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The Lipid Structure of the Glycosylphosphatidylinositol-anchored Mucin-like Sialic Acid Acceptors of Trypanosoma cruzi Changes during Parasite Differentiation from Epimastigotes to Infective Metacyclic Trypomastigote Forms
1995; Elsevier BV; Volume: 270; Issue: 45 Linguagem: Inglês
10.1074/jbc.270.45.27244
ISSN1083-351X
AutoresÁlvaro Acosta-Serrano, Sérgio Schenkman, Nobuko Yoshida, Angela Mehlert, J. Richardson, Michael A. J. Ferguson,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoThe major acceptors of sialic acid on the surface of metacyclic trypomastigotes, which are the infective forms of Trypanosoma cruzi found in the insect vector, are mucin-like glycoproteins linked to the parasite membrane via glycosylphosphatidylinositol anchors. Here we have compared the lipid and the carbohydrate structure of the glycosylphosphatidylinositol anchors and the O-linked oligosaccharides of the mucins isolated from metacyclic trypomastigotes and noninfective epimastigote forms obtained in culture. The single difference found was in the lipid structure. While the phosphatidylinositol moiety of the epimastigote mucins contains mainly 1-O-hexadecyl-2-O-hexadecanoylphosphatidylinositol, the phosphatidylinositol moiety of the metacyclic trypomastigote mucins contains mostly (~70%) inositol phosphoceramides, consisting of a C18:0 sphinganine long chain base and mainly C24:0 and C16:0 fatty acids. The remaining 30% of the metacyclic phosphatidylinositol moieties are the same alkylacylphosphatidylinositol species found in epimastigotes. In contrast, the glycosylphosphatidylinositol glycan cores of both molecules are very similar, mainly Manα1-2Manα1-2Manα1-6Manα1-4GlcN. The glycans are substituted at the GlcN residue and at the third αMan distal to the GlcN residue by ethanolamine phosphate or 2-aminoethylphosphonate groups. The structures of the desialylated O-linked oligosaccharides of the metacyclic trypomastigote mucin-like molecules, released by β-elimination with concomitant reduction, are identical to the structures reported for the epimastigote mucins (Previato, J. O., Jones, C., Gon¸alves, L. P. B., Wait, R., Travassos, L. R., and Mendo¸a-Previato, L.(1994) Biochem. J. 301, 151-159). In addition, a significant amount of nonsubstituted N-acetylglucosaminitol was released from the mucins of both forms of the parasite. Taken together, these results indicate that when epimastigotes transform into infective metacyclic trypomastigotes, the phosphatidylinositol moiety of the glycosylphosphatidylinositol anchor of the major acceptor of sialic acid is modified, while the glycosylphosphatidylinositol anchor and O-linked sugar chains remain essentially unchanged. The major acceptors of sialic acid on the surface of metacyclic trypomastigotes, which are the infective forms of Trypanosoma cruzi found in the insect vector, are mucin-like glycoproteins linked to the parasite membrane via glycosylphosphatidylinositol anchors. Here we have compared the lipid and the carbohydrate structure of the glycosylphosphatidylinositol anchors and the O-linked oligosaccharides of the mucins isolated from metacyclic trypomastigotes and noninfective epimastigote forms obtained in culture. The single difference found was in the lipid structure. While the phosphatidylinositol moiety of the epimastigote mucins contains mainly 1-O-hexadecyl-2-O-hexadecanoylphosphatidylinositol, the phosphatidylinositol moiety of the metacyclic trypomastigote mucins contains mostly (~70%) inositol phosphoceramides, consisting of a C18:0 sphinganine long chain base and mainly C24:0 and C16:0 fatty acids. The remaining 30% of the metacyclic phosphatidylinositol moieties are the same alkylacylphosphatidylinositol species found in epimastigotes. In contrast, the glycosylphosphatidylinositol glycan cores of both molecules are very similar, mainly Manα1-2Manα1-2Manα1-6Manα1-4GlcN. The glycans are substituted at the GlcN residue and at the third αMan distal to the GlcN residue by ethanolamine phosphate or 2-aminoethylphosphonate groups. The structures of the desialylated O-linked oligosaccharides of the metacyclic trypomastigote mucin-like molecules, released by β-elimination with concomitant reduction, are identical to the structures reported for the epimastigote mucins (Previato, J. O., Jones, C., Gon¸alves, L. P. B., Wait, R., Travassos, L. R., and Mendo¸a-Previato, L.(1994) Biochem. J. 301, 151-159). In addition, a significant amount of nonsubstituted N-acetylglucosaminitol was released from the mucins of both forms of the parasite. Taken together, these results indicate that when epimastigotes transform into infective metacyclic trypomastigotes, the phosphatidylinositol moiety of the glycosylphosphatidylinositol anchor of the major acceptor of sialic acid is modified, while the glycosylphosphatidylinositol anchor and O-linked sugar chains remain essentially unchanged. INTRODUCTIONTrypanosoma cruzi, the protozoan parasite that causes Chagas' disease in humans, has a complex life cycle alternating between the insect vector and the mammalian host. In the vector, it multiplies as noninfective epimastigotes that migrate to the hindgut and differentiate into infective metacyclic trypomastigotes. During the insect blood meal, the metacyclic trypomastigotes are deposited with the feces and urine near a skin wound, initiating the natural infection.T. cruzi is unable to synthesize sialic acids (SA), (1)The abbreviations used are: SAsialic acid(s)TStrans-sialidaseGPIglycosylphosphatidylinositolPIphosphatidylinositolES-MSelectrospray-mass spectrometryGC-MSgas chromatography-mass spectrometryHPTLChigh-performance thin layer chromatographyHPLChigh-performance liquid chromatographyAHM*[1-3H]2,5-anhydromannitolGlcNAc-olN-acetylglucosaminitolHexNAc-olN-acetylhexosaminitolLPPGlipopeptidophosphoglycanGuglucose units2-AEP2-aminoethylphosphonatePAGEpolyacrylamide gel electrophoresis. but it expresses a unique trans-sialidase (TS), which transfers α2-3-linked SA from host glycoproteins and glycolipids to acceptors containing terminal β-galactosyl residues present on the parasite surface (reviewed in (1Colli W. FASEB J. 1993; 7: 1257-1264Crossref PubMed Scopus (122) Google Scholar, 2Cross G.A.M. Takle G.B. Annu. Rev. Microbiol. 1993; 46: 385-411Crossref Scopus (144) Google Scholar, 3Briones M.R.S. Egima C.M. Acosta A. Schenkman S. Exp. Parasitol. 1994; 79: 211-214Crossref PubMed Scopus (11) Google Scholar, 4Schenkman S. Eichinger D. Pereira M.E.A. Nussenzweig V. Annu. Rev. Microbiol. 1994; 48: 499-523Crossref PubMed Scopus (274) Google Scholar)). Several studies characterizing the nature and structure of the SA acceptors have been published. These acceptors are abundant on the parasite surface and were first described as major surface glycoproteins of epimastigotes by Alves and Colli(5Alves M.J.M. Colli W. FEBS Lett. 1975; 52: 188-190Crossref PubMed Scopus (50) Google Scholar), who called them bands A, B, and C. Subsequently, a similar cell surface glycoprotein complex, called GP24, GP31, and GP37 was described by Ferguson et al.(6Ferguson M.A.J. Snary D. Allen A.K. Biochim. Biophys. Acta. 1985; 842: 39-44Crossref PubMed Scopus (16) Google Scholar), and Previato et al.(7Previato J.O. Andrade A.F. Pessolani M.C. Mendoça-Previato L. Mol. Biochem. Parasitol. 1985; 16: 85-96Crossref PubMed Scopus (175) Google Scholar) first described a 43-kDa SA acceptor. More recently, they have been called 38/43 glycoconjugates(8Previato J.O. Jones C. Gonçalves L.P.B. Wait R. Travassos L.R. Mendoça-Previato L. Biochem. J. 1994; 301: 151-159Crossref PubMed Scopus (105) Google Scholar), and the so called epimastigote lipophosphoglycan-like molecule could belong to the same family of molecules(9Singh B.N. Lucas J.J. Beach D.H. Costello C.E. J. Biol. Chem. 1994; 269: 21972-21982Abstract Full Text PDF PubMed Google Scholar). In metacyclic trypomastigote forms, the SA acceptors were reported originally as the 35/50-kDa antigens (10Yoshida N. Mortara R.A. Araguth M.F. Gonzalez J.C. Russo M. Infect. Immun. 1989; 57: 1663-1667Crossref PubMed Google Scholar, 11Mortara R.A. da Silva S. Araguth M.F. Blanco S.A. Yoshida N. Infect. Immun. 1992; 60: 4673-4678Crossref PubMed Google Scholar) that were subsequently defined as mucin-like glycoproteins(12Schenkman S. Ferguson M.A.J. Heise N. Cardoso de Almeida M.L. Mortara R.A. Yoshida N. Mol. Biochem. Parasitol. 1993; 59: 293-304Crossref PubMed Scopus (178) Google Scholar). In the trypomastigote forms found in mammals, the SA acceptors were described as a group of molecules that share the stage-specific epitope 3 (Ssp-3) (13Andrews N.W. Hong K. Robbins E.S. Nussenzweig V. Exp. Parasitol. 1987; 64: 474-484Crossref PubMed Scopus (198) Google Scholar), an epitope dependent on parasite sialylation(14Schenkman S. Jiang M.-S. Hart G.W. Nussenzweig V. Cell. 1991; 65: 1117-1125Abstract Full Text PDF PubMed Scopus (376) Google Scholar), and were also identified as mucin-like molecules that appear larger than the epimastigote and metacyclic mucins on SDS-polyacrylamide gel electrophoresis(15Acosta A. Schenkman R.P.F. Schenkman S. Braz. J. Med. Biol. Res. 1994; 27: 439-442PubMed Google Scholar, 16Almeida I.C. Ferguson M.A.J. Schenkman S. Travassos L.R. Biochem. J. 1994; 304: 793-802Crossref PubMed Scopus (195) Google Scholar). These trypomastigote mucins also contain some terminal α-galactosyl residues(16Almeida I.C. Ferguson M.A.J. Schenkman S. Travassos L.R. Biochem. J. 1994; 304: 793-802Crossref PubMed Scopus (195) Google Scholar). In summary, these mucin-like molecules are glycoproteins rich in threonine and serine that are linked to the parasite membrane via a glycosylphosphatidylinositol (GPI) anchor and that contain novel O-linked oligosaccharides. The O-linked oligosaccharides are attached to the protein via GlcNAc residues and act as SA acceptor sites for the parasite TS. The chemical structure of O-linked oligosaccharides of epimastigote mucins of G (8Previato J.O. Jones C. Gonçalves L.P.B. Wait R. Travassos L.R. Mendoça-Previato L. Biochem. J. 1994; 301: 151-159Crossref PubMed Scopus (105) Google Scholar) and Y (17Previato J.O. Jones C. Xavier M.T. Wait R. Travassos L.R. Parodi A.J. Mendonça-Previato L. J. Biol. Chem. 1995; 270: 7241-7250Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) strains have recently been elucidated. They are quite similar but differ in their average size and in some of the Gal linkages. The glycan structure of the GPI anchor of the epimastigote mucin (Y strain) has also been reported(17Previato J.O. Jones C. Xavier M.T. Wait R. Travassos L.R. Parodi A.J. Mendonça-Previato L. J. Biol. Chem. 1995; 270: 7241-7250Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar).Several lines of evidence suggest that the 35/50-kDa mucins of metacyclic-trypomastigotes are involved in host cell invasion. Monoclonal antibodies directed against the mucin, and the purified molecule itself, are able to inhibit parasite entry(10Yoshida N. Mortara R.A. Araguth M.F. Gonzalez J.C. Russo M. Infect. Immun. 1989; 57: 1663-1667Crossref PubMed Google Scholar, 18Ruiz R.C. Rigoni V.L. Gonzalez J. Yoshida N. Parasite Immunol. 1993; 15: 121-125Crossref PubMed Scopus (73) Google Scholar), and the 35/50-kDa antigens are capped and locally released during invasion (12Schenkman S. Ferguson M.A.J. Heise N. Cardoso de Almeida M.L. Mortara R.A. Yoshida N. Mol. Biochem. Parasitol. 1993; 59: 293-304Crossref PubMed Scopus (178) Google Scholar). Epimastigotes are unable to enter mammalian cells but express large amounts of mucins with similar size recognized by the same monoclonal antibodies. Therefore, we decided to investigate possible subtle structural differences between the mucins of these two stages. We found that, after differentiation of epimastigotes into metacyclic forms, the lipid portion is modified, while the oligosaccharide chains and the glycan structure of the GPI are conserved. This lipid change might correlate with the increased infectivity of metacyclic forms and the ability of the parasite to shed the mucins upon invasion of the host cell(12Schenkman S. Ferguson M.A.J. Heise N. Cardoso de Almeida M.L. Mortara R.A. Yoshida N. Mol. Biochem. Parasitol. 1993; 59: 293-304Crossref PubMed Scopus (178) Google Scholar).MATERIALS AND METHODSParasitesEpimastigotes of T. cruzi strain G (19Yoshida N. Infect. Immun. 1983; 40: 836-839Crossref PubMed Google Scholar) were grown at 28°C in liver-infusion tryptose medium (20Camargo E.P. Rev. Inst. Med. Trop. Sao Paulo. 1964; 6: 93-100PubMed Google Scholar) containing 10% fetal bovine serum. Metacyclic trypomastigotes were purified from cultures at stationary phase by passage through a diethylaminoethyl cellulose column, as described in (19Yoshida N. Infect. Immun. 1983; 40: 836-839Crossref PubMed Google Scholar). The purity of the metacyclic trypomastigote preparations were estimated by morphology and/or by complement-mediated lysis assay using normal human sera.Purification of MucinsMucins from epimastigotes and metacyclic trypomastigotes were extracted as described for the lipophosphoglycan of Leishmania donovani(21McConville M.J. Blackwell J.M. J. Biol. Chem. 1991; 266: 15170-15179Abstract Full Text PDF PubMed Google Scholar). Briefly, parasites (~7 × 1010 epimastigotes and ~8 × 1010 metacyclic trypomastigotes) were freeze-dried and placed in a sonicating water bath for 10 min with 50 ml of chloroform/methanol/water (1:2:0.8, by volume). After centrifugation (2,000 × g, 5 min) the insoluble material was re-extracted twice more, as described above, and the final insoluble pellet was used as a source of delipidated parasites. The pooled chloroform/methanol/water soluble fractions were evaporated under nitrogen, and the residue was extracted with 50 ml of butan-1-ol/water (2:1, by volume). The butan-1-ol phase, containing the lipid fraction (F1) was collected, and the aqueous phase (F2) was washed twice with 25 ml of water-saturated butan-1-ol and concentrated. The delipidated parasites were extracted by sonication (three times) with 25 ml of 9% butanol in water. The soluble material was pooled, concentrated, and freeze-dried to produce a polar fraction (F3). Most of the mucins from epimastigotes were recovered in F2, while mucins of metacyclic trypomastigotes were recovered in the F3 fraction. The mucins were resuspended in 0.5 ml of 0.1 M ammonium acetate in 5% propan-1-ol (v/v) (buffer A) and fractionated on an octyl-Sepharose column (10 × 0.5 cm), previously equilibrated in buffer A. The column was washed with 15 ml of buffer A and eluted with a linear gradient over 100 ml at a flow rate of 12 ml/h, starting with buffer A and ending with 60% (v/v) propan-1-ol in water. Fractions of 1 ml were assayed for reactivity with the monoclonal antibody 10D8 (10Yoshida N. Mortara R.A. Araguth M.F. Gonzalez J.C. Russo M. Infect. Immun. 1989; 57: 1663-1667Crossref PubMed Google Scholar) by using a chemiluminescent dot-blotting assay(16Almeida I.C. Ferguson M.A.J. Schenkman S. Travassos L.R. Biochem. J. 1994; 304: 793-802Crossref PubMed Scopus (195) Google Scholar). The immunoreactive material from the column was pooled, dried by rotatory evaporation, resuspended in water, and freeze-dried to remove traces of ammonium acetate. For the material submitted to lipid analysis, the samples were further partitioned between water and butan-1-ol to remove any remaining phospholipid and/or glycolipid contaminants. The aqueous phase was washed twice more with water-saturated butanol and freeze-dried again.Nitrous Acid Deamination and NaB3H4Reduction of GPI Neutral Glycans and Recovery of the Phosphatidylinositol MoietiesAbout 20 nmol of 35/50-kDa mucin of epimastigotes and metacyclic trypomastigotes, as judged by the myo-inositol content (see below) were freeze-dried, resuspended in 50 μl of 0.3 M sodium acetate, pH 4.0, and washed three times with 100 μl of butan-1-ol saturated with water. Then 25 μl of 1 M sodium nitrite was added to the aqueous phase (two times at 1-h intervals), and deamination was performed for 2 h at 37°C. The released phosphatidylinositol (PI) moieties were recovered by three extractions with 100 μl of butan-1-ol saturated with water and analyzed by electrospray-mass spectrometry (ES-MS), as described below. The deaminated molecules remaining in the aqueous phase were reduced with NaB3H4, as described in (22Ferguson M.A.J. Fukuda M. Kobata A. Glycobiology: A Practical Approach. IRL Press, Oxford1993: 349-383Google Scholar), and repurified by chromatography on a Sephadex G-10 column, where they eluted in the void volume. The deaminated and NaB3H4-reduced mucins were then dephosphorylated in 48% aqueous HF for 60 h at 0°C and re-N-acetylated, and the GPI neutral glycans were purified from radiochemical contaminants by downward paper chromatography and high voltage paper electrophoresis(22Ferguson M.A.J. Fukuda M. Kobata A. Glycobiology: A Practical Approach. IRL Press, Oxford1993: 349-383Google Scholar). The 3H-labeled neutral glycans were analyzed by Bio-Gel P4 chromatography and Dionex high-performance anion-exchange chromatography. The elution positions of the radiolabeled glycans were expressed in glucose units (Gu) and Dionex units, respectively, by linear interpolation of the elution position between adjacent glucose oligomer internal standards(22Ferguson M.A.J. Fukuda M. Kobata A. Glycobiology: A Practical Approach. IRL Press, Oxford1993: 349-383Google Scholar).Microsequencing of the GPI Glycan CoresThree aliquots (20,000 cpm) of the neutral glycan fractions recovered from the Bio-Gel P4 column were dried in speed-vac and subjected to digestion with jack bean α-mannosidase (Boehringer Mannheim), Aspergillus saitoi Manα1-2Man-specific α-mannosidase (Oxford Glycosystems) and by partial acetolysis. Digestions were carried out with 0.75 units of jack bean α-mannosidase in 30 μl of 0.1 M sodium acetate buffer, pH 5.0, for 16 h at 37°C or with 0.01 units of A. saitoi α-mannosidase in 10 μl of 0.1 M sodium acetate buffer, pH 5.0, for 16 h at 37°C. Acetolysis was performed as described in (23Schneider P. Ferguson M.A.J. Methods Enzymol. 1995; 250: 614-630Crossref PubMed Scopus (50) Google Scholar). The products were analyzed by high performance thin layer chromatography (HPTLC) on silica gel 60 plates (Merck) using solvent A, propan-1-ol/acetone/water (9:6:4, by volume). Radioactive glycans were detected with a radioactivity linear analyzer (Raytest RITA) and visualized by fluorography after spraying with En3Hance (DuPont NEN). A set of standards, terminating in [1-3H]2,5-anhydromannitol (AHM*), was prepared by partial acid hydrolysis (23Schneider P. Ferguson M.A.J. Methods Enzymol. 1995; 250: 614-630Crossref PubMed Scopus (50) Google Scholar) of authentic Manα1-2Manα1-2Manα1-6Manα1-4AHM*, prepared from T. cruzi lipopeptidophosphoglycan (LPPG)(24de Lederkremer R.M. Lima C. Ramírez M.I. Ferguson M.A.J. Homans S.W. Thomas-Oates J. J. Biol. Chem. 1991; 266: 23670-23675Abstract Full Text PDF PubMed Google Scholar). This so called Man4 ladder contains a mixture of Manα1-2Manα1-2Manα1-6Manα1-4AHM* (M4), Manα1-2Manα1-6Manα1-4AHM* (M3), Manα1-6Manα1-4AHM* (M2), Manα1-4AHM* (M1), and AHM*.Location of the Ethanolamine Phosphate and 2-Aminoethylphosphonate GroupsThis method was adapted from (23Schneider P. Ferguson M.A.J. Methods Enzymol. 1995; 250: 614-630Crossref PubMed Scopus (50) Google Scholar). Deaminated and NaB3H4-reduced mucins, 500,000 cpm (before dephosphorylation with aqueous HF), were subjected to partial acid hydrolysis in 500 μl of 0.1 M trifluoroacetic acid (100°C, 12 h). The samples were dried in a speed-vac and redried twice from 200 μl of water. The hydrolysates were then split into three portions and treated as follows: (i) dephosphorylated with aqueous HF (60 h, 0°C), (ii) treated with jack bean α-mannosidase prior to dephosphorylation with aqueous HF, or (iii) passed directly through a 0.2-ml QAE-Sephadex A-25 column. The products were analyzed by HPTLC using solvent A, as described above.Mild Alkaline Treatment of 35/50-kDa Mucins and Fractionation of OligosaccharitolsAbout 200 nmol of metacyclic 35/50-kDa mucin was freeze-dried twice, resuspended in 250 μl of 0.1 M NaOH containing 250 mM NaB2H4, and incubated for 24 h at 37°C. The sample was acidified with 1 M acetic acid, desalted by passage through 0.6 ml of AG 50-X12 (H+) (Bio-Rad), and dried, and boric acid was removed by co-evaporation with 250 μl of 5% acetic acid in methanol (three times) and with 250 μl of methanol (three times). Residual acetic acid was removed by co-evaporation with 50 μl of toluene (two times). Radiolabeled oligosaccharitols were prepared from freeze-dried mucins resuspended in 15 μl of 0.1 M of NaOH containing 36 mM NaB3H4 (13.6 Ci/mmol, DuPont NEN) and incubated for 24 h at 37°C, followed by desalting as described above. Released radiolabeled oligosaccharitols were purified from radiochemical contaminants by descending paper chromatography on Whatman 3MM paper in butan-1-ol/ethanol/water (4:1:0.6, by volume) and digested with 50 mMArthrobacter ureafaciens sialidase (Oxford Glycosystems) in 25 μl of 50 mM sodium acetate (pH 5.0) for 20 h at 37°C. After desalting (as described below for the exoglycosidase digests) the neutral oligosaccharitols were fractionated by HPLC using a 5-μm hydrophilic interaction Glycoplex™ column (200 × 46 mm, PolyLC Inc.). Briefly, samples were dissolved in 80% acetonitrile, water, and the column was eluted with an 80-ml gradient from 80 to 60% acetonitrile in water, at 1 ml/min. In preparative experiments, some of the radiolabeled material was added to the NaB2H4-reduced material to act as a tracer.Purified oligosaccharitols obtained by fractionation on the Glycoplex™ HPLC column were submitted to exoglycosidase digestion, as described below, or to mild acid hydrolysis (200 μl of 40 mM trifluoroacetic acid for 1 h at 100°C) to preferentially cleave Galf glycosidic bonds(25Turco S.J. Orlandi Jr., P.A. Homans S.W. Ferguson M.A.J. Dwek R.A. Rademacher T.W. J. Biol. Chem. 1989; 264: 6711-6715Abstract Full Text PDF PubMed Google Scholar), dried in a speed-vac, redried twice from 200 μl of water and re-N-acetylated as described in (22Ferguson M.A.J. Fukuda M. Kobata A. Glycobiology: A Practical Approach. IRL Press, Oxford1993: 349-383Google Scholar). The products were analyzed by Bio-Gel P4 chromatography.Exoglycosidase DigestionNeutral 3H-labeled oligosaccharitols were analyzed before and after exoglycosidase digestion by HPTLC using solvent B, butan-1-ol/acetone/water (6:5:4, by volume). Oligosaccharitols were digested with 0.75 units of coffee bean α-galactosidase (Boehringer Mannheim) in 30 μl of 0.1 M sodium citrate/phosphate buffer, pH 6.0, for 16 h at 37°C, with 2.5 units of fresh bovine testicular β-galactosidase (Boehringer Mannheim) in 20 μl of 0.1 M sodium citrate/phosphate buffer, pH 4.5, for 16 h at 37°C, or with 0.1 units of jack bean β-galactosidase (Oxford Glycosystems) in 25 μl of 50 mM sodium acetate buffer, pH 3.5, containing 38 μg/ml BSA, for 16 h at 37°C. All reactions were terminated by heating at 100°C for 5 min. The products were desalted by passage through a column of 0.2 ml of AG50X12 (H+), over 0.2 ml of AG3X4 (OH-) over 0.1 ml of QAE-Sephadex A-25. Eluates were dried, and the residual acetic acid was removed by co-evaporation with 50 μl of toluene (two times).Compositional Analysis and Methylation AnalysisAmino acids, ethanolamine, 2-aminoethylphosphonate, and glucosamine were quantified after strong acid hydrolysis (6 M HCl, 110°C, 16 h) and derivatization with phenylisothiocyanate using a Waters Pico-Tag system, as described(22Ferguson M.A.J. Fukuda M. Kobata A. Glycobiology: A Practical Approach. IRL Press, Oxford1993: 349-383Google Scholar). Gas chromatography-mass spectrometry (GC-MS) analyses were performed with a Hewlett-Packard 5890-5970 system. The myo-inositol content of samples was measured using selected ion monitoring after strong acid hydrolysis and trimethylsilyl derivatization(22Ferguson M.A.J. Fukuda M. Kobata A. Glycobiology: A Practical Approach. IRL Press, Oxford1993: 349-383Google Scholar). Monosaccharide and lipid contents were measured after methanolysis, re-N-acetylation, and trimethylsilyl derivatization(22Ferguson M.A.J. Fukuda M. Kobata A. Glycobiology: A Practical Approach. IRL Press, Oxford1993: 349-383Google Scholar). Methylation linkage analysis was performed as described(22Ferguson M.A.J. Fukuda M. Kobata A. Glycobiology: A Practical Approach. IRL Press, Oxford1993: 349-383Google Scholar).Electrospray-Mass SpectrometryES-MS data were obtained with a VG-Quattro triple-quadrupole mass spectrometer (Fisons Instruments, United Kingdom) coupled to a Michrom microbore HPLC system. Analysis of PI fractions was performed in negative ion mode, and aliquots of PI samples (20 μl of PI dissolved in chloroform/methanol/water (10:10:3, by volume)) were injected into the electrospray source at 5 μl/min. Source and spectrometer parameters were optimized using a standard of soybean PI(26Heise N. Cardoso de Almeida M.L. Ferguson M.A.J. Mol. Biochem. Parasitol. 1995; 70: 71-84Crossref PubMed Scopus (36) Google Scholar). The masses of the released O-linked oligosaccharitol components were determined in negative ion mode. Samples, dissolved in 20 μl of 50% acetonitrile, were injected into the electrospray source at 5 μl/min. Source and spectrometer conditions were optimized using a standard of maltoheptaose (Sigma).1H NMR-One-dimensional 500-MHz 1H NMR spectra of the individual oligosaccharitols were obtained using a Bruker AM 500 spectrometer equipped with a 5-mm triple resonance probe, and the samples were dissolved in 0.5 ml of 2H2O after repeated exchange in 2H2O. All experiments were performed at 300 K, and chemical shifts were referenced externally to acetone (2.225 ppm). Further assignments for oligosaccharitol c were deduced from two-dimensional 1H-1H experiments. Correlated spectroscopy and triple quantum-filtered correlated spectroscopy experiments were performed using a sweep width of 2,200 Hz, and 4,000 data points, and 512 increments were collected in f1. The rotating frame Overhauser effect spectroscopy experiment used 64 transients of 4,000 data points, and 1,024 increments were collected in f1. The spectral width collected was 4,400 Hz in each domain, and the mixing time was 500 ms. In the total correlation spectroscopy experiment, 64 transients of 4,000 datapoints were collected, and 512 experimental increments were collected in f1. The sweep width was 1,500 Hz, and the mixing time used was 203 ms.Trans-sialylation of Mucin O-Linked OligosaccharitolsPurified radiolabeled neutral oligosaccharitols, obtained after fractionation on the Glycoplex™ HPLC column, were dried in a speed-vac and redissolved in 0.02 M HEPES buffer (pH 7.0), 1 mM 3'-sialyllactose (SAα2-3Galβ1-4Glc) (Boehringer Mannheim), 0.2% bovine serum albumin (Ultrapure, Boehringer Mannheim) and incubated with purified T. cruzi TS(27Schenkman S. Chaves L.B. Pontes de Carvalho L. Eichinger D. J. Biol. Chem. 1994; 269: 7970-7975Abstract Full Text PDF PubMed Google Scholar). After 2 h at 37°C, the reaction was stopped by the addition of 1 ml of water, and the amount of nonsialylated products was quantified by passage through a 0.5-ml QAE-Sephadex A-25 column equilibrated in water. Sialylated products were recovered after washing the column with 8 ml of water and elution with 1 ml of 1 M ammonium formate. Alternatively, the products were adjusted to 5 mM sodium acetate buffer, pH 4.0, and chromatographed on a Mono Q column, as described in (28Kobata A. Endo T. Fukuda M. Kobata A. Glycobiology: A Practical Approach. IRL Press, Oxford1993: 79-102Google Scholar), to determine the extent of sialylation of each individual oligosaccharide.RESULTSThe mucins were purified from epimastigotes and metacyclic trypomastigotes by solvent extraction and octyl-Sepharose chromatography. The material recognized by the monoclonal antibody 10D8, specific for the sialic acid acceptors, eluted at 25% (v/v) propan-1-ol and appeared as two bands with apparent molecular masses of 35 and 50 kDa on SDS-polyacrylamide gel electrophoresis, as shown previously for the metacyclic trypomastigote sialic acid acceptors (12Schenkman S. Ferguson M.A.J. Heise N. Cardoso de Almeida M.L. Mortara R.A. Yoshida N. Mol. Biochem. Parasitol. 1993; 59: 293-304Crossref PubMed Scopus (178) Google Scholar). The significance of the double nature of the antigen is unknown, but it may reflect the presence of at least two different (O-glycosylated/GPI-anchored) gene products. The purified mucins were judged to be free of T. cruzi LPPG, which migrates near the front of an SDS-polyacrylamide electrophoresis gel, by silver staining and by Western blot analysis using an LPPG-specific antibody (data not shown). Based on the myo-inositol content of the recovered material (25 nmol/1010 cells), the metacyclic mucin is present at a minimum of 1.5 × 106 copies/parasite. The mucins eluted from the octyl-Sepharose column were subjected to compositional analysis, showing that amino acids (particularly Ser and Thr) and monosaccharides (Man, Gal, GlcNAc, and SA) together with myo-inositol, ethanolamine, 1-O-hexadecylglycerol, and fatty acids were present in both preparations in amounts similar to those reported previously(8Previato J.O. Jones C. Gonçalves L.P.B. Wait R. Travassos L.R. Mendoça-Previato L. Biochem. J. 1994; 301: 151-159Crossref PubMed Scopus (105) Google Scholar, 12Schenkman S. Ferguson M.A.J. Heise N. Cardoso de Almeida M.L. Mortara R.A. Yoshida N. Mol. Biochem. Parasitol. 1993; 59: 293-304Crossref PubMed Scopus (178) Google Scholar). In addition, a previously unidentified peak in the amino acid analyses (with a retention time of 3.2 min) was shown to co-elute with an authentic standard of 2-aminoethylphosphonate (2-AEP). The molar ratio of ethanolamine to 2-AEP was approximately 1:1 for both preparations.GPI Lipid StructureThe mucins (approximately 20 nmol of each, based on myo-inositol content) were subjected to nitrous acid deamination and extracted with butan-1-ol. The butan-1-ol extracts, containing the released PI moieties, were analyzed by ES-MS. The mucin isolated from epimastigotes produced one major pseudomolecular ion at m/z 795.5 and mi
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