Instituto de Microbiologia, CCS-Bloco I, Universidade Federal do Rio de Janeiro, 21944970-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.
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Abstract |
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Key words: FAB-MS spectrometry/mucin/NMR/sialic acid/Trypanosoma cruzi
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Introduction |
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Invasion of host cells by T. cruzi is a complex process probably involving different components on the parasite and host cell (Burleigh and Andrews, 1995, 1998). The T. cruzi cell surface sialoglycoproteins, known as mucin-like molecules, have been implicated in this process (Cross and Takle, 1993
; Ming et al., 1993
) and are thought to be involved in protection of the parasite from complement-mediated lysis (Kipnis et al., 1981
; Tomlinson et al., 1994
), in parasite escape from endosomes (Andrews, 1994
), in transition between developmental stages (Sher and Snary, 1982
), in induction of protective lytic antibodies (Almeida et al., 1994
), and in the production of proinflammatory cytokines by macrophages during infection (Camargo et al., 1997
; De Diego et al., 1997
; Almeida et al., 2000
).
The sialic acid present in these mucin-like molecules is derived from host sialyl-glycoconjugates (Previato et al., 1990a) and is transferred to terminal ß-Galp residues on T. cruzi surface glycoproteins by a transglycosylation reaction for sialic acid (Previato et al., 1985
). The sialic acid acceptors are highly O-glycosylated GPI-anchored glycoproteins encoded by the diverse MUC gene family (DiNoia et al., 1996
). The main sialic acid acceptors in epimastigote and trypomastigote metacyclic forms are mucin-type molecules in the 3550 kDa range (Schenkman et al., 1993
; Previato et al., 1985
, 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., 1994
; Serrano et al., 1995
). In cell-derived trypomastigotes the sialic acid is transferred to glycoproteins with molecular mass ranging from 60 to 200 kDa (Schenkman et al., 1991
) which carry the Ssp-3 epitope (Andrews et al., 1987
), which were subsequently called F2/3 glycoproteins (Almeida et al., 1994
).
The complete structures of the sialic acid acceptor O-linked oligosaccharide have been reported for epimastigotes of G (Previato et al., 1994) and Y (Previato et al., 1995
) strains, and metacyclic forms of G-strain (Serrano et al., 1995
). The oligosaccharides are O-glycosidically linked to threonine residues via an
-GlcNAc (Previato et al., 1998
) 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, 1978), a clone of which has also been chosen for complete genetic sequencing in the Trypanosoma genome project (Verdun et al., 1998
). These glycans are similar to those from the Y-strain, containing a ß-Galp substituent on the GlcNAc O-4, and lacking Galpß13 substituents on the 6-arm. Additional structures, including novel sialylated O-linked oligosaccharides, are described.
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Results |
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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)). 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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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 Galpß14GlcNAc-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., 1994; Jones et al., 2000
). 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 Galpß14[Galpß16]GlcNAc-ol. This oligosaccharide was observed in our study of the O-glycans from the Y-strain (Previato et al., 1995
). By analogy with Fraction A and the resolved reporter groups (Jones et al., 2000
), the minor disaccharide is assigned as Galpß14[Galpß16]ManNAc-ol.
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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 ß13Galp ß1- linkage were observed, in contrast to the tri-, tetra- and penta-galactosylated oligosaccharide alditols isolated from T. cruzi G-strain (Previato et al., 1994). Combining the NMR and mass spectrometric data indicate that the structure of this tetrasaccharide alditol is Galpß12Galpß12Galpß16[Galpß14]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 Galpß14GlcNAc-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 Galpß16 in Fraction B and the Galpß14 in Fraction C) and two
23-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., 1983
) (Figure 4). There was no evidence for Neu5Ac
26Galpß1- substructures (Vliegenthart et al., 1983
), and hence the two minor components were assigned as the two possible mono-
23 sialylated, digalactosylated species, Neu5Ac
23Galpß14[Galpß16]GlcNAc-ol and Neu5Ac
23Galpß16[Galpß14]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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Discussion |
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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., 1994; Serrano et al., 1995
), this is the first report of the isolation and structural characterization of sialylated O-glycans. While Neu5Ac
23Galpß14GlcNAc-ol was the major component isolated, both possible monosialylated digalactosylated oligosaccharides were also present (Table BII). The expression of Neu5Ac
23Galp1-4[Galpß16]GlcNAc-ol and Galpß14[Neu5Ac
23Galp1-6]GlcNAc-ol in approximately equal amounts suggests that the trans-sialidase of T. cruzi (Previato et al., 1985
; Schenkman et al., 1991
) 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., 1995
) 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). 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, 1978
). The G strain, isolated from opossum (Yoshida , 1983
) 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.
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Materials and methods |
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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, 1975). 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., 1963). 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 min1.
Methylation analysis
Permethylation of oligosaccharide-alditols was performed by the method of Ciucanu and Kerek (1984), modified by Previato et al. (1990b)
. 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., 1981
) 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., 2000), using the gradient described by Davies et al. (1992)
. 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). 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., 1994). Samples for NMR spectroscopy were deuterium exchanged by repeated lyophilization from D2O and dissolved in D2O before analysis. Chemical shifts (
) 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.
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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 3This paper is dedicated to the memory of Prof. Andre Verbert, our friend.
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References |
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