Structure of O-glycosidically linked oligosaccharides from glycoproteins of Trypanosoma cruzi CL-Brener strain: evidence for the presence of O-linked sialyl-oligosaccharides

Adriane R. Todeschini, Erika Xavier da Silveira, Christopher Jones3, Robin Wait1,4, José O. Previato and Lúcia Mendonça-Previato2

Instituto de Microbiologia, CCS-Bloco I, Universidade Federal do Rio de Janeiro, 21944–970-Cidade Universitária, Rio de Janeiro-RJ, Brasil, 3Laboratory for Molecular Structure, NIBSC, Blanche Lane, South Mimms, Herts, EN6 3QG, UK, and 4Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wilts, SP4 0JG, UK

Received on June 6, 2000; revised on July 5, 2000; accepted on July 5, 2000.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Glycoproteins on the cell surface of Trypanosoma cruzi are known to play important roles in the interaction of the parasite with the host cells. We previously determined the structures of the O-glycan chains from the sialoglycoproteins (mucin-like molecules) of the G- and Y-strains and observed significant differences between them. We now report the structures of the sialylated and nonsialylated O-linked oligosaccharides isolated from the cell surface glycoproteins of the myotropic CL-Brener strain grown in the presence of fetal calf serum. The structures of the O-linked oligosaccharide alditols obtained by reductive ß-elimination of the sialoglycoprotein were determined by a combination of methylation analysis, fast atom bombardment–mass spectrometry and nuclear magnetic resonance spectroscopy. The presence of a ß-galactopyranose substituent on the N-acetylglucosamine O-4 position shows that these O-linked oligosaccharides from CL-Brener strain belong to the same family as those isolated from mucins expressed by T. cruzi Y strain, a reticulotropic strain. In addition, novel O-glycans, including {alpha}2–3 mono-sialylated species are described.

Key words: FAB-MS spectrometry/mucin/NMR/sialic acid/Trypanosoma cruzi


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The intracellular parasite protozoan Trypanosoma cruzi, the causative agent of Chagas’ disease, has a dixenic life cycle involving morphologically distinct stages in mammalian and insect hosts (Brener, 1973Go). The taxon T. cruzi includes a wide range of different strains grouped into two major phylogenetic lineages based on rDNA markers (Souto et al., 1996Go).

Invasion of host cells by T. cruzi is a complex process probably involving different components on the parasite and host cell (Burleigh and Andrews, 1995Go, 1998). The T. cruzi cell surface sialoglycoproteins, known as mucin-like molecules, have been implicated in this process (Cross and Takle, 1993Go; Ming et al., 1993Go) and are thought to be involved in protection of the parasite from complement-mediated lysis (Kipnis et al., 1981Go; Tomlinson et al., 1994Go), in parasite escape from endosomes (Andrews, 1994Go), in transition between developmental stages (Sher and Snary, 1982Go), in induction of protective lytic antibodies (Almeida et al., 1994Go), and in the production of proinflammatory cytokines by macrophages during infection (Camargo et al., 1997Go; De Diego et al., 1997Go; Almeida et al., 2000Go).

The sialic acid present in these mucin-like molecules is derived from host sialyl-glycoconjugates (Previato et al., 1990aGo) and is transferred to terminal ß-Galp residues on T. cruzi surface glycoproteins by a transglycosylation reaction for sialic acid (Previato et al., 1985Go). The sialic acid acceptors are highly O-glycosylated GPI-anchored glycoproteins encoded by the diverse MUC gene family (DiNoia et al., 1996Go). The main sialic acid acceptors in epimastigote and trypomastigote metacyclic forms are mucin-type molecules in the 35–50 kDa range (Schenkman et al., 1993Go; Previato et al., 1985Go, 1994, 1995). Although only the metacyclic is the infective form, the O-linked glycans on the mucins from both developmental stages are identical (Previato et al., 1994Go; Serrano et al., 1995Go). In cell-derived trypomastigotes the sialic acid is transferred to glycoproteins with molecular mass ranging from 60 to 200 kDa (Schenkman et al., 1991Go) which carry the Ssp-3 epitope (Andrews et al., 1987Go), which were subsequently called F2/3 glycoproteins (Almeida et al., 1994Go).

The complete structures of the sialic acid acceptor O-linked oligosaccharide have been reported for epimastigotes of G (Previato et al., 1994Go) and Y (Previato et al., 1995Go) strains, and metacyclic forms of G-strain (Serrano et al., 1995Go). The oligosaccharides are O-glycosidically linked to threonine residues via an {alpha}-GlcNAc (Previato et al., 1998Go) and contain ß-Galp and ß-Galf substituents. These studies identified differences between the two strains. Only G strain expresses oligosaccharides containing a ß-Galf residue; and whilst the largest glycan isolated from Y-strain was a trisaccharide alditol, a pentasaccharide alditol was characterized from G-strain.

The structures of the O-oligosaccharide chains from mucins of different T. cruzi strains and developmental stages may reflect differences in infectivity and tissue tropism or different growth conditions, and their characterization may permit the identification of host cell receptors for T. cruzi molecules.

We now report the structure of the O-glycans from mucin-like molecules of a myotropic strain of T. cruzi, the CL-Brener strain (Melo and Brener, 1978Go), a clone of which has also been chosen for complete genetic sequencing in the Trypanosoma genome project (Verdun et al., 1998Go). These glycans are similar to those from the Y-strain, containing a ß-Galp substituent on the GlcNAc O-4, and lacking Galpß1–3 substituents on the 6-arm. Additional structures, including novel sialylated O-linked oligosaccharides, are described.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Isolation and chemical composition of sialoglycoproteins (mucins) from T. cruzi CL-Brener
T. cruzi CL-Brener cells were extracted with hot aqueous phenol, and a chloroform/methanol/water–insoluble carbohydrate–containing fraction recovered from the aqueous phase (Previato et al., 1985Go). When analyzed by SDS–PAGE, this gave a broad band at 38/43 kDa which strongly stained with periodate Schiff’s reagent. Carbohydrate analysis revealed Gal, Man, GlcNAc, Glc, Neu5Ac in an approximately molar ratio of 14:7:6:1:1.

Characterization of ß-eliminated reduced oligosaccharides
Oligosaccharide alditols released from the mucins by ß-elimination in the presence of sodium borohydride were fractionated by gel-filtration chromatography on Bio-Gel P-4. Five included saccharide fractions were obtained (referred to as fractions A to E in order of increasing molecular mass). The presence of mono-, di-, tri-, and tetra-saccharide alditols in Fractions A to D was demonstrated by FAB-MS of the peracetylated material, with protonated molecules (M+H)+ observed at m/z 722, 1010, 1298, and 1586. The presence of a terminal hexosaminitol (HexNAc-ol) was suggested by the observation of Y1 and Z1 fragments at m/z 392 in the spectrum of the monosaccharide alditol (Fraction A). The spectrum of the peracetylated tetrasaccharide alditol (Fraction D; Figure 1) contained abundant signals at m/z 331, 619, and 907, which were assigned as nonreducing terminal containing fragments (B-type carbenium ions using the nomenclature of Domon and Costello (1988)Go). The absence of a fragment ion at m/z 1195 (B4) suggests that this compound is branched at the HexNAc-ol residue, with the longer branch containing three hexoses. Similarly, the spectrum of the trisaccharide alditol (Fraction C) contained fragment ions at m/z 331(B1) and 619 (B2) but not m/z 907 (B3), again indicating a branched structure. The absence of the signals at m/z 392 and 374 (Y1 and Z1 from all fractions except A) is likewise consistent with branching at HexNAc-ol.



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Fig. 1. FAB-MS spectrum of the peracetylated tetrasaccharide-alditol (Fraction D) from mucins purified from T.cruzi CL-Brener strain.

 
NMR spectroscopy and HPLC analysis of the oligosaccharide alditols
Fraction A.
Together, the one-dimensional (1D) 500 MHz proton NMR spectrum and PGC-HPLC of Fraction A showed the presence of major and a minor monosaccharide alditol components (in an approximate ratio of 15:1), small amounts of GlcNAc-ol and traces of the disaccharide alditol which was the major component of Fraction B. The major and minor monosaccharide alditols were identified as Galpß1–4GlcNAc-ol and Galpß1–4ManNAc-ol respectively by comparison of the 1D spectrum with those of authentic reference compounds (Jones et al., 2000Go). The methylation analysis, showing the presence of terminal Galp, 4-O-substituted GlcNAc-ol and traces of 4-O-substituted ManNAc-ol, confirmed these structures. We showed previously that oligosaccharides containing ManNAc-ol arise by epimerization of GlcNAc residue during the reductive ß-elimination reaction (Jones et al., 2000Go).

Fraction B. HPLC analysis of Fraction B on PGC showed the presence of two disaccharide alditols in a ratio of 9:1, alongside traces of GlcNAc-ol and Galpß1–4GlcNAc-ol, the major component of Fraction A. The NMR spectrum of Fraction B, partially assigned from the 80 ms TOCSY spectrum (Table I), indicated that the major component contained two terminal ß-Galp residues. The 4,6-disubstitution of the GlcNAc-ol residue was apparent from the characteristic lowfield H-2 resonance at 4.33 p.p.m. and a double doublet at 4.20 p.p.m. arising from one of the GlcNAc-ol H-6s (Previato et al., 1994Go; Jones et al., 2000Go). The methylation analysis showed the presence of terminal Galp, and 4,6-di-O-substituted-GlcNAc-ol and ManNAc-ol residues. The major component is therefore Galpß1–4[Galpß1–6]GlcNAc-ol. This oligosaccharide was observed in our study of the O-glycans from the Y-strain (Previato et al., 1995Go). By analogy with Fraction A and the resolved reporter groups (Jones et al., 2000Go), the minor disaccharide is assigned as Galpß1–4[Galpß1–6]ManNAc-ol.


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Table I. Proton NMR assignments for nonsialylated and sialylated oligosaccharide alditols isolated from mucins of T. cruzi CL-Brener straina
 
Fraction C. PGC-HPLC analysis of this fraction showed the presence of one major and five minor components (5–10% of the major peak). The mass spectrometric and 1D 500 MHz 1H NMR data indicated that, compared to Fraction B, the major component contained an additional ß-Galp residue. Methylation analysis of Fraction C showed, in addition to methyl derivatives found from Fraction B, the presence of derivatives arising from an 2-O-substituted Galp residue. The anomeric proton resonance of the additional ß-Galp residue is at relatively lowfield (4.738 p.p.m.), whilst the anomeric proton resonance of the Galpß1–6 has been shifted downfield from 4.446 to 4.582 p.p.m. (Table I), consistent with 2-substitution of the Galpß1–6 residue. Thus, the structure of the major component Fraction C was assigned as Galpß1–2Galpß1–6[Galpß1–4]GlcNAc-ol. The presence of this compound was tentatively assigned in the mixture of oligosaccharide alditols from the Y-strain mucin (Previato et al., 1995Go).

Fraction D. The 500 MHz 1D 1H NMR spectrum of Fraction D showed a single major component, and four ß-Galp anomeric proton resonances were visible. The TOCSY spectrum (Figure 2) was assigned (Table I) and indicated the presence of two terminal ß-Galp spin systems and two 2-substituted ß-Galp spin systems. No lowfield Galp H-3 or H-4 resonances, typical of a Galp ß1–3Galp ß1- linkage were observed, in contrast to the tri-, tetra- and penta-galactosylated oligosaccharide alditols isolated from T. cruzi G-strain (Previato et al., 1994Go). Combining the NMR and mass spectrometric data indicate that the structure of this tetrasaccharide alditol is Galpß1–2Galpß1–2Galpß1–6[Galpß1–4]GlcNAc-ol, which is a novel O-glycan. Fraction E. Compositional and NMR analyses of Fraction E demonstrated the presence of Neu5Ac in addition to GlcNAc-ol and Gal. PGC-HPLC analysis showed the presence of a major and two minor components (Figure 3), the minor components being present in approximately equal amounts and eluting before the major component. Another early-eluting peak observed by PGC-HPLC was attributed to Galpß1–4GlcNAc-ol. The PGC-HPLC retention time of the major component and comparison of the 1D and 2D (TOCSY and DQFCOSY) NMR spectra with those of authentic material demonstrated that it was 3'-sialyl-lactosaminitol. The presence of a signal at m/z 1079 (M+H-60) in the FAB mass spectrum of the peracetylated fraction is consistent with this assignment. Minor spin systems in the TOCSY spectrum (ca. 30%) of the Fraction E (Figure 4) arose from two terminal Galp residues (corresponding closely in chemical shifts to the Galpß1–6 in Fraction B and the Galpß1–4 in Fraction C) and two {alpha}2–3-sialylated ß-Galp residues, identified by the lowfield chemical shift of the Galp H-3s at approximately 4.12 p.p.m. (Vliegenthart et al., 1983Go) (Figure 4). There was no evidence for Neu5Ac{alpha}2–6Galpß1- substructures (Vliegenthart et al., 1983Go), and hence the two minor components were assigned as the two possible mono-{alpha}2–3 sialylated, digalactosylated species, Neu5Ac{alpha}2–3Galpß1–4[Galpß1–6]GlcNAc-ol and Neu5Ac{alpha}2–3Galpß1–6[Galpß1–4]GlcNAc-ol. A signal at m/z 1367 (M+H-60) the FAB spectrum of the peracetylated sample supports this assignment. Other trace components were not characterized, but are probably attributable to oligosaccharides containing one and two additional galactose residues, since presumptive [M+H]-60 ions were observed in the FAB spectrum at m/z 1655 and 1943.



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Fig. 2. Partial 500 MHz 80 ms TOCSY spectrum of the tetrasaccharide-alditol (Fraction D) obtained at 30°C. The four ß-Galp and GlcNAc-ol spin systems are assigned as according to the structure Galpß1–4[Galpß1–2Galpß1–2Galpß1–6]GlcNAc-ol.

 


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Fig. 3. Analytical HPLC separation of sialylated oligosaccharide alditols (Fraction E) on PGC, eluted with an aqueous acetonitrile gradient and monitored by UV at 206 nm. Peaks are identified as follows: (1) lactosaminitol, (2) unidentified, (3) and (4) monosialylated digalactosylated alditols, and (5) 3'-sialyllactosaminitol.

 


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Fig. 4. Partial 500 MHz 1D 1H and 80 ms TOCSY NMR spectra of the sialylated oligosaccharide alditols (Fraction E). The 1D spectrum shows the region containing the anomeric proton resonances from the ß-Galp residues, and the TOCSY spectrum crosspeaks correlating to these anomeric resonances: the H-2, H-3 and H-4. For one trace the crosspeaks are labeled (2 = H-1/H-2 crosspeak, etc.: the shapes of the crosspeaks are characteristic). Spin systems from {alpha}2–3-sialylated Galp residues are characterized by the lowfield H-3 resonance, at ca. 4.1 p.p.m.: other spin systems arise from terminal residues. Galp(1) arises from 3'-sialyllactosaminitol, Galp(2) and Galp(4) from Galpß1- [Neu5Ac{alpha}2–3Galpß1–6]GlcNAc-ol, Galp(3) and Galp(6) from Neu5Ac{alpha}2–3 Galpß1–4[Galpß1–4]GlcNAc-ol, and Galp(5) arises from lactosaminitol.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
In this study we characterized O-linked glycans present on the mucins expressed on the cell surface of epimastigote forms of T. cruzi CL-Brener strain (Table BII). The oligosaccharides are linked to threonine through an {alpha}-GlcNAc residue (Previato et al., 1998Go). The {alpha}-GlcNAc can be substituted initially on the O-4 and then on the O-6 position by ß-Galp residues. This Galpß1–4[Galpß1–6]GlcNAc structure can be further elaborated by addition of one or two Galpß1->2 residues on the 6-arm. Monosialylation of the mono- and di-galactosylated species has been characterized.



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Table II. Proposed structures of nonsialylated and sialylated O-linked oligosaccharides isolated by reductive ß-elimination from mucins of T. cruzi CL-Brener strain

aManNAc-ol is a product of epimerization of GlcNAc during reductive ß-elimination (Jones et al., 2000Go).

1,2,3Minor structures present in Fraction A, B, and E, respectively.

 
We previously reported the structures of O-linked oligosaccharides in mucins from epimastigote forms of T.cruzi G (Previato et al., 1994Go), and Y (Previato et al., 1995Go). The O-linked oligosaccharides from CL-Brener strain are structurally similar to those found in Y strain. Both contain a ß-Galp on GlcNAc O-4, rather than the ß-Galf residue found in G-strain. Also, while the G strain modifies the Galpß1->6 arm by sequential galactopyranosylation at O-3 and O-2 to form branched structures (Galpß1–2[Galpß1–3]Galpß1–6...), in the CL-Brener strain, extension of the O-6 arm occurs by repetitive galactosylation at O-2 of the terminal ß-Galp to form a linear chain (Galpß1–2Galpß1–2Galpß1–6....). The presence of Galpß1–2 but not Galp ß1–3 residues was also observed in the Y-strain. Recently, NMR assignments of a range of model systems of glucosylated and galactosylated N-acetylhexosaminitols (Jones et al., 2000Go) shows that our previous conclusion (Previato et al., 1995Go) that the Y strain mucins express two families of O-glycans, containing either Galpß1–4GlcNAc-ol or Galpß1–3GlcNAc-ol substructure, was erroneous. The second family contains Galpß1–4ManNAc-ol, derived by epimerization of the GlcNAc during the reductive ß-elimination reaction (Jones et al., 2000Go).

Although sialic acid residues in mucins of T. cruzi have been implicated in key process of parasite host cells interaction and a series of O-glycan has been identified on the cell surface of infective trypomastigote forms (Almeida et al., 1994Go; Serrano et al., 1995Go), this is the first report of the isolation and structural characterization of sialylated O-glycans. While Neu5Ac{alpha}2–3Galpß1–4GlcNAc-ol was the major component isolated, both possible monosialylated digalactosylated oligosaccharides were also present (Table BII). The expression of Neu5Ac{alpha}2–3Galp1-4[Galpß1–6]GlcNAc-ol and Galpß1–4[Neu5Ac{alpha}2–3Galp1-6]GlcNAc-ol in approximately equal amounts suggests that the trans-sialidase of T. cruzi (Previato et al., 1985Go; Schenkman et al., 1991Go) has little selectivity for the site of sialic acid addition, and the absence of disialylated species supports our previous conclusion from in vitro studies (Previato et al., 1995Go) that the incorporation of the first sialic acid residue inhibits addition of a second residue on the same oligosaccharide.

The structural variation we found in O-linked oligosaccharide of mucin-type molecules between different T. cruzi strains could be related to the two phylogenetic lineages proposed by Souto et al. (1996)Go. However, there is no correlation between phylogenetic type and biological behavior, such as infectivity. Although Y and CL strains belong to the same lineage 1 (domestic cycle) the Y strain is 20 to 30-fold more infective than CL strain (Alcantara and Brener, 1978Go). The G strain, isolated from opossum (Yoshida , 1983Go) is associated with lineage 2 (sylvatic cycle). Our finding that both myotropic (CL-Brener) and reticulotropic (Y) T. cruzi strains express the same major O-linked oligosaccharides is also of biological significance, suggesting that differences in mucin-derived oligosaccharide chains are not responsible for differential tissue tropism.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Preparation of sialogycoproteins from T. cruzi CL-Brener strain
T. cruzi CL-Brener was grown in 200 ml of brain heart infusion (BHI) medium containing 10% fetal calf serum (FCS), and supplemented with 10 mg l–1 hemin and 20 mg l–1 folic acid. Cultures were incubated at 28°C with shaking (100 r.p.m.) for 5–7 days. This culture was used to inoculate 3 l flask containing 1 l of BHI/hemin medium containing 5% of FCS and cultured as above. Cells were harvested by centrifugation, washed three times with 0.9% NaCl, and frozen at –20°C. Frozen cells were thawed and then extracted with cold water, and the pellet was recovered by centrifugation. This extraction was repeated three times. The pellet was extracted with 45% (v/v) aqueous phenol at 75°C. The aqueous phase of the phenol extract was dialyzed, lyophilized, redissolved in water, and applied to a column of Bio-Gel P-100 (2 x 60 cm). Carbohydrate-containing material in the excluded volume was lyophilized and suspend in chloroform/methanol/water (10:10:3 v/v). The insoluble glycoproteins in this solvent mixture were analyzed by SDS–polyacrylamide gel electrophoresis as described by Laemmli (1970)Go.

Reductive ß-elimination of the sialogycoproteins
The sialoglycoproteins were treated with 0.01 M NaOH in the presence of 0.3 M NaBH4 at 37°C for 48 h. The solution containing the ß-eliminated oligosaccharide-alditols was neutralized and passed through Dowex 50W-X8 H+ form and lyophilized. Boric acid was removed by repeated additions of methanol and evaporation to dryness. The residue was dissolved H2O and fractionated on a Bio-Gel P-4 column (1 x 100 cm). Fractions of 1.3 ml were collected and elution monitored by spotting 5 µl portions onto a thin layer chromatography (TLC) plate and staining with orcinol-H2SO4 reagent (Humbel and Collaert, 1975Go). The purity of the oligosaccharide-alditols was determined by HPTLC using methanol:acetone:water (6:5:1 v/v) as mobile phase, and visualized with orcinol-H2SO4 as above.

Carbohydrate analysis
Intact sialoglycoproteins and purified oligosaccharide-alditols were methanolized with 0.5 M HCl in methanol for 18 h at 80°C, neutralized with silver carbonate and re-N-acetylated with acetic anhydride. The dried residue was trimethylsilylated by addition of bis(trimethylsilyl)-trifluoro-acetamide/pyridine (1:1 v/v) (Sweeley et al., 1963Go). The products were analyzed by gas-liquid chromatography (GC) on a DB-1 fused silica column (30 m x 0.25 mm i.d.) using hydrogen as the carrier gas. The column temperature was programmed from 120°C to 240°C at 2°C min–1.

Methylation analysis
Permethylation of oligosaccharide-alditols was performed by the method of Ciucanu and Kerek (1984)Go, modified by Previato et al. (1990b)Go. Permethylated samples were methanolyzed (0.5 M HCl in methanol, l8 h, 80°C), the products dried under a stream of nitrogen and acetylated with acetic anhydride/pyridine (9:1) for 24 h at 25°C, and analyzed by GC on a DB-1 fused silica column, as above. The O-acetylated, partially O-methylated methyl glycosides were identified by their retention time, GC-MS (Fournet et al., 1981Go) and quantified by peak area.

HPLC fractionation
The Bio Gel P-4 fractions were analyzed by HPLC on a porous graphitized carbon (PGC) column (4.6 x 100 mm; Life Sciences International, Basingstoke, UK) as previously described (Jones et al., 2000Go), using the gradient described by Davies et al. (1992)Go. Separations were monitored at 206 nm with typically 50 µg of carbohydrate loaded.

Reference compounds
The preparation and full NMR assignments of the authentic glucosylated and galactosylated N-acetylhexosaminitols, and the identification of structural reporter groups are described by Jones et al. (2000)Go. 3'-Sialyl-lactosaminitol was prepared by reduction of 3'-sialyl lactose (Dextra Laboratories, Reading, UK) with sodium borohydride, the reaction was neutralized and desalted, and the required product purified by HPLC on a PGC column.

Nuclear magnetic resonance spectroscopy (NMR)
NMR spectra were obtained on a Varian Unity 500 NMR spectrometer equipped with a 5 mm proton-detection triple resonance probe, at an indicated probe temperature of 30°C, as previously described (Previato et al., 1994Go). Samples for NMR spectroscopy were deuterium exchanged by repeated lyophilization from D2O and dissolved in D2O before analysis. Chemical shifts ({delta}) are expressed as p.p.m. downfield from external TSP-d4 at zero p.p.m.. Proton NMR spectra were assigned through a combination of DQF-COSY and 80 ms TOCSY spectra, with some additional assignments and information on the sequence and linkage of the sugar residues derived from ROESY spectra with l50 ms mixing times.

Fast atom bombardment (FAB)-MS
Samples were peracetylated by treatment with a mixture 1:1 of trifluoroacetic anhydride and acetic acid for 10 min at room temperature. The reagents were removed in a vacuum centrifuge, and the acetylated oligosaccharides were dissolved in 1 ml of chloroform and desalted by washing three times with an equal volume of distilled water. The chloroform was evaporated to dryness and the residue dissolved in methanol before FAB-MS analysis.

Positive-ion FAB mass spectra were recorded using a Kratos MS80RFA, equipped with an Ion Tech FAB gun using xenon atoms as the bombarding particles. Peracetylated samples were dissolved in a 1:1 mixture of glycerol and dithiothreitol/dithioerythritol (5:1, v/v) liquid matrix. The magnet was scanned at 10 s per decade, and about 10 scans were acquired and averaged using Mach-3 software (Kratos Analytical, Manchester, UK) to obtain the centroided spectra.


    Acknowledgments
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank L.J. Nascimento and O.A. Agrellos Filho for technical assistance. This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-Pronex, Padct); Financiadora de Estudos e Projetos (Finep); Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (Faperj); Fundação Universitária José Bonifácio (Fujb). We are grateful for support from the Sir Halley Stewart Trust. L.M.-P. is a Howard Hughes International Scholar.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
B, magnetic; E, electric; FAB, fast atom bombardment; Gal, galactose; Galf, galactofuranose; Galp, galactopyranose; Glc, glucose; GlcNAc, N-acetylglucosamine; HexNAc-ol, N-acetylhexosaminitol; HPLC, high performance liquid chromatography; N-acetylhexosaminitol; Man, mannose; m.s., mass spectra; Neu5Ac, N-acetylneuraminic acid; NMR, nuclear magnetic resonance; PGC, porous graphitized carbon; TOCSY, total correlation spectroscopy.


    Footnotes
 
1 Present address: Kennedy Institute for Rheumatology, 1 Aspenlea Road, London W6 8LH, UK Back

2 To whom correspondence should be addressed 3This paper is dedicated to the memory of Prof. Andre Verbert, our friend. Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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Brener, Z. (1973) Biology of Trypanosoma cruzi. Annu. Rev. Microbiol., 27, 347–382.[ISI][Medline]

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