzyme-binding site, interaction of inactive TS with α-2,6-sialyllactose and lacto-N-tetraose was studied. STD-TOCSY spectrum (Fig. 6) contains no signals arising from the lacto-N-tetraose, suggesting that a proper positioning of sialylated ligand is necessary for the β-galactoside interaction. To investigate whether inactive TS is specific for β-Galp residues, the enzyme was incubated with methyl α-mannoside (Fig. 7) or methyl β-melibiose (result not shown) in the presence of α-2,3-sialyllactose. Additional signals were not observed, demonstrating that, similar to its active analogue, inactive TS only interacts with terminal β-Galp residues. Taken together, our results show that correct positioning of the sialoside in the inactive TS-binding site is necessary for the protein to undergo the conformational change that allows the β-galactoside to bind (Fig. 8).Fig. 8Proposed events for the transfer reaction of T. cruzi TS.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Host/parasite interaction mediated by carbohydrates may be influenced by lectin properties. Lectins can behave either as receptors or ligands for parasite/host interactions, resulting in the recognition, attachment to, and invasion of host cells, and immunoregulation, enabling parasite replication and establishment of infection. T. cruzi genome contains hundreds of genes encoding a family of cell surface and enzymatically active and inactive TS and sialic acid acceptor glycoproteins (mucin-like molecules) (5Frasch A.C. Parasitol. Today. 2000; 16: 282-286Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Several reports (15Burleigh B.A. Woolsey A.M. Cell. Microbiol. 2002; 4: 701-711Crossref PubMed Scopus (162) Google Scholar) have suggested that these molecules could be either receptors or ligands during the process of T. cruzi infection. Previously, we showed that the enzymatically inactive TS is a sialic acid-binding lectin able to bind to and costimulate T cells through CD43 engagement, and that there is evidence for an additional binding site for β-galactopyranosyl residues, because the co-mitogenic effect was selectively abrogated by addition of N-acetyllactosamine (10Todeschini A.R. Nunes M.P. Pires R.S. Lopes M.F. Previato J.O. Mendonça-Previato L. DosReis G.A. J. Immunol. 2002; 168: 5192-5198Crossref PubMed Scopus (58) Google Scholar). This possibility prompted us to investigate the interaction of inactive TS with β-galactosides. By using NMR spectroscopy, we demonstrated that inactive T. cruzi TS has a carbohydrate recognition domain for β-Galp residue that is formed only after a conformational switch triggered by prior sialoside binding. As far as we know, this is the first report of a lectin that recognizes its ligands by a sequential ordered mechanism. The binding behavior of inactive TS may play a significant role in the process of T. cruzi adhesion, invasion, and host immunoregulation, allowing the parasitic infection to be established. Molecules of the TS family are glycosylphosphatidylinositol-anchored to the membrane and can be released into the serum in fairly high amounts during acute phase Chagas disease in humans (5Frasch A.C. Parasitol. Today. 2000; 16: 282-286Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Therefore, TS family members can act as soluble modifiers of immune responses. The bivalent nature of inactive TS demonstrated here would promote glycan cross-linking, which is believed to be essential for cellular signal transduction. Both soluble and membrane-bound inactive TS could interact with sialo- and asialo-glycoconjugates expressed by host cells leading to exacerbated and immunopathologic reactions in Chagas disease. The finding that inactive TS has two carbohydrate binding domains may explain some apparently contradictory results on the involvement of sialyl and galactosyl epitopes in T. cruzi/host cell interaction. Whereas Schenkman et al. (16Schenkman S. Jiane M.S. Hart G.W. Nussenweig V. Cell. 1991; 65: 1117-1125Abstract Full Text PDF PubMed Scopus (381) Google Scholar) have shown that sialylation of Ssp-3 epitope of mammalian cell-derived trypomastigotes is required for target cell recognition, Yoshida et al. (17Yoshida N. Dorta M.L. Ferreira A.T. Oshiro M.E.M. Mortara R.A. Serrano-Acosta A. Favoreto Jr., S. Mol. Biochem. Parasitol. 1997; 84: 57-67Crossref PubMed Scopus (44) Google Scholar) reported that the removal of sialic acid from the surface of insect-derived metacyclic trypomastigotes enhances parasite/host interaction. The removal of sialic acid from T. cruzi glycoproteins and the concomitant exposure of cryptic β-Galp residues would favor inactive TS interaction with both host sialoglycoconjugates and terminal β-Galp-containing glycoproteins on the parasite surface, thus enhancing T. cruzi-host adhesion. This phenomenon was well characterized for CD22, a mammalian sialic acid-binding lectin (18Razi N. Varki A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7469-7474Crossref PubMed Scopus (217) Google Scholar, 19Collins B.E. Blixt O. Bovin N.V. Danzer C.P. Chui D. Marth J.D. Nitschke L. Paulson J.C. Glycobiology. 2002; 12: 563-571Crossref PubMed Scopus (54) Google Scholar, 20Crocker P.R. Varki A. Trends Immunol. 2001; 22: 337-342Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar). CD22 is masked on the surface of murine B cells, as evidenced by enhanced binding of specific sialyl probes after sialidase treatment of B cells (19Collins B.E. Blixt O. Bovin N.V. Danzer C.P. Chui D. Marth J.D. Nitschke L. Paulson J.C. Glycobiology. 2002; 12: 563-571Crossref PubMed Scopus (54) Google Scholar). The removal of sialic acid and concomitant exposure of β-Galp residues from host cell glycans, which occurs as a result of the T. cruzi TS reaction, may therefore be physiologically significant by promoting parasite adherence to and penetration of host cells (7Ming M. Chuenkova M. Ortega-Barria E. Pereira M.E.A. Mol. Biochem. Parasitol. 1993; 59: 243-252Crossref PubMed Scopus (100) Google Scholar, 8Ciavaglia M.C. De Carvalho T.U. De Souza W. Biochem. Biophys. Res. Commun. 1993; 193: 718-721Crossref PubMed Scopus (42) Google Scholar, 9Schenkman R.P.F. Vandekerckhove F. Schenkman S. Infect. Immun. 1993; 61: 898-902Crossref PubMed Google Scholar). The findings that inactive TS recognizes its ligands with the same specificity as its active analogue may bring insights into the mechanism of catalytic transglycosylation by T. cruzi TS. T. cruzi TS is an unusual sialidase with a predominant transglycosylase activity (21Vandekerckhove F. Schenkman S. Pontes de Carvalho L. Tomlinson S. Kiso M. Yoshida M. Hasegawa A. Nussenzweig V. Glycobiology. 1992; 2: 541-548Crossref PubMed Scopus (124) Google Scholar). The catalytic mechanism of trypanosomal sialidase (11Todeschini A.R. Mendonça-Previato L. Previato J.O. Varki A. Van Halbeek H. Glycobiology. 2000; 10: 213-221Crossref PubMed Scopus (47) Google Scholar) seems to be similar to viral sialidase (22Burmeister W.P. Henrissat B. Bosso C. Cusack S. Ruigrok R.W. Structure. 1993; 1: 19-26Abstract Full Text PDF PubMed Scopus (152) Google Scholar) which is thought to hydrolyze the sialyl glycosidic bound through an oxocarbenium ion intermediate, with formation of trace amount of 2-deoxy-2,3-didehydro-N-acetylneuraminic (Neu5Ac2en) as a by-product. The predominance of transglycosylation over hydrolysis can be explained by the presence of distinct binding sites for acceptor and donor substrates in the TS catalytic pocket. Our data support this hypothesis; we demonstrated that inactive TS binds α2,3-sialyllactose and lacto-N-tetraose or methyl-β-lactose in a sequential ordered manner to form a ternary complex. In the same way as we have shown for inactive TS, Buschiazzo et al. (23Buschiazzo A. Amaya M.F. Cremona M.L. Frasch A.C. Alzari P.M. Mol. Cell. 2002; 10: 757-768Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar) demonstrated recently that sialic acid binding induces a conformational modification in the crystal structure of active TS, allowing the acceptor substrate (lactose) to bind. The authors, however, were unable to show the existence of two distinct binding sites when monoclinic crystals were soaked in lactose and Neu5NAc2en, indicating that the lactose-binding site is too narrow to accommodate the lactose moiety of the donor and acceptor substrates simultaneously. Our results, using inactive TS incubated with α2,6-sialyllactose in the presence of lacto-N-tetraose, show that incorrect fitting of sialoside into the binding site of inactive TS does not trigger β-Galp binding. These data suggest that Neu5NAc2en interaction with active TS (23Buschiazzo A. Amaya M.F. Cremona M.L. Frasch A.C. Alzari P.M. Mol. Cell. 2002; 10: 757-768Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar) is not sufficient to induce the required conformational rearrangement to accommodate acceptor and donor substrates simultaneously. Consistent with our hypothesis are the surface plasmon resonance results showing that lactose binds to an inactive mutant of TS (Asp59 → Asn) in the presence of α2,3-sialyllactose (23Buschiazzo A. Amaya M.F. Cremona M.L. Frasch A.C. Alzari P.M. Mol. Cell. 2002; 10: 757-768Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). Although the exact catalytic mechanism of trans-sialylation is still obscure, data obtained in this study may provide some clues. Correct binding of sialic acid donor to TS may trigger a conformational change in the enzyme that creates the conditions for formation of a ternary complex. Acceptor binding would displace the water molecules from TS catalytic cleft before formation of the oxicarbonium ion (11Todeschini A.R. Mendonça-Previato L. Previato J.O. Varki A. Van Halbeek H. Glycobiology. 2000; 10: 213-221Crossref PubMed Scopus (47) Google Scholar, 23Buschiazzo A. Amaya M.F. Cremona M.L. Frasch A.C. Alzari P.M. Mol. Cell. 2002; 10: 757-768Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar) or sialyl-enzyme intermediate (24Watts A. Damager I. Amaya M.L. Buschiazzo A. Alzari P. Frasch A.C. Withers S.G. J. Am. Chem. Soc. 2003; 125: 7532-7533Crossref PubMed Scopus (186) Google Scholar) takes place, facilitating the selective transfer reaction (Fig. 8). Thus, high efficiency of transfer of Neu5Ac from α2–3-sialyllactose over 4-methylumbelliferylsialic acid to lactose by active TS could be explained by this model. Natural sialosides as α2–3-sialyllactose would fit correctly in the TS catalytic pocket, inducing the acceptor donor to bind and increasing transference rates, whereas synthetic donors, as 4-methylumbelliferyl-sialic acid and p-nitrophenyl sialic acid, would not be able to trigger a sufficient shifting in the enzyme framework to allow acceptor binding, being better substrates for hydrolysis reaction (25Harrison J. Kartha K.P.R. Turnbull B.W. Scheuer S.L. Naismith J.H. Schenkman S. Field R.A. Bioorg. Med. Chem. Lett. 2001; 11: 141-144Crossref PubMed Scopus (24) Google Scholar). Taken together, our results suggest that enzymatically inactive molecules of the TS family are sialic acid- and β-Galp-binding proteins that can play an important role during the T. cruzi infection, and that inhibition of TS activity based on sialic acid analogues may transform T. cruzi TS in a β-Galp-binding lectin. Therefore, polyvalent galactosides/sialosides provide intriguing possibilities for the design and synthesis of selective TS inhibitors. We thank Dr. A. C. C. Frasch from Universidad Nacional de General, San Martin, Argentina, for the gift of inactive TS-expressing plasmid. We also thank the Centro Nacional de Ressonância Magnética Nuclear, UFRJ, Brasil, for the NMR facilities.
Cryptococcus neoformans is a fungus that mainly affects the respiratory system and the central nervous system. One of the main virulence factors is the capsule, constituted by the polysaccharides glucuronoxylomannan (GXM) and glucuronoxylomanogalactan (GXMGal). Polysaccharides are immunomodulators. One of the target cell populations for modulation are macrophages, part of the first line of defense, important for innate and adaptive immunity. It has been reported that macrophages can be modulated to act as a "Trojan horse" taking phagocytosed yeasts to strategic sites or having their machinery activation compromised. The scarcity of information on canine cryptococcosis led us to assess whether the purified capsular polysaccharides from C. neoformans would be able to modulate the microbicidal action of macrophages. In the present study, we observed that the capsular polysaccharides, GXM, GXMGal or capsule total did not present toxic effects for the DH82 macrophage cell line. However, it was possible to demonstrate that phagocytic activity was decreased after treatment with polysaccharides. In addition, yeasts recovered from macrophages treated with the polysaccharides, after phagocytosis, could be cultured, showing that their viability was not altered. The polysaccharides led to a reduction in ROS production and the expression of IL-12 and IL-6. We observed that GXMGal inhibits MHC class II expression and GXM reduces ERK phosphorylation. In contrast, GXMGal and GXM were able to increase the PPAR- expression. Furthermore, our data suggests that capsular polysaccharides can reduce the microbicidal activity of canine macrophages DH82.
The effects of capsular polysaccharides, galactoxylomannan (GalXM) and glucuronoxylomannan (GXM), from acapsular (GXM negative) and encapsulate strains of Cryptococcus neoformans were investigated in RAW 264.7 and peritoneal macrophages. Here, we demonstrate that GalXM and GXM induced different cytokines profiles in RAW 264.7 macrophages. GalXM induced production of TNF-α, NO and iNOS expression, while GXM predominantly induced TGF-β secretion. Both GalXM and GXM induced early morphological changes identified as autophagy and late macrophages apoptosis mediated by Fas/FasL interaction, a previously unidentified mechanism of virulence. GalXM was more potent than GXM at induction of Fas/FasL expression and apoptosis on macrophages in vitro and in vivo. These findings uncover a mechanism by which capsular polysaccharides from C. neoformans might compromise host immune responses.
The work reported herein describes the synthesis and the assessment of the trypanocidal activity of thirteen new 1,2,4-triazole-3-thiones obtained from natural piperine, the main constituent of the dry fruits of Piper nigrum. It is part of a research program aiming to use abundant and easily available natural products as starting materials for the design and synthesis of new molecules potentially useful as antiparasitic drugs. The variously substituted triazole derivatives were synthesized from the natural amide in four steps with the use of microwave irradiation on overall yields ranging from 32% to 51%. The cyclohexyl substituted derivative showed the best trypanocidal profile on proliferative forms of Trypanosoma cruzi (Y strain), with IC50s = 18.3 and 8.87 mM against epimastigotes and amastigotes, respectively.
Cancer and parasitic diseases, such as leishmaniasis and Chagas disease, share similarities that allow the co-development of new antiproliferative agents as a strategy to quickly track the discovery of new drugs. This strategy is especially interesting regarding tropical neglected diseases, for which chemotherapeutic alternatives are extremely outdated. We designed a series of (E)-3-aryl-5-(2-aryl-vinyl)-1,2,4-oxadiazoles based on the reported antiparasitic and anticancer activities of structurally related compounds. The synthesis of such compounds led to the development of a new, fast, and efficient strategy for the construction of a 1,2,4-oxadiazole ring on a silica-supported system under microwave irradiation. One hit compound (23) was identified during the in vitro evaluation against drug-sensitive and drug-resistant chronic myeloid leukemia cell lines (EC50 values ranging from 5.5 to 13.2 µM), Trypanosoma cruzi amastigotes (EC50 = 2.9 µM) and Leishmania amazonensis promastigotes (EC50 = 12.2 µM) and amastigotes (EC50 = 13.5 µM). In silico studies indicate a correlation between the in vitro activity and the interaction with tubulin at the colchicine binding site. Furthermore, ADMET in silico predictions indicate that the compounds possess a high druggability potential due to their physicochemical, pharmacokinetic, and toxicity profiles, and for hit 23, it was identified by multiple spectroscopic approaches that this compound binds with human serum albumin (HSA) via a spontaneous ground-state association with a moderate affinity driven by entropically and enthalpically energies into subdomain IIA (site I) without significantly perturbing the secondary content of the protein.
ABSTRACT Systemic sporotrichosis is an emerging infection potentially fatal for immunocompromised patients. Adhesion to extracellular matrix proteins is thought to play a crucial role in invasive fungal diseases. Here we report studies of the adhesion of Sporothrix schenckii to the extracellular protein fibronectin (Fn). Both yeast cells and conidia of S. schenckii were able to adhere to Fn as detected by enzyme-linked immunosorbent binding assays. Adhesion of yeast cells to Fn is dose dependent and saturable. S. schenckii adheres equally well to 40-kDa and 120-kDa Fn proteolytic fragments. While adhesion to Fn was increased by Ca 2+ , inhibition assays demonstrated that it was not RGD dependent. A carbohydrate-containing cell wall neutral fraction blocked up to 30% of the observed adherence for the yeast cells. The biochemical nature of this fraction suggests the participation of cell surface glycoconjugates in binding by their carbohydrate or peptide moieties. These results provide new data concerning S. schenckii adhesion mechanisms, which could be important in host-fungus interactions and the establishment of sporotrichosis.
IPC (inositol phosphorylceramide) synthase is an enzyme essential for fungal viability, and it is the target of potent antifungal compounds such as rustmicin and aureobasidin A. Similar to fungi and some other lower eukaryotes, the protozoan parasite Trypanosoma cruzi is capable of synthesizing free or protein-linked glycoinositolphospholipids containing IPC. As a first step towards understanding the importance and mechanism of IPC synthesis in T. cruzi, we investigated the effects of rustmicin and aureobasidin A on the proliferation of different life-cycle stages of the parasite. The compounds did not interfere with the axenic growth of epimastigotes, but aureobasidin A decreased the release of trypomastigotes from infected murine peritoneal macrophages and the number of intracellular amastigotes in a dose-dependent manner. We have demonstrated for the first time that all forms of T. cruzi express an IPC synthase activity that is capable of transferring inositol phosphate from phosphatidylinositol to the C-1 hydroxy group of C6-NBD-cer {6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-amino]hexanoylceramide} to form inositol phosphoryl-C6-NBD-cer, which was purified and characterized by its chromatographic behaviour on TLC and HPLC, sensitivity to phosphatidylinositol-specific phospholipase C and resistance to mild alkaline hydrolysis. Unlike the Saccharomyces cerevisiae IPC synthase, the T. cruzi enzyme is stimulated by Triton X-100 but not by bivalent cations, CHAPS or fatty-acid-free BSA, and it is not inhibited by rustmicin or aureobasidin A, or the two in combination. Further studies showed that aureobasidin A has effects on macrophages independent of the infecting T. cruzi cells. These results suggest that T. cruzi synthesizes its own IPC, but by a mechanism that is not affected by rustmicin and aureobasidin A.
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