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Volume 270, Number 13, Issue of March 31, 1995 pp. 7241-7250
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structural Characterization of the Major Glycosylphosphatidylinositol Membrane-anchored Glycoprotein from Epimastigote Forms of Trypanosoma cruzi Y-strain (*)

(Received for publication, November 23, 1994; and in revised form, January 9, 1995)

José O. Previato (1),  Christopher Jones (2),  Marcia T. Xavier (1),  Robin Wait (3),  Luiz R. Travassos (4),  Armando J. Parodi (5),  Lucia Mendonça-Previato (1)(§)

From the  (1)Departamento de Microbiologia Geral, Universidade Federal do Rio de Janeiro, 21944970, Brazil, Cidade Universitária, CCS-Bloco I, Rio de Janeiro, RJ, Brazil, the (2)Laboratory of Molecular Structure, NIBSC, Potters Bar, Herts EN6 3QG, United Kingdom, (3)Center for Applied Microbiology and Research, Porton Down, Salisbury SP4 OJG, United Kingdom, the (4)Disciplina de Biologia Celular, Escola Paulista de Medicina, 04023-062, São Paulo, SP, Brazil, and the (5)Instituto de Investigaciones Bioquímica Fundación Campomar, Buenos Aires, Argentina

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have investigated the structure of the glycosylphosphatidylinositol (GPI) anchor and the O-linked glycan chains of the 40/45-kDa glycoprotein from the cell surface of the protozoan parasite Trypanosoma cruzi. This glycoconjugate is the major acceptor for sialic acid transferred by trans-sialidase of T. cruzi Y-strain, epimastigote form. The GPI anchor was liberated by treatment with hot alkali, and the phosphoinositol-oligosaccharide moiety was characterized and shown to have the following structure.

Usually the glucosamine was 6-O-substituted with 2-aminoethylphosphonate, and 2-aminoethylphosphonate was also present on the third mannose residue distal to glucosamine, partially replacing the ethanolamine phosphate. The beta-eliminated reduced oligosaccharide chains showed that two novel classes of O-linked N-acetylglucosamine oligosaccharide were present. The first series had the structures Galpbeta1-3GlcNAc-ol; Galpbeta1-6(Galpbeta1-3)GlcNAc-ol; and Galpbeta1-2Galpbeta1-6(Galpbeta1-3)GlcNAc-ol, whereas the other series had a 1-4 linkage to N-acetylglucosaminitol and had structures Galpbeta1-4GlcNAc-ol, Galpbeta1-6(Galpbeta1-4)GlcNAc-ol, and Galpbeta1-2Galpbeta1-6(Galpbeta1-4)GlcNAc-ol. We have also investigated the kinetics of in vitro sialylation of these O-linked oligosaccharides by the T. cruzi trans-sialidase and have shown that incorporation of one molecule of sialic acid hinders entry of a second molecule when two potential acceptor sites are present.

INTRODUCTION

The flagellate protozoan Trypanosoma cruzi, the causative agent of Chagas' disease in man, infects several million people throughout the Americas. Characterization of cell surface glycoconjugates in the various developmental stages of the parasite (1) is important because these compounds mediate a number of biological processes related to parasitism and to the pathogenesis of this infection. Glycoproteins have been implicated in the invasion of host cells(2, 3) , parasite escape from endosomes into the cytoplasm of infected cells(4) , morphological transitions(5) , and induction of protective lytic antibodies(6) . In a previous paper we demonstrated sialylation of galactose residues in cell surface macromolecules of T. cruzi by a novel trans-sialidase reaction, rather than by the more usual CMP-sialic acid-dependent sialyltransferase(7) . The biological significance of the trans-sialidase reaction emerged when it was shown that sialylation of T. cruzi cell surface components was necessary for invasion of host cells(3, 8) .

In trypomastigote forms of T. cruzi the sialic acid residues are transferred predominantly to O-linked oligosaccharide chains of 60-200-kDa GPI(^1)-anchored glycoproteins, collectively designated the F2/3 complex(6) . These glycoproteins are equivalent to the highly glycosylated proteins carrying the Ssp-3 epitope which is recognized by monoclonal antibody 3C9(3) . In epimastigotes (9) and in metacyclic trypomastigotes (10) derived from axenic cultures, which do not express the Ssp-3 epitope, sialic acid residues are transferred to O-linked carbohydrate chains of GPI-anchored glycoproteins which migrate on polyacrylamide gel electrophoresis as a broad band of apparent molecular masses in the 35-50-kDa range. Although there is considerable evidence for the participation of sialylated epitopes in the attachment and invasion of mammalian cells by the trypomastigote forms of T. cruzi, until recently the structures of the sialic acid acceptors were unknown. We recently showed (11) that the sialic acid acceptors of epimastigote forms of T. cruzi G-strain were oligosaccharides glycosidically linked to threonine and/or serine residues mainly via N-acetylglucosamine rather than the more usual N-acetylgalactosamine.

In the present paper we extend our analyses to O-linked oligosaccharides isolated from the major GPI membraneanchored glycoprotein in epimastigote forms of the Y-strain of T. cruzi (the 40/45-kDa glycoprotein), which differ from the oligosaccharides of strain G. We have also characterized the GPI anchor of this glycoprotein, which differs from previously characterized anchors in that it contains glucosamine substituted with 2-aminoethylphosphonate. Also unusual is the fact that in a proportion of molecules the third sugar unit distal to glucosamine, in the tetramannose chain, is substituted by aminoethylphosphonate rather than by the more typical ethanolamine phosphate.

EXPERIMENTAL PROCEDURES

Materials

[^14C]Acetic anhydride (50 Ci/mol) was purchased from American Radiolabeled Chemicals (St. Louis, MO). Sialyllactose, Clostridium perfringens neuraminidase, sugars, fatty acids, glycerol, and thioglycerol were from Sigma. trans-Sialidase, isolated from T. cruzi trypomastigotes(12) , was a gift of Dr. A. Frasch, Instituto de Investigaciones Bioquímicas, Fundacion Campomar, Buenos Aires. Deuterium oxide (99.9%) was obtained from Goss Scientific (Ingatestone, United Kingdom).

Extraction and Purification of 40/45-kDa Glycoprotein

Approximately 10 cells of T. cruzi, Y-strain (epimastigote forms) were grown in brain heart infusion medium (without fetal calf serum) supplemented with 10 mg/liter hemin, (BHI-hemin medium) as described previously(11) . Frozen cells were thawed, extracted with cold water, and the pellet recovered by centrifugation. This was repeated three times. The residue remaining after the final centrifugation was extracted with 45% (v/v) aqueous phenol at 75 °C. The aqueous phase was dialyzed, freeze-dried, dissolved in water, and applied to a 2 times 100-cm column of Bio-Gel P-100 (100-200 mesh). The carbohydrate fraction obtained in the excluded volume was lyophilized, extracted several times with chloroform/methanol/water (10:10:3, v/v/v) and then boiled in 80% aqueous ethanol. The resulting insoluble product was dissolved in 10 mM sodium phosphate buffer (pH 7.2) containing 0.9% NaCl and applied to a concanavalin A-Sepharose 4B column (1.5 times 30 cm) equilibrated with the same buffer. The bound material was eluted with 0.1 M methyl-alpha-D-mannopyranoside and repurified by gel filtration chromatography on a Bio-Gel P-6 DG column (2.5 times 100 cm). The resulting 40/45-kDa glycoprotein was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, according to Laemmli(13) , using a 15% acrylamide gel, and the bands were visualized by staining with periodate-Schiff reagent(14) .

Isolation of Phosphoinositol Oligosaccharide (PI-oligosaccharide) from GPI Membrane Anchor

To isolate the PI-oligosaccharides, the 40/45-kDa glycoprotein (80 mg) was subjected to alkaline degradation in 8 ml of 1 M NaOH for 6 h at 100 °C. The pH was adjusted to 7.0 with 50% acetic acid, and the solution was passed through Dowex 50W-X8 (25-50 mesh H form), and the eluate was lyophilized. The residue was dissolved in 0.05 M acetic acid and purified on a Bio-Gel P-4 (extra fine) column (1 times 120 cm). Fractions of 1.5 ml were collected and assayed by spotting 10-µl portions onto a thin layer chromatography (TLC) plate and staining with orcinol-H(2)SO(4)(15) , ninhydrin(16) , and molybdate (17) reagents for the detection of respectively carbohydrate, nitrogen, and phosphorus.

Reductive beta-Elimination of O-Linked Oligosaccharides

Alkali-catalyzed beta-elimination of serine- and threonine-linked carbohydrate was performed on 40/45-kDa glycoprotein (40 mg) in 0.01 M NaOH in the presence of 0.3 M NaBH(4) (4 ml) at 37 °C for 24 h. The solution was neutralized with 2 M acetic acid and passed through Dowex 50W-X8 (25-50 mesh H form) and lyophilized. Boric acid was removed by repeated additions of methanol and evaporation to dryness. The residue was dissolved in 0.05 M acetic acid and fractionated on a Bio-Gel P-4 (extra fine) column (1 times 150 cm). Fractions of 1.0 ml were collected and monitored by spotting 5-µl portions onto a TLC plate and staining with orcinol-H(2)SO(4) reagent (15) and H(2)SO(4) spray(18) .

Carbohydrate Analysis

For analysis of neutral sugars and N-acetylated amino sugars, samples were methanolyzed with 0.5 M HCl in methanol containing xylitol as internal standard, (18 h at 80 °C), followed by neutralization with silver carbonate, re-N-acetylation with acetic anhydride, and trimethylsilyl derivatization. The products were analyzed by gas-liquid chromatography (GC) on a DB-1 fused silica column (30 m times 0.25 mm internal diameter) using hydrogen (0.7 times 10^5 pascals) as the carrier gas. The column temperature was programmed from 120 to 240 °C at 2 °C/min. For analysis of inositol and glucosamine, samples were treated with 3 M HCl (containing xylitol as internal standard) for 18 h at 80 °C. The dried methanolysates were hydrolyzed with 6 M HCl for 18 h at 100 °C, reduced with sodium borohydride, acetylated (acetic anhydride/pyridine, 9:1), and analyzed by GC as described above, except that the column temperature was programmed from 120 to 240 °C at 3 °C/min.

Lipid Analysis

For analysis of fatty acids and alkylglycerols, the 40/45-kDa glycoprotein was methanolyzed with 0.5 M HCl in methanol for 18 h at 80 °C. The solution was extracted with heptane, and the resulting mixture of fatty acid methyl esters and alkylglycerols was analyzed by GC after trimethylsilylation, using a DB-1 fused silica column (30 m times 0.25 mm internal diameter) and hydrogen carrier gas at 0.7 times 10^5 pascals. The column temperature was programmed from 180 to 310 °C at 5 °C/min. Peaks were identified by their retention times and by GC-MS.

Phosphatidylinositol-Phospholipase C Digestion of 40/45-kDa Glycoprotein

40/45-kDa glycoprotein (5 mg) was dissolved in 1.0 ml of Tris-HCl buffer (pH 7.2), containing 0.1% (w/v) sodium deoxycholate and incubated with PI-phospholipase C from Bacillus thuringiensis (3 units). After 24 h at 37 °C the enzymatic reaction was stopped by the addition of 1.0 ml of chloroform. The lipids were analyzed in the aqueous and chloroform layers under the conditions described above.

Methylation Analysis

Permethylation of beta-eliminated oligosaccharide-alditols was performed by the method of Ciucanu and Kerek(19) . The permethylated sample was methanolyzed with 0.5 M HCl in methanol for 18 h at 80 °C. The methanolysates were dried in 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 (30 m times 0.25 mm internal diameter) using hydrogen as the carrier gas at 0.7 times 10^5 pascals. The column oven was temperature-programmed from 110 to 240 °C at 2 °C/min. The O-acetylated partially O-methylated methyl glycosides were identified by their typical retention time, GC-MS, and quantified by their areas.

Labeling of beta-Eliminated Oligosaccharide-Alditols

Oligosaccharide-alditols (200 µg) obtained from the 40/45-kDa glycoprotein were heated in 0.5 ml of 1 M KOH at 100 °C for 90 min and the solutions cooled and neutralized with perchloric acid. The insoluble precipitate was removed by centrifugation, and the supernatants were dried. The resulting deacetylated oligosaccharides were dissolved in 0.5 ml of saturated NaHCO(3) and re-N-acetylated by the addition of 200 µCi of [^14C]acetic anhydride(20) . After 60 min at 25 °C, 5 µl of unlabeled acetic anhydride was added and the solutions kept for a further hour at the same temperature. The solutions were neutralized with acetic acid and applied to a 1.2 times 57-cm Sephadex G-10 column equilibrated with 7% 2-propanol. Substances in the void volume were submitted to paper electrophoresis in 0.1 M pyridine acetate buffer (pH 6.5) for 80 min at 28 V/cm. Labeled oligosaccharide-alditols were eluted from the origin.

trans-Sialidase and Neuraminidase Assays

For trans-sialidase assay the incubation mixtures contained, in a total volume of 50 µl, 2 mM sialyllactose, 50 mM of PIPES buffer (pH 7.0), enzyme corresponding to 10^5 parasites and 100,000 cpm of oligosaccharide-alditols. After 2 h at 37 °C, solutions were heated for 5 min at 100 °C and submitted to paper electrophoresis in 0.1 M pyridine acetate buffer, pH 6.5, for 80 min at 28 V/cm. For neuraminidase assay, sialylated oligosaccharide-alditols were eluted from papers, dissolved in 50 mM sodium acetate buffer (pH 5.0), and incubated for 2 h at 37 °C with 0.25 unit of C. perfringens neuraminidase in a total volume of 50 µl. After heating the solutions for 5 min at 100 °C, they were submitted to paper electrophoresis in pyridine-acetate buffer as above.

Nuclear Magnetic Resonance Spectroscopy (NMR)

NMR spectra were obtained on a Varian Unity 500 NMR spectrometer (Varian Associates, Palo Alto, CA) equipped with either a 5-mm proton-detecting triple resonance probe or a 5-mm broadband probe, at an indicated probe temperature of 30 °C. Standard pulse sequences were used throughout. Proton spectra were referenced to internal acetate anion at 1.908 ppm and carbon spectra to internal acetone at 31.5 ppm. The P spectrum was referenced to external 85% H(3)PO(4) at 0 ppm. The proton NMR spectrum was assigned through a combination of a DQF-COSY (21, 22) and a TOCSY spectrum(23, 24) . Carbon-13 assignments were made from an HMQC spectrum (25) obtained without carbon decoupling. Some additional assignments and information on the sequence and linkage of the sugar residues were derived from a ROESY spectrum (26) obtained with 150-ms mixing time.

Fast Atom Bombardment Mass Spectrometry (FAB-MS)

FAB mass spectra were recorded using a Kratos MS80RFA spectrometer equipped with an Ion Tech FAB gun using xenon atoms as the bombarding particles. Underivatized samples were dissolved in 30% acetic acid to a concentration of about 10 µg/µl, and 1 µl was mixed with an equal volume of a 1:1 mixture of glycerol and dithiothreitol (5:1) on the stainless steel target. Peracetylated samples were analyzed using 3nitrobenzyl alcohol as liquid matrix. The instrument was operated at 4 kV accelerating voltage, and conventional FAB spectra were obtained at a scan rate of 30 s per decade of mass at a resolution of 1000. The spectra were mass assigned using the reference peaks of cesium iodide.

Sample were peracetylated for FAB-MS by treatment for 10 min at room temperature with a 2:1 (v/v) mixture of trifluoroacetic anhydride and acetic acid. The reagents were removed in a stream of nitrogen, and the residue was dissolved in 1 ml of chloroform and washed three times with an equal volume of water. The chloroform was evaporated under nitrogen, and the peracetylated oligosaccharides were dissolved in 5 µl of methanol prior to analysis.

Other Analytical Methods

Total neutral sugars were analyzed by the phenol-H(2)SO(4) procedure(27) ; protein was determined by the method of Lowry et al.(28) ; phosphorus was assayed by the Ames (29) and Bartlett (30) methods, hexosamine by modification of the Elson-Morgan reaction(31) , and sialic acid by the thiobarbituric acid method(32) . Amino acid analysis was performed according to Fauconnet and Rochemont(33) .

RESULTS

Analysis by SDS-polyacrylamide gel electrophoresis of a Bio-Gel P-100-purified phenol water extract of T. cruzi cells revealed two Schiff positive bands at 40/45 and 20/30 kDa. The 40/45-kDa components were purified to apparent homogeneity by selective extraction and affinity chromatography on a concanavalin A-Sepharose column.

Chemical analysis of the purified 40/45-kDa glycoprotein revealed the presence of neutral sugars (45%), protein (7%), hexosamine (10%), phosphorus (1%), and lipids. Galactose (Gal), mannose (Man), N-acetylglucosamine (GlcNAc), and N-acetylgalactosamine (GalNAc) were detected by carbohydrate analysis in the molar ratio of 3.0:1.5:1.0:0.1, together with traces of inositol (Ins) and sialic acid. Amino acid analysis indicated that the predominant amino acids were threonine, alanine, and aspartic acid, which together comprised about 65% of the total, the balance being mainly glutamic acid, glycine, proline, and lysine.

On acid methanolysis the glycoprotein released methyl palmitate, methyl stearate, and a monoalkylglycerol in the molar ratio of 0.9:0.2:1.0. The monoalkylglycerol was trimethylsilylated and identified by GC-MS as a hexadecylglycerol. The presence of fragment ions at m/z 205 (CHOSi(CH(3))(3)-CH(2)OSi(CH(3))(3)) in the mass spectrum, together with the absence of m/z 218 and 191, suggested that the alkyl chain is exclusively located on the sn-1-position of glycerol. GC and GC-MS analyses of the products obtained by digestion of 40/45-kDa glycoprotein with PI-phospholipase C, following partition between chloroform and water, revealed that all glycerolipid moiety was present only in the chloroform layer. In the non-enzymatic control, the glycerolipid moiety was present only in the aqueous layer. The molar ratio of alkylglycerol and polypeptide chain (calculated by amino acid analysis) in the 40/45-kDa glycoprotein was 0.9:1.0.

Structural Analysis of PI-oligosaccharide Isolated from the GPI Anchor of the 40/45-kDa Glycoprotein

The elution volume on Bio-Gel P-4 of the PI-oligosaccharide obtained by hot alkaline degradation of the 40/45-kDa glycoprotein was equivalent to that of an alpha(14)-linked octa-D-glucopyranosyl oligosaccharide alditol. Chemical analysis of the PI-oligosaccharide revealed mannose, glucosamine (GlcN), inositol, and phosphorus in the molar ratio of 5.0:1.1:1.0:3.2 and the presence of ethanolamine (EtN) and 2-aminoethylphosphonate (AEP). In agreement with this data, the 202-MHz P NMR spectrum (Fig. 1) comprised four resonances assigned as OPO(2)CH(2)CH(2)NH(2) (22 ppm), Ins-2-OPO(3) (4.5 ppm), Ins-1-OPO(3) (2.5 ppm), and OPO(3)CH(2)CH(2)NH(2) (0 ppm) in the area ratio 1.3:0.2:0.8:0.6. We have previously reported migration of the phosphate from position 1 to position 2 of the inositol ring during base cleavage(34) .

Figure 1: P NMR spectroscopy of PI-oligosaccharide. The 202-MHz P NMR spectrum of the PI-oligosaccharide from the GPI anchor of T. cruzi 40/45-kDa glycoprotein is shown.


Four major signals were observed in the anomeric region of the one-dimensional 500-MHz proton NMR spectrum of the PI-oligosaccharide (Fig. 2) in an approximate area ratio 1:2:1:2. A lower field anomeric resonance (5.711 ppm) was recognized as alpha-GlcN H-1 by its coupling to a high-field H-2 resonance and by the pattern of cross-peaks in the TOCSY spectrum (Fig. 3). An additional alpha-GlcN spin system observed in the TOCSY spectrum was attributed to the (14)GlcNalpha(1-6)-Ins-2-phosphate resulting from phosphate migration during base cleavage; this was also the reason for the presence of two inositol phosphate spin systems. The other major spin systems were assigned as alpha-Man residues. The anomeric configurations of the mannose residues were determined from the values of ^1J measured from the HMQC(35, 36) spectrum. These varied between 172 and 174 Hz and are characteristic of equatorial anomeric protons (i.e. alpha-linked D-Man residues)(37) . At a higher field, characteristic resonances were observed attributable to AEP (at 3.22 and 2.04 ppm) and to EtNP (at 3.29 ppm). These assignments are summarized in Table 1.

Figure 2: ^1H NMR spectroscopy of PI-oligosaccharide. The 500-MHz one-dimensional proton NMR of the PI-oligosaccharide from GPI anchor of 40/45-kDa glycoprotein of T. cruzi is shown. The resonances are labeled according to the convention used in the text and Table 1.


Figure 3: TOCSY spectrum of the PI-oligosaccharide. The partial 500-MHz TOCSY spectrum of the PI-oligosaccharide from the GPI anchor of T. cruzi 40/45-kDa glycoprotein is shown. This region of the spectrum shows the correlations between the anomeric protons and the other sugar residue protons. The arrows indicate the traces on the H-1 resonances of the major residues. The boxed area indicated the approximate region where the H-1/H-2 cross-peak of a (13)Manalpha(1-4)GlcN or (13,6)Manalpha(1-4)GlcN is expected. The low intensity ``cross-peak'' present is not symmetrical about the diagonal and is considered to be artifactual.


Inter-residue NOEs were observed in the ROESY experiment between the alpha-GlcN H-1 and H-1 and H-6 of Ins-1-phosphate (Fig. 4), and between alpha-Man(2) H-1 and alpha-GlcN H-4. In the HMQC spectrum, the anomeric proton of alpha-Man(3) correlated to a relatively high-field C-1 resonance (99.22 ppm), diagnostic of a (1, 2, 3, 4, 5, 6) -linkage. The NOEs to resonances at 4.001, 3.837, and 3.767 ppm of alpha-Man(3) were not rigorously assigned, but were consistent with inter-residue NOEs to H-5, H-6, and H-6` of the adjacent residues, as observed in other systems(38) , including GPI anchor structures(39) . The value of the chemical shift of the Man(2) H-2 (4.063 ppm) is similar to that found in the corresponding residue of the GPI anchor from yeast (39) and the lipopeptidophosphoglycan (LPPG) of T. cruzi(40) rather than to its value (typically 4.23 ppm) when present in (13)- or (13,6)-linked structures(34, 38, 41) , indicating a linear rather than a branched oligosaccharide. None of the minor alpha-Man H-1/H-2 cross-peaks in the TOCSY spectrum (Fig. 3) suggested the presence of either branched (13,6)Manalpha(1-4)GlcN or linear (13)Manalpha(1-4)GlcN substructures. In the HMQC spectrum the H-2 of alpha-Man(3) correlated to a low-field C-2 resonance (79.38 ppm) and inter-residue NOEs were observed between alpha-Man(1) H-1 and H-1 and H-2 of alpha-Man(3). Similarly, alpha-Man(1) H-2 correlated with a low-field C-2 resonance (79.18 ppm), and inter-residue NOEs were assigned as indicating that alpha-Man(4) H-1 is close in space to both alpha-Man(1) H-1 and H-2. The low-field chemical shift of the alpha-GlcN H-5 is consistent with the presence of a phosphorylated substituent at O-6 position, probably AEP by analogy with LPPG(42) , leading to the following partial structure.

Figure 4: Scheme showing the chemical shift and inter-residue NOE data used to assign linkages and sequence in the PI-oligosaccharide from GPI anchor of T. cruzi 40/45-kDa glycoprotein.


A weak cross-peak was observed in the ROESY spectrum between two resonances at 5.05 and 5.308 ppm assigned to the H-1s of two residues arbitrarily designated alpha-Man(4a) and alpha-Man(1a). This cross-peak could be due to either a minor tetramannose species in which the subterminal mannose is substituted with AEP rather than EtNP (i.e. Man(4a)alpha(1-2)Man(6AEP) (1a) (alpha1), or alternatively, to an alpha(1-2) extension to the tetramannose chain by Man(4a) as in the yeast GPI anchor(39) . A molecular ion consistent with the presence of the latter was detected by mass spectrometry (see below).

The location of the EtNP substituent was not apparent from the NMR data and could not be established by methylation analysis, since GPI structures containing four hexoses and two phosphate ester of EtNP or 2-AEP are difficult to methylate. (^2)Methylated derivatives eventually obtained from this preparation originated from a co-purified high mannose contaminant, derived from the 40/45-kDa glycoprotein. In agreement with this observation, the NMR spectrum showed minor resonances corresponding to a high mannose oligosaccharide such as would arise from elimination of an Asn-linked chain(43) . The terminal alpha-Manp units of this contaminant contribute to the intensity of the resonance at 5.05 ppm (Fig. 2).

The most significant feature in the FAB mass spectrum of the PI-oligosaccharide was a cluster of signals in the m/z 1300-1400 region. These were assigned as multiply cationized forms of two GPI species, both with the composition (hexose)(4)-hexosamine-inositol-phosphate, but varying in substitution pattern. The major species ([M + H] = 1300.4, [M + Na] = 1322.4, [M - H + Na(2)] = 1344.3) has one AEP and one EtNP substituent, whereas the other ([M + H] = 1284.3, [M + Na] = 1306.4, [M - H + Na(2)] = 1328.4) has two AEP substituents. The calculated values of the protonated molecules of these two species are, respectively, 1300.339 and 1284.345. A minor signal at m/z 1484.5 may correspond to the sodium-cationized molecule ([M + Na]) of a homologue of the major species (m/z 1300) containing an additional hexose residue.

Because of high levels of salt contamination, and the presence of high mannose species, the signal to noise ratio of the spectrum was poor, and it was consequently difficult to assign with confidence any fragment ions. The characteristic Y(2) fragment ion(34, 41, 44, 45) , which (in salt-free samples) is diagnostic of the substituent on glucosamine, was not observed at m/z 529, 545, or 422; however an ion at m/z 551 (549 in the negative ions spectrum) may correspond to a sodium-cationized analogue of m/z 529, in which case an AEP substituent is located on glucosamine. The signals at m/z 689, 851, and 1013, although differing by the residue mass of hexose, could not be rationalized as fragmentation products of the PI-oligosaccharide, although they could correspond to sodium-cationized molecules of oligohexoses. This was verified by FAB-MS after treatment with trifluoroacetic anhydride/acetic acid, which resulted in a spectrum containing peracetylated oligohexose ions. In order to improve the quality of the FAB-MS data, the sample was desalted with Dowex 50W-X8 and reanalyzed. Two protonated molecules were now obtained, at m/z 1300.3 (major) and 1284.3 (minor), as expected. An abundant Y(2) fragment ion at m/z 529 confirmed that the glucosamine was substituted primarily with AEP. The mass increment of 285 between the fragments assigned as Y(5) (m/z 1138) and Y(4) (m/z 853) suggests that the EtNP substituent of the major species is located on the third mannose distal to glucosamine. A corresponding signal at m/z 1122 indicated that this residue is substituted with AEP in the analogue containing two AEP groups.

Structural Analysis of beta-Eliminated Oligosaccharide-alditols Isolated from 40/45-kDa Glycoprotein

Oligosaccharide-alditols were released from the 40/4-kDa glycoprotein by alkaline borohydride treatment and separated by chromatography on a Bio-Gel P-4 column. Four fractions were obtained in the molar ratio of 0.3:5.0:3.0:0.4. The elution volumes of these fractions, designated I, II, III, and IV were similar to those of(1, 2, 3, 4) -linked alpha-D-glucopyranosyl oligosaccharide-alditols containing two, three, four, and five glucose residues, respectively.

The beta-eliminated saccharide-alditols were methanolyzed, trimethylsilylated, and analyzed by GC. Fractions II, III, and IV contained galactose, GlcNAc-ol and GalNAc-ol in molar ratio of 1.0:1.0:0.09, 2.0:1.0:0.1, and 3.0:1.0:0.08, respectively, whereas fraction I contained only GlcNAc-ol. The protonated molecules (M + H) obtained by FAB-MS identified Fractions II (m/z 386), III (m/z 548), and IV (m/z 710) as, respectively, N-acetylhexosaminitol-containing mono-, di-, and trisaccharides. The products obtained in the methylation analysis of the monosaccharide alditol (fraction II) corresponded to terminal galactose, 3-O- and 4-O-substituted GlcNAc-ol in the molar ratio 1.0:0.4:0.6. The proton NMR spectrum of this fraction (Fig. 5A) contained two major anomeric resonances in a 6:4 ratio at 4.502 and 4.494 ppm (^3J about 8 Hz), typical of beta-Galp units and two major N-acetyl methyl resonances near 2.0 ppm from the GlcNAc-ol residues. The 80-ms TOCSY spectrum, the C NMR assignments, and comparison with the spectra of oligosaccharides isolated from T. cruzi G-strain (11) enable an almost full assignment of the proton NMR spectrum of this fraction (Table 2). These data define the monosaccharide alditol fraction as a mixture of oligosaccharides of structures Galpbeta1-3GlcNAc-ol and Galpbeta1-4GlcNAc-ol.

Figure 5: ^1H NMR spectroscopy of the O-linked oligosaccharides. 500-MHz ^1H NMR spectra of O-linked oligosaccharide from 40/45 kDa glycoprotein of T. cruzi are shown. A, monosaccharide-alditols; B, disaccharide-alditols; and C, trisaccharide-alditols.


The proton NMR spectrum of the disaccharide-alditol (fraction III) differed from that of fraction II in several respects (Fig. 5B). Two beta-Galp anomeric resonances were present in an approximate 6:4 ratio; although 0.06-0.09 ppm downfield of those in the previous spectrum, their chemical shifts and those of the ring protons were still consistent with a nonreducing terminal location. A third anomeric resonance was present at 4.450 ppm (^3J about 8 Hz) with approximately the combined intensity of the other two anomerics. This signal was assigned to an additional nonreducing terminal beta-Galp residue by means of an 80-ms TOCSY experiment. The location of this residue was deduced from the following arguments: (a) the proton (Fig. 5B) and carbon chemical shifts of the additional anomeric resonance (4.450 ppm ^1H and 104.41 ppm C) (Table 2) are very similar to those of the Galpbeta1-6GlcNAc-ol system found in the T. cruzi G-strain oligosaccharides (4.431 and 104.41 ppm, respectively(11) ); (b) the resonances assigned as GlcNAc-ol H-6 and H-6` are shifted downfield compared with the corresponding signals in the monosaccharide-alditol; (c) the high-field resonances corresponding to C-6 of GlcNAc-ol in the monosaccharide-alditol (at 63.53 and 63.27 ppm) are replaced by signals at 70.83 and 70.92 ppm (Table 2). These data suggest that the disaccharide-alditol is a 4:6 mixture of Galpbeta1-3(Galpbeta1-6)GlcNAc-ol and Galpbeta1-4(Galpbeta1-6)GlcNAc-ol and agree with the detection in the methylation analysis of nonreducing end units of galactose, 3,6- and 4,6-di-O substituted GlcNAc-ol in a molar ratio of 2.0:0.4:0.6.

In the methylation analysis of the trisaccharide-alditol (fraction IV), in addition to 3,6-di-O- and 4,6-di-O-substituted GlcNAc-ol (in a molar ratio of 0.4:0.6), derivatives corresponding to nonreducing terminal galactose and 2-O-substituted galactose were detected in 2:1 molar ratio. The one-dimensional proton spectrum of the trisaccharide-alditol (Fig. 5C) was complex and lacked the anomeric doublet assigned as Galpbeta1-6GlcNAc-ol in the disaccharide-alditol, consistent with the addition of a beta-galactose residue to that branch. These data show that the trisaccharide-alditol is a mixture of two compounds: Galpbeta1-2Galpbeta1-6(Galpbeta1-3)GlcNAc-ol and Galpbeta1-2Galpbeta1-6(Galpbeta1-4)GlcNAc-ol. The structures of the major O-linked GlcNAc oligosaccharides from the 40/45-kDa glycoprotein of T. cruzi Y-strain are summarized Table 3.

In Vitro Sialylation of Oligosaccharide-alditols from the 40/45-kDa Glycoprotein

The monosaccharide-alditols Galpbeta1-3GlcNAc-ol and Galpbeta1-4GlcNAc-ol, disaccharide-alditols Galpbeta1-6(Galpbeta1-3)GlcNAc-ol and Galpbeta1-6(Galpbeta1-4)GlcNAc-ol, and trisaccharide-alditols Galpbeta1-2Galpbeta1-6(Galpbeta1-3)GlcNAc-ol and Galpbeta1-2Galpbeta1-6(Galpbeta1-4)GlcNAc-ol were tested as potential sialyl acceptors. The oligosaccharides were labeled in the acetyl groups with ^14C and incubated with sialyllactose and trans-sialidase. On high voltage paper electrophoresis a single negatively charged peak was obtained from the monosaccharide-alditol mixture (Fig. 6, upper panel A), whereas two peaks appeared in the case of di- (Fig. 6, upper panel B) and trisaccharide-alditols (Fig. 6, upper panel C). The more intense of these exhibited the same migration behavior as that obtained with the monosaccharide-alditols, whereas the minor peak migrated more rapidly. Reincubation of the slow migrating substances (Fig. 6, upper panels B and C) with trans-sialidase and sialyllactose yielded a small amount of the fast migrating one (Fig. 6, lower panels A and B). These results suggest that incorporation of one sialyl unit strongly hinders entry of another. A neutral product was obtained on incubating either peak with C. perfringens (Fig. 6, panels D, E, and F).

Figure 6: Formation of mono- and disialyl-oligosaccharide-alditols from beta-eliminated oligosaccharides of T. cruzi 40/45-kDa glycoprotein. Upper panel, the labeled mono- (A), di (B), and trisaccharide-alditols (C) were incubated with sialyllactose and T. cruzitrans-sialidase and run on paper electrophoresis in pyridine-acetate buffer, pH 6.5. The charged compound in A, the slow migrating oligosaccharide in B, and the fast migrating compound in C were eluted, incubated with C. perfringens neuraminidase, and run on paper electrophoresis as above (D, E, and F, respectively). Lower panel, the slow migrating compounds obtained in upper panels B and C were eluted and reincubated with trans-sialidase and sialyllactose and run on paper chromatography (lower panels A and B, respectively).


DISCUSSION

In the present study we have determined the structure of the GPI anchor of the 40/45-kDa glycoprotein of T. cruzi epimastigotes and of the oligosaccharides O-linked to the protein. We have also shown that these latter oligosaccharides act as acceptors for sialic acid transferred via the trans-sialidase reaction(7) .

The PI-oligosaccharide of the anchor was characterized by NMR spectroscopy, FAB mass spectrometry, and compositional analysis. Our results show that the major GPI species contains an EtNP group attached to a mannotetraose backbone of sequence Manalpha(1-2)Manalpha(1-2)Manalpha(1-6)Manalpha(1. This tetramannosyl chain is (14)-linked to a non-acetylated GlcN residue substituted at O-6 by AEP, a feature that has not been reported previously in a GPI-protein anchor. The GlcN unit is glycosidically linked to the O-6 position of a myo-inositol phospholipid, containing 1-O-alkyl-2-O-acyl-sn-glycerol. The EtNP was shown by FAB-MS to be located on the third mannose distal to inositol, in agreement with the structure of all GPI-protein anchors characterized so for(46) .

Apart from the AEP substituent, the structure of this compound is identical with that of the GPI anchor of the 1G7 antigen isolated from metacyclic forms of T. cruzi(47) . The presence of AEP in the 1G7 anchor cannot, however, be excluded, because the structure was determined after dephosphorylation with aqueous hydrofluoric acid(47) .

Interestingly, T. cruzi epimastigotes synthesize a free glycoinositolphospholipid called LPPG(48) , the PI-oligosaccharide core of which (42) is similar to the GPI anchors of T. cruzi glycoproteins. It differs, however, in that the tetramannosyl chain is further substituted by galactofuranosyl residues and by the absence of EtNP. This suggests that T. cruzi may use the same set of glycosyltransferases for the synthesis of both LPPG and glycoprotein-linked GPI anchors. The lipid moiety of LPPG, however, is a ceramide (42, 49) and thus diverging from the protein-linked GPI anchors.

It has been suggested that GPI-containing glycoconjugates, including LPPG from T. cruzi, the heterogeneous lipophosphoglycan of Leishmania species, and the free glycoinositolphospholipids of various trypanosomatids(50) , may have evolved from primitive GPI anchors linked via EtNP to protein. In the 40/45-kDa GPI anchor, however, FAB mass spectra showed that EtNP can be partially replaced by AEP. An alternative hypothesis therefore is that the GPI anchors of ancestral Kinetoplastida might have contained AEP, which was replaced by EtNP in the course of evolution.

The O-linked oligosaccharides of 40/45-kDa glycoproteins of the Y-strain of T. cruzi differ from those of the G-strain (11) in several aspects. Although linked to threonine and/or serine via GlcNAc, they are shorter and do not contain beta-galactofuranosyl units. They form, after beta-elimination, two series of mono-, di-, and trisaccharide-alditols in which the beta-galactopyranosyl branch is either (13)- or (14)-linked. The short oligosaccharide chains of the 40/45-kDa glycoprotein of Y-strain epimastigotes described in this paper differ from the analogous structures of cell-derived trypomastigotes of the same strain(6) . In the latter glycoprotein (called F2/3 or Ssp-3), the shortest O-linked oligosaccharide is the trisaccharide Galpalpha(1-3)Galpalpha(1-4) GlcNAc, but chains as long as 10 sugar units have been detected. In contrast to the highly glycosylated components of epimastigotes (from both the G- and the Y-strains) and of metacyclic trypomastigotes, the corresponding glycoproteins from cell-derived trypomastigotes are unique in that they contain terminal alpha-Galp units(6) . The 3-O substitution of GlcNAc described in the present study seems to occur only in the oligosaccharides of epimastigotes. In cell-derived trypomastigotes, a high proportion of the O-linked oligosaccharide chains not containing alpha-Galp are substituted by sialic acid units, giving rise to an epitope recognized by monoclonal antibody 3C9. This monoclonal antibody, however, does not react with the glycoproteins of epimastigotes and metacyclic trypomastigotes of the Y-strain(3) . This suggests that sialic acid substitution of O-linked oligosaccharides in these molecules is not sufficient per se to generate the epitope characteristic of the Ssp-3 (or F2/3) glycoproteins of cell-derived trypomastigotes which is recognized by the 3C9 antibody. Differences in the glycosylation of these molecules in T. cruzi parallels the varying patterns of O-linked glycosylation observed as a function of cell lineage and stage of differentiation in mammalian cells(51) .

It has been demonstrated previously that most of the sialic acid transferred by the trans-sialidase reaction from fetal calf serum donor molecules is incorporated into the 40/45-kDa glycoprotein of T. cruzi (Y-strain) epimastigotes(7) . This is another example from a eukaryotic system of a transglycosylation reaction that is independent of nucleotide phosphate sugars; oligosaccharide biosynthesis via similar reactions have previously been noted in fungi (52, 53) .

Non-sialylated molecules were obtained by culturing epimastigotes in the absence of serum; these were used as acceptors for in vitro sialic acid transfer by trans-sialidase from trypomastigotes, with sialyllactose as a donor substrate. The oligosaccharide acceptors specificity of T. cruzitrans-sialidase has been studied previously(54, 55) . Only beta-linked terminal galactosyl units are susceptible to sialylation by the enzyme. Internal beta-linked or terminal alpha-linked residues cannot serve as acceptors. Fucosylation of the residues adjacent to an otherwise susceptible galactopyranose also abolishes sialylation. Although it has been claimed that the trans-sialidase reaction is unaffected by chain length and the nature of (unbranched) vicinal residues, it seems clear from the present data that sialylation of one residue in T. cruzi glycans modulates the susceptibility of nearby sites to sialylation, as shown by the kinetics of in vitro sialic acid incorporation into the oligosaccharides of the 40/45-kDa glycoprotein. These results suggest that the potential use of the trypanosome trans-sialidase as an effective and economical replacement of the alpha(2-3) sialyl transferase for in vitro sialylation (56) may be limited by an inability to efficiently sialylate complex multiantennary substrates.

Because of the low activity of the trans-sialidase in epimastigotes, some workers (56, 57) have suggested that sialic acid is absent from the surface of these forms; however, it should be noted that the 40/45-kDa glycoprotein isolated from epimastigotes grown in the presence of fetal calf serum has similar composition and properties to the recently characterized ``sialylated lipophosphoglycan-like molecules'' from epimastigote forms of T. cruzi(58) .

It is unclear whether the structural differences in the mucin-like molecules of epimastigotes from different strains of T. cruzi are a common feature of this species or whether they are in any way related to the infectivity of the corresponding trypomastigote stage. Further analyses with additional strains of this parasite should answer this question.

FOOTNOTES

*
This work was supported by grants from Financiadora de Estudos e Projetos (FINEP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/RHAE, PADCT), Conselho de Ensino de Pós-Graduação (CEPG/UFRJ); by the United Nations Development Program/World Bank/World Health Organization Special Program for Research and Training in Tropical Diseases and the Swedish Agency for Research Cooperation with Developing Countries (SAREC); and by the British Council/CNPq (for funding reciprocal visits of C. J., R. W., and J. O. P. between the United Kingdom and Brazil). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Fax: 55-21-270-8647.

(^1)
The abbreviations used are: GPI, glycosylphosphatidylinositol; AEP, 2-aminoethylphosphonic acid; DQF-COSY, double quantum-filtered correlation spectroscopy; EtNP, ethanolaminephosphate; FAB, fast atom bombardment; GC, gas-liquid chromatography; GlcN, glucosamine; GlcNAc, N-acetylglucosamine; GlcNAc-ol, N-acetylglucosaminitol; GalNAc-ol, N-acetylgalactosaminitol; HMQC, heteronuclear multiple quantum coherence; LPPG, lipopeptidophosphoglycan; MS, mass spectrometry; PI, phosphoinositol; ROESY, rotating frame nuclear Overhauser enhancement spectroscopy; TOCSY, total correlation spectroscopy; Ins, inositol; PIPES, 1,4-piperazinediethanesulfonic acid; NOE, nuclear Overhauser effect.

(^2)
J. O. Previato and L. Mendonça-Previato, unpublished observation.

ACKNOWLEDGEMENTS

We are grateful to Orlando Augusto Agrellos Filho and Lucy Jacinto do Nascimento for excellent technical assistance. We thank Maria Cristina M. P. Lima (Mass Spectrometry Laboratory at Núcleo de Pesquisa de Produtos Naturais/Universidade Federal do Rio de Janeiro) for GC-MS analysis.

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