Glycobiology Advance Access originally published online on June 2, 2005
Glycobiology 2005 15(10):895-904,; doi:10.1093/glycob/cwi084
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© Published by Oxford University Press 2005.
Structures of the O-linked oligosaccharides of a complex glycoconjugate from Pseudallescheria boydii
2 Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, CCS, Bloco I, Ilha do Fundão, Rio de Janeiro, RJ 21941-970, Brazil; 3 Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná (UFPR), Curitiba, PR 81531-990, Brazil; 4 Faculty of Medicine, Kennedy Institute of Rheumatology Division, Imperial College London, 1, Aspenlea Road, Hammersmith, London W6 8LH, UK; and 5 Laboratory for Molecular Structure, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK
1 To whom correspondence should be addressed; e-mail: eliana.bergter{at}micro.ufrj.br
Received on November 8, 2004; revised on May 20, 2005; accepted on May 23, 2005
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Abstract |
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Nonreducing O-linked oligosaccharides were obtained from the peptidorhamnomannan of mycelia of Pseudallescheria boydii by alkaline ß-elimination under reducing conditions. They were separated by gel filtration chromatography to give three oligosaccharide fractions. The major oligosaccharide from fraction 1 was characterized by a combination of techniques including electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI MS/MS), matrix-assisted laser desorption ionization mass spectrometry (MALDI MS), nuclear magnetic resonance (NMR), and methylation gas–liquid chromatography-mass spectrometry (GC-MS) analysis. It was branched, with a principal chain of
-Rhap-(1 
3)--Rhap-(1 3)--Manp-(1 2)-Man-ol substituted at O-6 of mannitol with an -Glcp-(1 4)-ß-Galp group. Species containing one and two additional -Glcp-(1 4) substituents in the rhamnose branch were also present. The major component of fraction 2 was a substructure of oligosaccharide-1, lacking a hexose from the Glc-Gal branch. Fraction 3 contained a mixture of smaller, unbranched, oligosaccharides. In hapten inhibition tests, fractions 1 and 2 blocked the reaction between peptidorhamnomannan (PRM) and rabbit anti-P. boydii mycelium hyperimmune serum by 
75%, whereas fraction 3 inhibited by 55%. Key words: ELISA / ESI MS / MS / MALDI MS / methylation-GC-MS / Pseudallescheria boydii mycelia
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Introduction |
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Pseudallescheria boydii is an emerging fungal pathogen, frequently found in soil and polluted water, which causes localized and disseminated infections in both immunocompetent and immmunocompromised hosts (Rippon, 1998)
. P. boydii in tissue specimens resembles other fungal species and is not easily distinguished from Aspergillus. Novel O-Linked oligosaccharides have been obtained from cell wall glycoproteins of a variety of fungi, including Saccharomyces cerevisiae (Ballou, 1990), Sporothrix schenckii (Lopes-Alves et al., 1992), Candida albicans (Gemmil and Trimble, 1999), and Aspergillus fumigatus (Leitão et al., 2003). These O-linked glycans may contain novel antigens and thus are potentially diagnostically useful.
Recently, a peptidorhamnomannan (PRM) isolated from mycelial forms of P. boydii was characterized chemically and immunologically (Pinto et al., 2001). It reacted strongly with an antiserum against P. boydii mycelium, and this interaction was only weakly inhibited by the PRM from S. schenckii or by peptidogalactomannan from A. fumigatus, suggesting that P. boydii expresses antigens which are related to, but are not cross reactive with S. schenckii peptidopolysaccharide and the major Aspergillus glycoconjugate. This rhamnose-containing antigen could, therefore, be diagnostically useful, for example in cases of mixed allergic bronchopulmonary fungal disease due to P. boydii and Aspergillus (Lake et al., 1990).
In this study, we have investigated the PRM of P. boydii for the presence of O-linked oligosaccharides. Several low-weight oligosaccharides were obtained from the PRM by ß-elimination under reducing conditions, and their structures were determined using methylation analysis,1H and 13C nuclear magnetic resonance (NMR) spectroscopy, matrix-assisted laser desorption ionization mass spectrometry (MALDI MS), and electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI MS/MS). In addition, the antigenicity of the oligosaccharides was evaluated by investigating their ability to inhibit the reaction between PRM and rabbit anti-P. boydii mycelium hyperimmune serum.
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Results |
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Alkaline reductive ß-elimination and fractionation of resulting oligosaccharides
PRM was submitted to ß-elimination under reducing conditions (Leitão et al., 2003)
to release the O-linked oligosaccharides as their alditol derivatives, thus avoiding degradation by "peeling" reactions. The products were separated by gel filtration chromatography on Biogel P-2, giving four fractions (Figure 1). The components eluting in the void volume had strong absorbance at 280 nm, were rich in carbohydrate, and presumably correspond to the peptide moiety bearing sugar chains resistant to base-catalyzed elimination, probably N-linked rhamnomannans.
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The three fractions eluting in the oligosaccharide range of the column (based on calibration with glucose oligomers) were designated, in the order of elution, fractions 1, 2, and 3, and were collected and freeze-dried.
Characterization of oligosaccharides from fraction 1
Monosaccharide composition.
The MALDI mass spectrum of fraction 1 (Figure 2a) contained sodium and potassium cationized molecules at m/z 983.5 and 999.6 respectively, consistent with a hexasaccharide comprising three hexose, two methylpentose, and one hexitol residues. Minor signals were present at m/z 821.5 and 837.6, attributable to a pentasaccharide with one fewer hexose residues, and at m/z 1145.5 and 1161.7, consistent with addition of a further hexosyl unit to the hexasaccharide. Electrospray ionization MS (Figure 2b) also detected hexasaccharide and pentasaccharide molecular ions, together with an additional sodiated molecule at m/z 659.3, consistent with one hexose, two methylpentose, and one hexitol residues.
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The monosaccharide composition of this fraction was Gal : Glc : Man : Rha, (from high-performance thin-layer chromatography [HPTLC]) which were present in a 1.0:1.2:2.1:1.9 molar ratio (gas–liquid chromatography-mass spectrometry [GC-MS] of derived alditol acetates). Together with the MS data, this indicates that the major component in this fraction is a hexasaccharide consisting of one galactose, one glucose, two mannose, and two rhamnose monosaccharide residues. The ratio of monosaccharides deviates somewhat from the theoretical values for a hexasaccharide, attributable to the presence of a small proportion of truncated oligosaccharides and a heptasaccharide containing an additional Glc unit, and also to partial acid-mediated destruction of each aldose at different rates.
Methylation analysis.
The GC-MS analysis of O-methylalditol acetates derived via sodium borodeuteride reduction in an intermediate step was in agreement with a predominant hexasaccharide structure. Six main O-methylalditol acetates were found, with their respective retention times (in minutes) and percentage compositions as follows: 2,3,4-Me3-Rha (7.85, 13%), 2,4-Me2-Rha (9.91, 15%), nondeuterated 2,3,4,6-Me4-Man units (9.99, 20%), monodeuterated 2,3,4,6-Me4-Glc (10.10, 22%), 2,4,6-Me3-Man (13.24, 14%), and 2,3,6-Me3-Gal (13.93, 11%). There was also a smaller peak of 2,3,6-Me3-Glc (14.30, 6%) (Table I).
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NMR spectroscopy.
A single ß-anomeric signal in the 1H NMR spectrum (Figure 3a) at 4.52 ppm was accompanied by -anomeric peaks at 5.08 and 4.98 ppm at about twice its integrated intensity: the peak at 4.98 ppm clearly consisted of two incompletely resolved signals. A total correlation spectroscopy (TOCSY) spectrum identified five separate monosaccharide residues associated with these anomeric signals, from their characteristic patterns of signals and 1H-1H coupling constants. Two almost completely overlapped anomeric signals at 5.08 ppm were assigned to -Rha and -Man; with a further -Rha (H1 at 4.99 ppm), -Glc (H1 at 4.98 ppm), and ß-Gal (H1 at 4.52 ppm). These results are consistent with monosaccharide analysis of the whole glycoconjugate fraction, which identified mannose, rhamnose, galactose, and glucose as constituent residues (Pinto et al., 2001). 1H chemical shifts are recorded for these residues in Table I. The 13C NMR spectrum of all three oligosaccharides was weak, due to scarcity of sample. Fraction 1 gave the best signal-to-noise ratio, and its 13C spectrum is shown in Figure 4. Five anomeric signals can readily be identified. Assignment of the 13C spectrum from heteronuclear single quantum coherence (HSQC) cross peaks corresponding to assigned signals in the 1H spectrum was straightforward, and 13C chemical shifts are also listed in Table II.
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An expansion of the rotating frame Overhauser enhancement spectroscopy (ROESY) spectrum of fraction 1 is shown in Figure 5. Cross peaks may be seen between anomeric protons and ring protons of the same and adjacent residues. ROESY cross peaks across glycosidic linkages were used to infer the sequence of monosaccharide residues in the oligosaccharide and to determine the positions of glycosylation. A cross peak is seen from H1 of -Glc to H4 and H6 of ß-Gal. H1 of -Rha1 (the terminal unit) is close to H3 of -Rha2 (the adjacent unit), and H1 of -Rha2 to H2 or H3 of -Man (the cross peaks Rha2 H1-H3 and Rha2 H1-Man H3 are coincident: see Table II). These results imply the two partial sequences -Glc-(14)-ß-Gal and -Rha-(13)--Rha-[12(or 3)]--Man. Long-range HSQC cross peaks between H1 of Rha1 and C3 of Rha2; H1 of Rha2 and C3 of Man; and C1 of Glc to H4 of Gal confirmed these sequences (data not shown) and resolve the ambiguity in the Rha2-Man linkage arising from the ROESY spectrum. ROESY cross peaks are seen between H1 of -Man and a ring proton at 3.83 ppm (13C, 82.5 ppm) and between H1 of Gal and a proton signal at 4.23 ppm (13C, 74.5 ppm). As methylation analysis has established the presence of a 2,6-di-O-substituted mannitol residue, these signals may be assigned to mannitol H2/C2 and H6/C6 respectively. The remaining assignments for the mannitol residue are tentative. The structure implied by NMR and methylation analysis is shown in Figure 6.
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Tandem mass spectrometry.
The sodium cationized molecules at m/z 983.4, 821.4 and 659.3 in the electrospray mass spectrum of fraction 1 were selected for collision induced decomposition, and MS/MS analysis. The parent ion at m/z 659.3 loses methylpentose forming m/z 513.3; a further methylpentose loss gives m/z 367.2, which in turn eliminates hexose to give m/z 205.1 ([hexitol + Na]+); consistent with a linear Rha-Rha-Hex-Mannitol structure. The parent ion at m/z 821.4 (Figure 7a) loses either methylpentose (forming m/z 675.3) or hexose (giving m/z 659.3), which is consistent with the presence of two branches, one terminated by rhamnose and the other by hexose. Loss of a further methylpentose gives a fragment ion at m/z 529.3, which loses one (m/z 367.2), and then a second hexose, to give a sodiated hexitol fragment at m/z 205.1, consistent with the structure Rha-Rha-Hex-(Hex)-Mannitol (O-2 in Table III).
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On collisional activation, the sodiated molecule at m/z 983.4 loses one hexose (forming m/z 821.3), then two methylpentoses (giving m/z 529.3), and another hexose forming a sodiated hexose-hexitol fragment at m/z 367.2, which supports the structure Rha-Rha-Hex-(Hex-Hex)-Mannitol determined by NMR for O-1 (Table III).
A minor sodium cationized molecule was present at m/z 1145.6, consistent with addition of a hexose residue to oligosaccharide O-1. Tandem mass spectrometry suggested a biantennary structure with terminal hexose and methylpentose residues, but did not unambiguously localize the additional hexose to one or the other branch.
Permethylated oligosaccharides frequently produce more informative mass spectra than native samples, providing higher sensitivity, improved detection of minor species, unambiguous assignment of branching, and reliable discrimination between Y type fragment ions and truncated molecules, which for underivatized oligosaccharides are isobaric.
The ESI mass spectrum of methylated fraction 1 contained sodiated molecules at m/z 841.4, 1045, 1249, and 1453, originating from permethylation of the underivatized species at m/z 659, 821, 983, and 1145 (Table III). An additional signal, not corresponding to any species detected in the underivatized fraction, was observed at m/z 1657.6. Figure 7b–d show tandem mass spectra of some of these species. The tandem mass spectrum of permethylated O-1 is entirely consistent with the structure deduced from the NMR evidence. The loss of 188 and 218 from the sodiated molecule (giving the fragments m/z 1061 and m/z 1031) demonstrates the presence of both terminal rhamnose and terminal hexose. The sequential elimination of 188, 174, and 204 to give the fragment at m/z 683 supports the presence of a Rha-Rha-Hex branch, whereas the loss of the terminal hexose (218) from m/z 683, followed by loss of another (internal) hexose to give a sodiated mannitol fragment at m/z 261 is evidence for the dihexosyl structure of the other branch.
Signals at m/z 1453 and 1657 in the ESI spectrum of permethylated fraction 1 are consistent with species containing one (oligosaccharide O-1a) or two (oligosaccharide O-1b) additional hexose residues compared to O-1. Tandem mass spectra of these oligosaccharides are shown in Figure 7c and d. Both spectra contained fragments at m/z 261, 465, and 683, implying that the dihexosyl branch was conserved in both oligosaccharides and that therefore the additional hexose residues are present in the rhamnose branch. This is in agreement with observation of the fragment series m/z 1469, 1295, 1091, 887, and 683 in O-1b which is evidence for Rha-Rha-Hex-Hex-Hex. Similarly, O-1a lost terminal rhamnose, followed by sequential elimination of internal rhamnose and two hexoses, establishing the substructure Rha-Rha-Hex-Hex. The presence of these heptasaccharide and octasccharide components would explain the detection of 2,3,6-Me3-Glc in the methylation analysis (Table I). A minor anomeric signal at 5.00 ppm (H2 3.57 ppm) in the 1H NMR spectrum could originate from 4-O-substituted Glc, but no further corroborative assignments were possible.
The mass spectrometry evidence supports the NMR and methylation analysis results and leads unambiguously to the structure shown in Figure 6 for the predominant component of fraction 1, which also contains a proportion of related pentasaccharide and heptasaccharide structures (Table III).
Characterization of fractions 2 and 3
The 1H NMR spectra of oligosaccharide fractions 2 (Figure 3b) and 3 (Figure 3c) are more complex than those of fraction 1. Fraction 2 gives a spectrum with anomeric signals of Rha-1, Rha-2 and Man clearly present, but with Glc and Gal signals of much reduced intensity. The TOCSY spectrum (not shown) traces subspectra for each residue similar to that for O-1. The anomeric region of the proton spectrum, however, contains additional minor resonances not seen in the spectrum of O-1. H1 signals at 5.20 and 5.11 ppm, connected in the correlation spectroscopy and TOCSY spectra with H2 at 4.28 and 4.15 ppm respectively, are consistent with mannose or rhamnose residues. An additional ß-anomeric signal at 4.45 ppm is also present. Gel filtration chromatography (Figure 1) indicates a lower molecular mass than for fraction 1. Fraction 2, therefore, probably contains truncated substructures of O-1, the additional anomeric signals arising from terminal residues in the truncated species. Mass spectrometric analysis of the permethylated material suggested that this fraction comprised a mixture in which the components designated O-2 and O-3a (Table III) predominated.
Fraction 3 was a mixture of smaller oligosaccharides, and full NMR analysis of the structures present was not practicable. However, the two most prominent signals in the 1H spectrum, at 5.06 and 4.97 ppm respectively, are identifiable from a TOCSY spectrum (not shown) as corresponding to the terminal (Rha-1) and internal (Rha-2) rhamnose spin systems of O-1 and O-2. No signals from either ß-Gal or -Glc are visible in the proton spectra. Minor anomeric signals at 5.11 ppm (H2 at 4.15 ppm) and 4.99 ppm (H2 at 4.11 ppm) may arise from terminal rhamnose or mannose.
Fraction 3 is, therefore, likely to consist largely of oligosaccharides containing the Rha-Rha-Man substructure as in O-1 and O-2, but lacking the Glc-Gal branch.
Electrospray ionization mass spectrometry revealed cationized molecules at m/z 659.16, 513.13, and 365.7 in underivatized fraction 3; permethylation shifted these to 841.4, 667.3, and 477.18, respectively. The species at m/z 365 was consistent with a sodiated dihexose, and on collisional activation it underwent glycosidic cleavage, producing a sodiated Y1 fragment at m/z 203.
Tandem mass spectrum of the parent ion at m/z 513 exhibited losses of methylhexose, followed by hexose, leaving a sodiated mannitol fragment at m/z 205, suggesting the structure Rha-Hex-mannitol (O-3a; Table III).
The species at m/z 659 fragmented by sequential loss of two rhamnoses and a hexose, consistent with the unbranched structure Rha-Rha-Hex-Mannitol (O-3, Table III), which was confirmed by analysis of its permethylated derivative (Figure 7e). This lost terminal rhamnose (188), giving a fragment at m/z 653, followed by loss of a second (internal) rhamnose, forming m/z 479, and then hexose, leaving a sodiated methyl mannitol fragment at m/z 275. B-type nonreducing terminal-containing ions at m/z 211, (B1) 385 (B2), and 589 (B3) confirmed these assignments.
The heterogeneity of fraction 3 precluded full methylation analysis, but the presence of 1,2,3,4,6-penta-O-methyl mannitol, (and the absence of 2,3,4,6 tetra-O-methyl mannitol) is consistent with the predominance of unbranched structures.
Hapten inhibition test.
The antigenic role of the O-linked oligosaccharides was determined by a hapten inhibition test (Figure 8). Fractions 1 and 2 showed the greatest inhibitory effects (60–70%). A 50% inhibition was observed with fraction 3.
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Discussion |
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The complex glycoconjugate (PRM) from P. boydii consists of a peptide chain substituted with both O-linked and N-linked glycans (Pinto et al., 2001)
. O-Linked oligosaccharides were released by ß-elimination under mild alkaline reducing conditions in the presence of sodium borohydride, and structurally and immunologically characterized. At least six O-linked oligosaccharides were present; the most abundant being a hexasaccharide (oligosaccharide-1) the primary structure of which was determined by ESI MS/MS, MALDI MS, NMR, and methylation analysis (Figure 6). Two structures containing additional hexose residues in the rhamnose branch were partially characterized by tandem MS of their permethyl derivatives, but were not abundant enough for detailed NMR characterization. Fractions 2 and 3 contained a number of components related to oligosaccharide-1 but lacking some nonreducing terminal residues (Table III).
Some structural features of these O-linked oligosaccharides appear to be shared with the polysaccharide components of PRM (probably N-linked glycans), for example, chains terminated by (13)-linked -Rhap residues. This is also true for the O-linked oligosaccharides of A. fumigatus (Leitão et al., 2003) and suggests that the O- and N-linked glycans of P. boydii may be elaborated by the same glycosyltransferases, as occurs in S. cerevisiae (Lussier et al., 1999).
Rhamnose-containing structures appear to be the immunodominant epitopes in the rhamnomannans of P. boydii, S. schenckii and Ceratocystis stenoceras, particularly if they are present as (13)-linked -Rhap side chain units (Lloyd and Travassos, 1975; Pinto et al., 2001). Antibodies recognizing this structure may, therefore, recognize both the N-linked high molecular weight polysaccharides and the O-linked oligosaccharides in the PRM molecules. To verify this, we compared the antigenicity of de-O-glycosylated PRM with that of intact PRM using rabbit anti-P. boydii serum. The results consistently showed that 80% of the reactivity was lost after alkaline ß-elimination of PRM (data not shown). Similar results were obtained with the PRM from A. fumigatus (Leitão et al., 2003) and with PRM from S. schenckii (Penha and Bezerra, 2000). Besides the contribution of O-linked oligosaccharides to the antigenicity of PRM, recent data suggest that O-glycosylation is critical for fungal adhesion to host cells, because de-O-glycosylated PRM efficiently inhibits the adhesion P. boydii conidia to epithelial cells (HEp2) and prevents endocytosis (Pinto et al., 2004).
The immunodominance of the O-linked oligosaccharide chains was evaluated testing their ability to inhibit of reactivity between the PRM and anti-P. boydii rabbit antiserum in an enzyme-linked immunosorbent assay (ELISA) hapten system (Figure 8). Up to 75% inhibition was obtained with the oligosaccharide fractions 1 and 2 when the amounts of inhibitor and antigen in the ELISA well were the same. Fraction 3 inhibited 55% of the reaction when the same amount of antigen was used.
Our results show that O-glycosidically linked oligosaccharides are present in the PRM from the mycelial wall of P. boydii. The isolated oligosaccharide alditols blocked recognition between rabbit sera and intact PRM in a dose-dependent manner. Thus, O-glycosidically linked oligosaccharide chains, despite being the less abundant carbohydrate component of the P. boydii PRM, may account for a significant part of the antigenicity, associated with the rhamnomannan component of P. boydii PRM.
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Materials and methods |
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Microorganism and growth conditions
P. boydii, isolated from eumycotic mycetoma, was kindly supplied by Dr. Bodo Wanke from the Evandro Chagas Hospital, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil. It was maintained in Difco peptone, 10 g/L; Difco yeast extract, 5 g/L; Difco agar, 20 g/L; and glucose, 40 g/L. Cells were grown on Sabouraud solid slants and inoculated into Erlenmeyer flasks (500 mL) containing culture medium (200 mL) and incubated for 7 days at 25°C with shaking. Cultures were then transferred to the same medium (3 L) and incubated for 7 days at the same temperature with shaking; the mycelium was filtered, washed with distilled water, and stored at –20°C.
Extraction and fractionation of the PRM
The PRM fraction was obtained as described elsewhere (Pinto et al., 2001). Briefly, crude glycopeptides were extracted from intact P. boydii mycelia in phosphate buffer (50 mM, pH 7.2, at 100°C for 2 h) and purified by hexadecyltrimethylammonium bromide (Cetavlon, Merck, Darmstadt, Germany) fractionation. The mother liquors from Cetavlon precipitation were adjusted to pH 8.8 in the presence of borate and the resulting precipitates recovered by centrifugation to give the major PRM fraction. The PRM was dialyzed against distilled water and freeze-dried.
Reductive alkaline ß-elimination of PRM
PRM was de-O-glycosylated by mild alkaline treatment under reducing conditions (0.1 M NaOH, 0.5 M NaBH4, 25°C, 24 h) (Leitão et al., 2003). The reaction medium was then neutralized by addition of HOAc, deionized in an Amberlite IR 120-P column and freeze-dried. Borate salts were removed as trimethyl borate by repeated evaporation with methanol. The product was applied to a Biogel P-2 column (H2O, 20 mL/h, 2 x 140 cm, Bio-Rad, Hercules, CA). De-O-PRM was recovered in the void volume. The liberated oligosaccharide-alditol fractions were recovered in the column fractionation range. The degree of purification of the oligosaccharide fractions (1, 2 and 3) was determined by HPTLC on silica gel-60 plates (0.25 mm, Merck, Darmstadt, Germany) developed with n-butanol-EtOH-H2O (2:1:1 v/v) and visualized by orcinol-sulphuric acid.
Monosaccharide analysis
The O-linked oligosaccharides were hydrolyzed with 3 M trifluoroacetic acid at 100°C for 3 h; the resulting monosaccharides were characterized by HPTLC and quantified by GC as alditol acetate derivatives (Sawardeker et al., 1965) using an OV-225 fused silica capillary column (30 m x 0.25 mm internal diameter) with temperatures programmed from 50 to 220°C at 50°C/min, then hold.
Methylation analysis
The analysis was carried out by the method of Tischer et al. (2002) using a modification of the methylation of Ciucanu and Kerek (1984). Oligosaccharide-1 (1.0 mg) was dissolved in one drop of H2O, to which Me2SO (1.0 mL) and then MeI (1 mL) were added. After addition of powdered NaOH (0.3 g), the mixture was vigorously agitated in a vortex for 30 min and then left for 16 h. After acidification with HOAc, H2O was added and the per-O-methylated product extracted with CHCl3, which was washed three times with H2O. After evaporation, the residue was dissolved in H2SO4–H2O (1:1 v/v) for 1 h at 0°C, and the solution then diluted to 1M H2SO4, which was heated at 100°C for 18 h. The mixture of partially O-methylated aldoses was reduced with NaBD4, and then acetylated with Ac2O-pyridine to give O-methylated alditol acetates. These were examined by GC-MS on a capillary column of DB-225 (30 m x 0.25 mm internal diameter), programmed from 50°C at 40°C/min to 210°C (constant temperature). The fragments were identified by their retention times and electron impact spectra (Jansson et al., 1976).
Per-O-methylation of oligosaccharide alditols for ESI MS/MS
This was carried out on 5 µg samples, using the above procedure of Ciucanu and Kerek (1984).
NMR spectroscopy
One- and two-dimensional 1H and 13C NMR spectroscopy was carried out with a Varian Unity 500 MHz spectrometer, using pulse sequences and software supplied by the manufacturer, with additional spin-echo sequences in the ROESY and TOCSY experiments. 1H-13C correlated spectra were carried out using the WHSQC sequence (Wider and Wuthrich, 1993). ROESY spectra were obtained using a mixing time of 150 ms, and TOCSY spectra were carried out with a mixing time of 120 ms. Proton–carbon correlated spectra were recorded using the WHSQC sequence, optimized for JCH = 150 Hz for one-bond 1H-13C cross peaks and optimized for JCH = 7.7 Hz for longer-range cross peaks. Samples (1–5 mg) were taken up in 0.7 mL 99.8% D2O (Apollo Scientific Ltd., Stockport, Cheshire, UK), and spectra were recorded at 30°C except where indicated otherwise.
MALDI MS and ESI MS/MS
MALDI mass spectra were recorded with a Micromass TofSpec 2E spectrometer, equipped with a 337 nm nitrogen laser. The instrument was operated in the positive ion reflectron mode at 20 kV accelerating voltage with time lag focussing enabled. Samples were dissolved in 5% aqueous HCO2H and 0.75 µL of the mixture was mixed with an equal volume of 2.5% dihydroxybenzoic acid matrix solution (20 mg/mL in 50% aqueous EtOH) and air-dried on the stainless steel target.
For nano-electrospray MS samples were dissolved in 50% (v/v) MeOH 0.1% (v/v) aqueous HCO2H, loaded into palladium coated borosilicate nanoelectrospray needles (Protana, Odense, Denmark) and mounted in the source of a Micromass Q-TOF hybrid quadrupole/orthogonal acceleration time of flight spectrometer. Stable electrospray was obtained at capillary voltage between 1200 and 1800 V. The collision gas was argon, and collision energies and argon pressure were tuned to optimize the fragmentation pattern of individual precursor ions.
Hapten inhibition tests
Wells of flat-bottomed polyvinyl microtitre plates (Hemobag, Campinas, São Paulo, Brazil) were coated with P. boydii PRM (1 µg/well). Oligosaccharide solutions ranging from 1 to 100 ng were separately mixed with the same volume of rabbit sera against whole P. boydii hyphae and preincubated for 1 h at 37°C. The immunodominance of each reduced O-linked oligosaccharide was evaluated by ELISA (Leitão et al., 2003).
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Acknowledgements |
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We are grateful to Dr. T. Frenkiel, National Institute for Medical Research, for adapting the WHSQC sequence for the Varian spectrometer and Maria de Fátima Ferreira Soares, Universidade Federal do Rio de Janeiro, for technical assistance. The research was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo a Pesquisa no Estado do Rio de Janeiro (FAPERJ) and CAPES-Prodoc. RW thanks the Wellcome Trust, the Medical Research Council and the Arthritis Research Campaign.
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Abbreviations |
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ELISA, enzyme-linked immunosorbent assay; ESI MS, electrospray/quadrupole time-of-flight mass spectrometry; GC-MS, gas–liquid chromatography-mass spectrometry; HSQC, heteronuclear single quantum coherence; HPTLC, high-performance thin-layer chromatography; HSQC, heteronuclear single quantum coherence; MALDI MS, matrix-assisted laser desorption ionization mass spectrometry; NMR, nuclear magnetic resonance; PRM, peptidorhamnomannan; ROESY, rotating frame Overhauser enhancement spectroscopy; TOCSY, total correlation spectroscopy
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References |
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Ballou, C.E. (1990) Isolation, characterization, and properties of Saccharomyces cerevisiae mnn mutants with nonconditional protein glycosylation defects. Methods Enzymol., 185, 440–470.[Medline]
Ciucanu, I. and Kerek, F. (1984) A simple and rapid method for the permethylation of carbohydrates. Carbohydr. Res., 131, 209–217.[CrossRef]
Gemmil, T.R. and Trimble, R.B. (1999) Overview of N- and O-linked oligosaccharide structures found in various yeast species. Biochim. Biophys. Acta, 1426, 227–237.[Medline]
Jansson, P.-E., Kenne, L., Liedgren, H., Lindberg, B., and Lönngren, J. (1976) A practical guide to the methylation analysis of carbohydrates. Chem. Commun. University Stockholm, 8, 1–75.
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