ß-Galactofuranose-containing O-linked oligosaccharides present in the cell wall peptidogalactomannan of Aspergillus fumigatus contain immunodominant epitopes

Eduardo A. Leitão1,3, Vera C.B. Bittencourt3, Rosa M.T. Haido4, Ana P. Valente5, Jasna Peter-Katalinic6, Matthias Letzel7, Lauro M. de Souza8 and Eliana Barreto-Bergter2,3

3 Instituto de Microbiologia Professor Paulo de Góes, CCS Universidade Federal do Rio de Janeiro (UFRJ), 21944-970, Rio de Janeiro, RJ, Brasil; 4 Instituto Biomedico, Universidade do Rio de Janeiro, 20211-040, Rio de Janeiro, RJ, Brasil; 5 Centro Nacional de Ressonância Magnética Nuclear, Departamento de Bioquímica, ICB/UFRJ, 21941-590, Rio de Janeiro, RJ, Brasil; 6 Institute for Medical Physics and Biophysics, University of Münster, Robert-Koch Str 31, D-48149, Münster, Germany; 7 University of Bielefeld, Bielefeld, Germany; and 8 Departamento de Bioquímica, Universidade Federal do Paraná (UFPR), D-33501, 81531-990, Curitiba, Paraná, Brazil

Received on January 16, 2003; revised on May 28, 2003; accepted on May 30, 2003


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
O-linked oligosaccharide groups ranging from di- to hexasaccharide were ß-eliminated by mild alkaline treatment under reducting conditions from the peptidogalactomannan of Aspergillus fumigatus mycelial cell wall. The resulting reduced oligosaccharides, which were the minor components of the peptidogalactomannan fraction, were fractionated to homogeneity by successive gel filtration and high-performance liquid chromatography. Their primary structures were determined based on a combination of techniques including gas chromatography, ESI-QTOF-MS, 1H COSY and TOCSY, and 1H-13C HMQC NMR spectroscopy and methylation analysis, to be: {alpha}-Glcp-(1 -> 6)-Man-ol, ß-Galf-(1 -> 6)-{alpha}-Manp-(1 -> 6)-Man-ol, ß-Galf-(1 -> 5)-ß-Galf-(1 -> 6)-{alpha}-Manp-(1 -> 6)-Man-ol and ß-Galf-(1 -> 5)-[ß-Galf-(1 -> 5]3-ß-Galf-(1 -> 6)-Man-ol. The ß-Galf containing oligosaccharides have not been previously described as fungal O-linked oligosaccharides. The peptidogalactomannan is antigenic and was recognized by human sera of patients with aspergillosis when probed by ELISA, but de-O-glycosylation rendered a 50% decrease in its reactivity. Furthermore, when tested in a hapten inhibition test, the isolated oligosaccharide alditols were able to block, on a dose-response basis, recognition between human sera and the intact peptidogalactomannan. The immunodominant epitopes were present in the tetra- and hexasaccharide, which contain a ß-Galf-(1 -> 5)-ß-Galf terminal group. These results suggest that the O-glycosidically linked oligosaccharide chains, despite being the less abundant carbohydrate component of the A. fumigatus peptidogalactomannan, may account for a significant part of its antigenicity, other than the known activity associated with the galactomannan component.

Key words: Aspergillus fumigatus / ELISA / ESI-QTOF-MS / NMR / O-linked {alpha}-Glcp and {alpha}-Galf-oligosaccharides


    Introduction
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Aspergillus fumigatus is the major etiological agent of several respiratory tract–related diseases, including allergic manifestations, airway cavity colonization, and the almost always lethal invasive aspergillosis (Kwon-Chung and Bennet, 1992Go).

In the search for a structure that could be helpful in the diagnosis of the different forms of aspergillosis, much attention has been paid to the study of Aspergillus cell wall antigens, considering their high immunogenicity (Hearn et al., 1991Go; Hearn and Sietsma, 1994Go). Cell wall polysaccharides and glycoproteins with diagnostic potential have been identified in several fungi, including species of Aspergilli sp. (Notermans and Soentoro, 1996; Latgé, 1999Go). The structure of galactomannans from the aspergilli cell wall, associated or not with a protein counterpart, have long been studied (Barreto-Bergter et al., 1980Go, 1981aGo; Latgé et al., 1992Go). The carbohydrate portion of these molecules contains ß-Galf-bearing chains that are regarded to be the immunodominant epitopes (Bennet et al., 1984Go), especially when they are (1 -> 5)-linked (Notermans et al., 1998Go). Monoclonal antibodies have been raised against these structures and are used with some success for detecting circulating antigens (Stynen et al., 1992Go).

Recently, a peptidogalactomannan (pGM) fraction, obtained by hot buffer extraction of the mycelial cell wall of A. fumigatus, was shown to react with rabbit sera raised against intact A. fumigatus cells and with sera from aspergillosis patients (Haido et al., 1998Go), as demonstrated by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence. The importance of the carbohydrate moiety for the antigenicity of the pGM was thus highlighted.

O-glycosylation is a form of protein modification that consists of covalent attachment of carbohydrate chains, usually oligosaccharides, to the hydroxylated amino acids serine or threonine. O-linked oligosaccharides have been obtained from glycoprotein preparations isolated from the cell wall of fungi, such as Saccharomyces cerevisiae (Ballou, 1990Go), Sporothrix schenckii (Lopes-Alves et al., 1992Go), Fusarium sp. (Jikibara et al., 1992Go), Pichia pastoris (Duman et al., 1998Go), Candida albicans (Gemmil and Trimble, 1999Go), and Aspergillus niger (Gunnarsson et al., 1984Go), among others. To date, all the O-linked oligosaccharides described in fungi were demonstrated to be attached to the protein moiety by a mannosyl residue, distinct from the O-glycosylation usually seen in mammalian cells, where an N-acetyl-galactosaminyl unit is linked to Ser/Thr. For example, a family of protein O-mannosyl transferases have been described in S. cerevisiae as the enzymes responsible for the donation of a mannose residue to Ser or Thr in a nascent protein, located in the endoplasmic reticulum (Strahl-Bolsinger et al., 1999Go).

The importance of glycosylation in general and of fungal O-mannosylation in particular remains to be established and is a matter of speculation (Varki, 1993Go). To detect the occurrence of O-glycosylation in a growing number of medically important fungi may be an important step toward the better understanding of their physiology and may be useful for providing potential targets for drug development. From a diagnostic point of view, O-linked glycans could be useful if they contain novel antigens.

We investigate the presence of the O-linked oligosaccharides in the pGM fraction of A. fumigatus mycelial cell wall, as suggested previously (Haido et al., 1998Go). Four low-weight molecules were ß-eliminated under mild reducing alkaline conditions from the pGM, and their structures were thoroughly characterized by gas-liquid chromatography (GC), electrospray/quadrupole-time-of-flight mass spectrometry (ESI-QTOF-MS), and nuclear magnetic resonance (NMR). Three of them turned out to be novel, ß-Galf-containing molecules. The de-O-glycosylated pGM (de-O-pGM) was also recovered and studied. In addition, the antigenic role of these oligosaccharides against sera of patients with aspergillosis was examined.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Isolation of cell wall pGMs of A. fumigatus
The crude glycoprotein obtained from mycelial cell wall of A. fumigatus by hot buffer extraction was purified by selective precipitation with Cetavlon/Na2B4O7 at pH 8.8, yielding a pGM fraction. pGM represented 12% (w/w) of the crude glycoprotein preparation.

Release of O-linked oligosaccharides by mild alkaline treatment, isolation of de-O-pGM, and oligosaccharides
pGM was submitted to ß-elimination under reducing conditions (Yen and Ballou, 1974Go), to release the O-linked oligosaccharides as oligosaccharide-alditols, thus avoiding peeling reactions. The products were then fractionated separated by gel filtration chromatography on BioGel P-2 (2 x 140 cm), to give four fractions (Figure 1). A major one with strong absorbance at 280 nm and a high total carbohydrate content was eluted in the void volume, corresponding to the de-O-pGM. This fraction accounts for more than 90% of the total carbohydrate content of the original pGM and corresponds to the peptide moiety still glycosylated with sugar chains that were resistant to mild alkali pretreatment, probably as N-linked galactomannans.



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Fig. 1. Oligosaccharide-alditol separation from de-O-pGM and initial purification by Bio Gel P-2 gel filtration. Solvent: H2O (10 ml/h). Fraction size: 2 ml/tube. Carbohydrate profile was probed by the phenol–sulfuric acid method. Protein profile was estimated by reading at 280 nm. G2–G6, glucose oligomers; V0, void volume.

 
The three distinct fractions were eluted in the oligosaccharide range of the column (di- to pentasaccharide, based on glucose oligomers). The oligosaccharide fractions were named OA (minor), OB, and OC (major), in order of elution. The three fractions were pooled and recovered. Fractions OC and OB were rechromatographed by gel filtration in BioGel P-2 (1 x 180 cm). OA turned out to be a mixture of two oligosaccharide-alditols (OA1 and OA2) that could not be resolved by BioGel P-2 or P-4 gel filtration but were successfully isolated by LiChrosorb-NH2 high-performance liquid chromatograpy (HPLC). The four oligosaccharide-alditols (OC, OB, OA1, and OA2) were homogenous, as determined by high-performance thin-layer chromatography (HPTLC) (Figure 2).



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Fig. 2. HPTLC plate of O-linked oligosaccharides from A. fumigatus. S, hydrolyzed dextran; OC, disaccharide-alditol; OB, trisaccharide-alditol; OA1, tetrasaccharide-alditol; OA2, hexasaccharide-alditol. Sugars were detected by orcinol–sulfuric acid spray reagent.

 
Characterization of pGM and de-O-pGM
The structures of pGM and de-O-pGM were compared. Both fractions had a similar composition (Table I). Methylation analysis of pGM and de-O-pGM (Table II) showed the presence of nonreducing end, 2-O- and 6-O-substituted and 2,6-di-O-substituted units mannopyranosyl residues as expected for a (1 -> 6)-linked mannan main chain, substituted at O-2 with (1 -> 2)-linked manno-oligosaccharides, as previously described by Haido et al. (1998)Go. Nonreducing end and 5-O-substituted galactofuranosyl units were detected, corresponding to the 5-O-linked galactofuranosyl side chains, typical for A. fumigatus galactomannans (Haido et al., 1998Go). Residues of nonreducing end Glcp units were detected in a minor proportion in pGM, which were significantly diminished (from 2% to 1%) after ß-elimination.


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Table I. Chemical composition of pGM and de-O-pGM fractions of A. fumigatus

 

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Table II. GC-MS analysis of O-methylalditol acetates derived from methylation analysis of pGM and de-O-pGM of A. fumigatus

 
These results were confirmed by 1H, 13C heteronuclear multiple quantum coherence (HMQC) NMR spectroscopy. The anomeric region of pGM was complex, showing seven cross-peaks, whose assignments are proposed in Table III. These signals correspond to {alpha}-Manp units that are nonreducing ends (103.7/5.05 ppm), 2-O-substituted (102.1/5.26 ppm), and 2,6-di-O-substituted (100.6/5.09 ppm). Two cross-peaks at lower field relative to 13C are typical of the presence of ß-Galf residues: a major signal at 108.4/5.19 ppm corresponding to 5-O-substituted ß-Galf and ß-Galf nonreducing end and a signal at 109.3/5.02 ppm that corresponds to ß-Galf residues directly linked to O-6 of {alpha}-Manp (Barreto-Bergter et al., 1980Go). The anomeric region in the HMQC spectrum of the de-O-pGM contained only five signals that are almost identical to those already described. However, it lacks two prominent signals, one at 100.3/4.94 ppm, corresponding to nonreducing end {alpha}-Glcp units and another, unassigned, at 103.3/4.88 ppm. The absence of the anomeric signal at 100.3/4.94 ppm in the de-O-pGM HMQC spectra (not shown) is accompanied by the absence of two signals that resonate far from the anomeric region, namely, at 74.0/3.55 ppm and 71.9/3.42 ppm, corresponding to C4/H4 and C2/H2 of terminal {alpha}-Glcp units. This confirms the removal of these residues by the alkaline treatment.


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Table III. 1H- and 13C-NMR assignment for the anomeric region of pGM and de-O-pGM signals

 
Analysis of oligosaccharide-alditols
Monosaccharide composition
Monosaccharide analysis data of the oligosaccharide-alditols are shown in Table IV. All the oligomers contained mannitol, indicating that mannose is the linkage point to the peptide moiety. The oligosaccharide OC also contains glucose, and its presence in the main fraction explains the loss of the signals corresponding to {alpha}-Glcp in the de-O-pGM HMQC spectrum. The oligosaccharide OB contains mannitol, mannose, and galactose in the proportion 1:1:1, whereas oligosaccharide OA1 has the same monosaccharides but contains an extra galactosyl residue. Fraction OA2, in contrast, appears to be a galacto-oligosaccharide containing Gal:Man-ol in an approximate ratio of 5:1.


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Table IV. Monosaccharide analysis of the oligosaccharide-alditol

 
ESI-QTOF MS
The ESI-QTOF MS1 spectra, run in either the positive and/or the negative ion mode provided information regarding the size of oligosaccharide chains in analyzed fractions (data not shown). In fragmentation analysis, using a tandem QTOF (MS/MS) approach, a number of informative fragment ions were observed arising from cleavage of glycosidic linkages (Figure 3). In particular, Y series fragment ions (Domon and Costello, 1988Go) from the reducing end, abundant in reduced oligosaccharide ions, were clearly defined in these spectra.






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Fig. 3. Structures of ß-eliminated oligosaccharides and the principal ions observed on ESI-QTOF-MS.

 
Fraction OC is a disaccharide-alditol, as determined by the negative ion MS/MS spectrum (Figure 3). Using the molecular [M-H]- ion at m/z 343 as a precursor ion, B, C, Y, and Z glycosidic cleavage ions were observed in MS/MS, besides a number of daughter ring cleavage ions. B1 was at m/z 161, Z1 at m/z 163, C1 at m/z 179, and Y1 at m/z 181. Most prominent ring cleavage ions were at m/z 89 (0,3A1) and 119 (0,2A1), indicating the hexose moiety at the nonreducing end.

In the positive ion MS/MS spectrum of the trisaccharide alditol OB (Hex2Hex-ol) using the ion [M + Na]+ at m/z 529 as precursor, highly abundant sequence ions Y1 at m/z 205 and Y2 at m/z 367 were obtained, whereas the B1/B2 and Z1/Z2 were only of low abundance (Figure 3). Complementary data for glycosidic fragment ions B1, Z1, C1, Z2, and Y2 were obtained from a negative ion MS/MS fragmentation experiment (not shown) using the molecular [M-H]- ion at m/z 505 as a precursor, beside the diagnostic ring cleavage ion 2,4A2 at m/z 221.

The fragmentation pattern of the tetrasaccharide alditol OA1 (Hex3-Hex-ol) in the negative ion mode ESI-QTOF, using the molecular [M-H]- ion at m/z 667 as precursor, gave a complete series of diagnostic ion pairs, Y1/Z1 at m/z 181 and 163, Y2/Z2 at m/z 343 and 325, and Y3/Z3 at m/z 505 and 487 (Figure 3). The ring cleavage ions were of the 2,4A type at m/z 221 (A2) and 383 (A3). B1 and C1 were at m/z 161 and 179, respectively.

In the positive ion QTOF MS/MS of the hexasaccharide alditol OA2 ( Hex5-Hex-ol), Y-type ions of high abundance were well defined at m/z 367 (Y2), 529 (Y3), 691 (Y4), and 853 (Y5), besides several A-type cleavage ions. The doubly charged ion at 516.3 could originate from the K+ adduct, namely, [M + Na + K]2+. B-type ions were here also of low abundance (Figure 3). In negative ion MS/MS (not shown), however, formation of clear-cut diagnostic ion pairs, Y1/Z1 at m/z 181 and 163, Y2/Z2 at m/z 343 and 325, Y3/Z3 at m/z 505 and 487, Y4/Z4 at m/z 667 and 649, Y5/Z5 at m/z 829 and 811, respectively, was observed. 2,4A-type ring cleavage ions were at m/z 221 (A2), 383 (A3), 545 (A4), and 707 (A5), respectively. 0,2A3 was at m/z 443.

Methylation analysis of GlcMan-ol OC
Methylation analysis of OC provided partially O-methyl alditol acetates of 2,3,4,6-Me4-Glc and 1,2,3,4,5-Me5-Man (GC-MS). The latter was synthesized from 6-O-{alpha}-mannopyranosyl-mannitol and its retention time and electron impact (e.i.) breakdown pattern were different from those of 1,2,3,5,6-Me5-Man and 1,2,3,4,6-Me5-Man, prepared from 4-O-ß-mannopyranosyl-mannitol and 2-O-{alpha}-mannopyranosyl-mannitol, respectively.

NMR spectroscopy of the disaccharide-alditol, OC
OC was analyzed by 1H (observed) 13C HMQC NMR, which correlates at a high sensitivity directly linked protons and carbons. The signals arising from the nonreducing glucosyl end unit had the {alpha}-configuration because its C-1 signal appeared at high field of 99.4 ppm (Hall and Johnson, 1969Go). The {alpha}-configuration was also deduced from the 3J1,2 coupling constant (3.4 Hz) obtained from a 1D NMR spectrum of OC (data not shown). The O-substitution position on the mannitol units was shown by HMQC, which contained doublets at 62.0/3.73 and 3.84 ppm from the C-6 of the {alpha}-Glcp unit and another at 64.5/3.83 typical of free-CH2OH groups. 6-O-substituted mannitol unit occurs at 72 ppm. Because there was no signal of 2-O- and 3-O-substituted mannitol signals in 80 ppm region, it can be concluded that the substitution was at O-6 (Table V).


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Table V. 1H and 13C NMR chemical shifts (ppm) for the disaccharide alditol (fraction OC, -{alpha}Glcp-(1 -> 6)-Man-ol) from A. fumigatus

 
Methylation analysis of trisaccharide-alditol, OB
Methylation analysis, due to the complexity of the GC spectrum (which contained many contaminants produced in the analysis process, resulting in many overlapping peaks), characterized only the acetate of 1,2,3,4,5-Me5-Man-ol.

NMR spectroscopy of the trisaccharide-alditol OB
In the HMQC spectrum of OB it was possible to assign C-1 signals at low field, arising from residues of terminal ß-Galf units namely, at C1/H1 at 109.2/5.02 ppm. Typical signals were also present of C2/H2 at 82.0/4.10 ppm, C3/H3 at 78.3/4.05 ppm, and C4/H4 at 84.0/3.98 ppm (Table VI). These signals could be readily identified in the correlation spectroscopy (COSY) spectrum (not shown). The anomeric signal shift is compatible with that expected for a terminal ß-Galf unit substituting the O-6 of a mannosyl residue (Barreto-Bergter et al., 1980Go, 1981aGo,bGo). An intermediate {alpha}-Manp unit gives rise to the remaining anomeric signal present in the HMQC spectrum (102.6/4.97 ppm). 6-O-substituted Manp residue is responsible for the C-6 signal at 68.0/3.64 ppm.


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Table VI. 1H and 13C NMR chemical shifts (ppm) for the trisaccharide-alditol (fraction OB, ß-Galf-(1 -> 6)-{alpha}-Manp-(1 -> 6)-Man-ol) from A. fumigatus

 
Methylation analysis of OA1
In this case it was possible to detect GC-MS peaks of 2,3,6-Me3-Gal and 1,2,3,4,5-Me5-Man, which did not suffer from overlapping.

NMR spectroscopy of OA1
The HMQC spectrum of the oligosaccharide alditol OA1 closely resembles that of OB but with an additional anomeric signal can be seen at 108.7/5.19 ppm, corresponding to a terminal ß-Galf unit linked to O-5 of a ß-Galf unit. The substitution point in the intermediate ß-Galf residue is revealed by the presence of a signal at 77.4/3.93 ppm, absent in the OB spectrum, that is precisely the substituted position 5 (Barreto-Bergter et al., 1980Go). Another feature of the spectrum of OA1, when compared with that of OB, is that there are two sets of signals corresponding to C-2, C-3, and C-4 of the ß-Galf; with the signals of positions 2 and 3 being only distinguishable in the proton dimension and the signals of position 4 being only recognizable in the 13C dimension, as previously pointed out by Gorin and Mazurek (1976)Go. These data allow us to show that the oligosaccharide alditol OA1 is actually an extension of OB by the addition of an extra ß-Galf residue at the O-5 of the adjacent ß-Galf (Table VII).


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Table VII. 1H and 13C NMR chemical shifts (ppm for the tetrasaccharide-alditol (fraction OA1, ß-Galf-(1 -> 5)-ß-Galf-(1 -> 6)-{alpha}-Manp-(1 -> 6)-Man-ol) from A. fumigatus

 
Methylation analysis of OA2
As with the methylation data of OA1, only 1,2,3,4,5-Me5 Man and 2,3,6-Me3-Gal were detected (GC-MS).

NMR spectroscopy of OA2
The anomeric region of the HMQC spectrum of OA2 showed only three signals, located at low field in the 13C spectrum. The chemical shifts arising of the OA2 are summarized in Table VIII. These were from a ß-Galf-(1 -> 5)-[ß-Galf-(1 -> 5)]3 group at 108.7 ppm and 108.8 ppm and ß-Galf linked to mannitol units at 109.7 ppm. This indicates that besides the mannitol residue, only ß-Galf units are present in this oligosaccharide-alditol. Two of these signals are identical to those present in the spectrum of OA1. The anomeric signal at 108.8 is mostly prominent than the others and should correspond to three external ß-Galf units relative to Man-ol. If compared with that present in the spectrum of OA1, the C-5 signal at 77.6/3.91 ppm present in the spectrum of OA2 was larger, also indicating that the five galactosyl residues form a ß-(1 -> 5)-linked chain. In addition, signals at 78.3/4.08, 83.6/4.07, and 62.8/3.77 ppm, corresponding, respectively, to positions 3, 4, and 6 of internal ß-Galf were also greater (Svensson et al., 1986Go). These data show that the oligosaccharide OA2 has a linear ß-(1 -> 5)-linked galactofuranosyl chain attached to the O-6 of the mannitol residue.


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Table VIII. 1H and 13C NMR chemical shifts (ppm) for the hexasaccharide-alditol (fraction OA2, ß-Galf-(1 -> 5)-(ß-Galf- (1 -> 5)3-ß-Galf-(1 -> 6)-Man-ol) from A. fumigatus

 
Immunochemical analysis
The intact pGM and de-O-pGM were analyzed by a direct ELISA method. Intact pGM reacted strongly with aspergillose patient sera, whereas the de-O-glycosylated molecule showed a reactivity about 50% lower (Figure 4).



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Fig. 4. Reactivity of pGM (open circles) and de-O-pGM (closed squares) with sera from patients with aspergillosis and with normal human sera (closed triangles). Serum dilutions were 1/400 to 1/3200.

 
Hapten inhibition test
The antigenic role of the O-linked was determined by a hapten inhibition test (Figure 5). The disaccharide OC, {alpha}-D-Glcp-(1 -> 6)-Man-ol, and the trisaccharide OB, ß-D-Galf-(1 -> 6)-{alpha}-D-Manp-(1 -> 6)-Man-ol were weak inhibitors (less than 50% inhibition at 250 ng). The tetrasaccharide OA1, ß-D-Galf-(1 -> 5)-ß-D-Galf-(1 -> 6)-{alpha}-D-Manp-(1 -> 6)-Man-ol and hexasaccharide OA2, ß-D-Galf-(1 -> 5)[-ß-D-Galf-(1 ->]3-ß-D-Galf-(1 -> 6)-Man-ol, showed the greatest inhibitory effects. The 50% inhibition was observed with OA1 and OA2 by using concentrations 10 times lower than that of the antigen adsorbed in the ELISA well. A 90% inhibition was obtained with 2.5 µg/well of each OA1 and OA2.



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Fig. 5. Inhibition of the pGM reactivity to sera from patients with aspergillosis by O-linked oligosaccharide fractions (hapten). OC, closed diamonds; OB, closed squares; OA1, closed triangles; OA2, closed circles.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
This investigation was carried out to determine whether O-linked oligosaccharides were present in pGMs from the cell wall of A. fumigatus mycelia. O-linked oligosaccharides were selectively released from pGM by ß-elimination under mild alkaline reductive conditions in the presence of sodium borohydride, isolated, and then structurally and immunologically characterized.

The ß-eliminated products were fractionated by gel filtration chromatography, giving rise to a major high-molecular-weight fraction (de-O-pGM) and minor low-weight fractions, consisting of other reduced oligosaccharides.

NMR and methylation analysis of the glycan portion of pGM and de-O-pGM showed that they are very similar and consisted mainly of galactomannan-type polysaccharides containing a (1 -> 6)-linked {alpha}-mannan main chain substituted at O-2 by {alpha}-mannosyl to mannotriosyl side chains and by immunodominant (1 -> 5)-linked ß-Galf side chains attached to O-6 of mannosyl residues, as previously described (Haido et al., 1998Go). This comparative analysis showed that galactomannans are the major constituents of pGM and dominate the profile of this fraction.

Four O-linked oligosaccharides were present in pGM. A disaccharide and a trisaccharide were purified by gel filtration. A tetrasaccharide and a hexasaccharide could only be resolved by HPLC using an amine-bonded phase. Complete analysis on the nature of the oligosaccharide was carried out by GC, ESI-QTOF-MS and NMR, and methylation analysis.

GC analysis revealed the type of component monosaccharides. MS analysis by MS/MS was run in the positive and the negative ion mode in parallel to explore the fragmentation behavior of reduced oligosaccharide moieties. The data obtained by this approach revealed the size of the reduced glycans, the composition in terms of hexoses, and their sequence, by differentiation of the reducing and the nonreducing end. To determine the type of hexoses involved, the linkage sites and the type of the sugar ring was not accessible by this approach. These data were obtained directly determined by methylation and NMR analysis.

The disaccharide is the major low-weight constituent of pGM, and its structure containing {alpha}-Glcp attached (1 -> 6) to Man-ol has been previously described as one of the minor O-linked oligosaccharides attached to A. niger glucoamylases G1 and G2 (Gunnarsson et al., 1984Go). The presence of a major {alpha}-Glcp-containing oligosaccharide in pGM shows that glucosyl residues are actually present as part of the pGM fraction and not a mere contaminant. This finding explains the lack of cross-peaks relative to the presence of terminal {alpha}-Glcp in the HMQC spectrum of de-O-pGM while they are present in that of pGM.

The remaining oligosaccharides represent novel structures, to our knowledge. The trisaccharide and the tetrasaccharide contain Man-ol, {alpha}-Manp, and ß-Galf units. They have related structures, with the tetrasaccharide being an extension of the trisaccharide by the attachment of a second ß-Galf residue in O-5 of the subterminal ß-Galf unit. The hexasaccharide, on the other hand, contains only Man-ol and an extended (1 -> 5)-linked ß-Galf chain.

All the oligosaccharides described have a mannosyl residue at the reducing end unit, corresponding to the former attachment point to the protein moiety. This finding is in accordance with all reports to date concerning fungal protein O-glycosylation, referred to as protein O-mannosylation by Strahl-Bolsinger and co-workers (1999)Go. It seems that A. fumigatus follows that general rule.

An interesting observation is that the oligosaccharides contained some structural features that were also present in the polysaccharides (supposedly N-linked glycans), for instance, chains terminated by (1 -> 5)-linked ß-Galf residues and having one of them substituting O-6 of mannosyl residues. This suggests that the same glycosyl transferases that modify N-glycans may also modify O-glycans in A. fumigatus, as occurs in S. cerevisiae (Lussier et al., 1999Go).

Galactofuranose-containing groups are regarded as the immunodominant epitopes present in the galactomannan of A. fumigatus, particularly when they are present as (1 -> 5)-linked ß-Galf side chains (Bennet et al., 1984Go; Latgé, 1999Go). The presence of these groups in the O-linked oligosaccharides called attention to the fact that the antigenicity of the intact pGM could be partly associated with O-glycans in addition to the galactomannan. To answer this question, the antigenicity of de-O-pGM was tested against sera of patients with aspergillosis and compared with the antigenicity of intact pGM. The results consistently showed that about half of the reactivity was lost after the ß-elimination of pGM. The peptidorhamnomannan from the pathogenic fungus Sporothrix schenckii has the same behavior when tested against either anti-S. schenckii rabbit antiserum (Lopes-Alves et al., 1994Go) or against sera from sporotrichosis patients (Penha and Bezerra, 2000Go).

To evaluate the immunodominance of the O-linked oligosaccharide chains, they were tested in a ELISA hapten system as inhibitors of the reactivity between the pGM and the sera of patients with aspergillosis. Inhibitions as high as 65% were obtained with the tetra (3)- and the hexasaccharide (4) when the amount of inhibitor used was the same as that of antigen adsorbed in the ELISA well. Increasing the amount of inhibitor to 10 times higher caused inhibition levels of 90% for the tetrasaccharide and the hexasaccharide. These haptens, that contain ß-Galf-(1 -> 5)-ß-Galf groups are therefore potent inhibitors of recognition between patients sera and pGM. The trisaccharide, which contains only one ß-Galf residue, was able to inhibit 50% of the reaction when the same amount of the antigen was used. {alpha}-Glcp-(1 -> 6)-Man-ol only reduced recognition by 35%.

Our results show clearly that O-glycosidically linked oligosaccharides are present in the pGM from mycelial cell wall of A. fumigatus. This type of molecule is described herein for the first time in this medically important fungus. Each contains immunodominant epitopes that can be recognized by sera of patients with aspergillosis and should therefore be taken into account in the search for circulating antigens in fluids these patients. It is possible that the low-molecular-weight peptides containing O-linked glycan chains are present in body fluids as has already been proven in the case of the A. fumigatus galactomannan.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Materials
Reagents were obtained from Merck (Darmstadt, Germany) or Sigma (St. Louis, MO). Milli-Q water was obtained using a Milli-Q Academic System (Millipore, Bedford, MA). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was carried out using a Mini-Protean II System (BioRad, Hercules, CA).

Microorganism and growth conditions
A. fumigatus strain NCPF 2109 (AF 2109) was supplied by Dr. C.K. Campbell, Mycology Reference Laboratory, Bristol Public Health Laboratory, U.K. The fungus was maintained in a culture medium containing (g {bullet} L-1): Difco (Detroit MI) peptone, 10; Difco yeast extract, 5; Difco agar, 20; and glucose, 20. Cells were grown on Sabouraud solid slants and inoculated into Erlenmeyer flasks (500 ml) containing the culture medium (200 ml), which was incubated for 7 days at 25°C with orbital shaking. Cultures were then transferred to the same medium (1 L) and incubated for further 7 days at the same temperature with orbital shaking; the mycelium was filtered, washed with distilled water, and stored at -10°C.

Extraction and fractionation of the pGM
The pGM fraction was obtained as described elsewhere (Haido et al., 1998Go). Briefly, crude glycopeptides were extracted from intact AF 2109 mycelia in phosphate buffer (50 mM, pH 7.2, at 100°C for 2 h) and purified by hexadecyltrimethylammonium bromide (Cetavlon) 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 pGM fraction. The pGM was dialyzed against distilled water and lyophilized.

Reductive alkaline ß-elimination of pGM
pGM was de-O-glycosylated by mild alkaline treatment under reducing conditions (0.1 M NaOH, 0.5 M NaBH4, 25°C, 24 h (Yen and Ballou, 1974Go). The reaction medium was then neutralized by addition of glacial acetic acid, deionized in a Amberlite IR120-P (Sigma) 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). De-O-pGM was recovered in the void volume. The liberated oligosaccharide-alditol fractions were recovered in the column fractionation range. Eluate fractions (2 ml) were probed for protein by their absorbance at 280 nm and for total sugar content by the phenol-sulfuric acid method (Dubois et al., 1956Go). The oligosaccharide fractions were subjected to further fractionation either by individual elution on Bio Gel P-2 (1 x 180 cm) or by LiChrosorb-NH2 HPLC.

Isolation of the oligosaccharide fraction OA by HPLC
OA was fractionated by HPLC using a LiChrosorb-NH2 column (4 x 250 mm, 5 µm; Merck) coupled to an ÄKTA Purifier Liquid Chromatograph (Pharmacia-Amersham, Uppsala, Sweden). The initial eluant was acetonitrile/water (80:20, v/v), pumped at 1 ml/min for 20 min, followed by a linear gradient to acetonitrile:water (65:35, v/v) for 40 min. Fractions of 400 µl were collected, dried, resuspended in 1 ml water, and probed for the presence of carbohydrates after 1-µl aliquots were spotted onto a TLC plate and detected by the orcinol-sulfuric acid reagent (Fukuda, 1989Go).

HPTLC
The degree of purification of the oligosaccharide-alditol fractions was determined by HPTLC on silica gel 60 plates (0.25 mm; Merck) developed with n-butanol:ethanol:water (2:1:1, v/v) and visualized by the orcinol–sulfuric acid reagent. Oligosaccharide homologs from dextran hydrolysis were used as molecular weight references (Yamashita et al., 1982Go).

Sugar analysis
pGM, de-O-pGM, and 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., 1965Go) using an OV-225 fused silica capillary column (30 m x 0.25 mm ID), with temperatures programmed from 50 to 220°C at 50°C/min.

Methylation analysis of PGM and de-O-pGM
PGM and de-O-pGM were methylated by the lithium methyl sulfinyl carbanion method according to Parente et al. (1985)Go. The products were hydrolyzed with 3 M trifluoroacetic acid at 100°C for 3 h and converted to their partially O-methylated alditol acetates, which were subjected to GC-MS using a DB-225 column (30 m x 0.25 mm, 50°C to 220°C, 2°C/min) (Barreto-Bergter et al., 1981bGo).

Methylation analysis of oligosaccharides
This was carried out by the method of Tischer et al. (2002)Go, staring with a modification of the per-O-methylation procedure of Ciucanu and Kerek (1984)Go. Each oligosaccharide (~1 mg) was dissolved in a drop of H2O, which was diluted with Me2SO (1 ml) and then MeI (1 ml). Powdered NaOH (~0.3 g) was added and the mixture agitated vigorously with a vortex for 30 min and then left overnight. Water was added, followed by HOAc and the per-O-methylated product was extracted with CHCl3, which was washed three times with H2O. After evaporation, the residue was dissolved in 1,4-dioxan (0.2 ml) and 2 M H2SO4 (0.4 ml) added; the mixture was maintained at 100°C for 8 h. The resulting mixture of partially O-methylated aldoses and mannitol was treated with NaBD4 and then acetylated with Ac2O-pyridine to give O-methylalditol acetates. These were examined by GC-MS on a column of DB-255 (30 m x 0.25 mm ID) programmed from 50°C (1 min) at 40°C/min to 210°C (constant temperature).

Synthesis and GC-MS characteristics of penta-O-methylmannitols
To characterize penta-O-methylmannitols, the three possible isomers were prepared. Sodium borohydride-reduced ß-Manp-(1 -> 4)-Man, {alpha}-Manp-(1 -> 2)-Man, {alpha}-Manp-(1 -> 6)-Man were subjected to methylation analysis to give acetates of 1,2,3,5,6, 1,2,3,4,6-, and 1,2,3,4,5-Me5Man-ol, respectively. Retention time and fragmentation data of penta-O-methyl mannitol acetates are:

General analytical procedures
Total carbohydrate was determined by the phenol–sulfuric acid method (Dubois et al., 1956Go) and protein by the Folin phenol reagent method (Lowry et al., 1951Go).

NMR
1H and 13C spectra were recorded using either a Bruker DRX 600 with a triple resonance probe or a Bruker DRX-400 equipment. PGM and de-O-pGM (5 mg each) were dissolved in 0.5 ml D2O (99.9%, Merck) and analyzed at 60°C. The oligosaccharide-alditols were dissolved in 0.5 ml D2O and examined at 40°C. All spectra were recorded with HOD suppression by presaturation. COSY, TOCSY, and 1H/13C HMQC spectra were recorded using states-time proportion phase incrementation for quadrature detection in the indirect dimension. TOCSY spectra were run with a spin-lock field of about 10 kHz and a mixing time of 80 ms. HMQCs were globally optimized with alternating phase rectangular pulses for decoupling. All chemical shifts were relative to external trimethylsilylpropionic acid and 13C-methanol ({delta}c = 0). Manufacturer pulse sequences were used.

ESI-QTOF-MS
Nano-ESI MS analyses were performed using a QTOF mass spectrometer (Micromass, Manchester, UK) in the positive and negative ion mode. A Z-spray atmospheric pressure ionization source was used, with the source temperature set to 30°C and the desolvation gas (N2) flow rate set to 50 L h-1. Purified dried gel filtration and LiChrosorb-NH2 fractions, OC, OB, OA1, and OA2 at an estimated concentration of 10 pmol/µl dissolved in methanol were loaded into homemade nanospray capillaries. A potential of 1.1 kV was applied to the capillary tip, and the cone voltage was set to 40 V. Under these conditions the estimated flow rate of the sample into the analyzer was about 50 nl min-1. For the fragmentation analysis collision-induced-dissociation tandem MS studies, a doubly charged precursor ion was selected in the quadrupole analyzer and partially fragmented in the hexapole collision cell, with the pressure of the collision gas (Ar) 2.7 x 10-5 mBar and collision energy typically at 20 eV. Data acquisition was optimized to give the highest possible resolution and signal-to-noise ratio even in the case of low-abundant signals. Up to 50 TOF pulses were accumulated for each spectrum shown. Acquisition and analysis of data were carried out using the MassLynx Windows NT PC data system. NaI was used as a mass standard for instrument calibration. The mass accuracy of all measurements was within 0.2 m/z units (Th).

ELISA
Wells of flat-bottomed polyvinyl microtitre plates (Falcon-Becton & Dickinson, Franklin Lakes, NJ) were coated with 100 µl of a 2.5 µg/ml solution of pGM and maintained for 1 h at 37°C and overnight at 4°C. After plate washing with 0.05% phosphate buffered saline–Tween 20, the nonspecific sites were blocked by addition of 5% skim milk in 0.1% phosphate buffered saline–Tween 20. Serial dilutions of human sera in blocking buffer (100 µl) were added to the wells and the antibody binding was measured using goat anti-human IgG antibody conjugated to horseradish peroxidase (Sigma) at 1/2000 (100 µl). Each step was followed by a 1 h/37°C incubation and washings with 0.1% phosphate buffered saline–Tween 20. The substrate used was 0.4 mg/ml o-phenylenediamine and 0.4 µl/ml H2O2 in 0.01 M sodium citrate buffer, pH 5.0. The enzyme reaction was followed by measuring the absorbance at 490 nm using an automated reader (BioRad ELISA Reader) 20 min after terminating the reaction with 50 µl 1.5 M H2SO4.

Hapten inhibition tests
Oligosaccharide solutions ranging from 0.01 to 16 times the relative carbohydrate pGM concentration (250 ng/well) were separately mixed with the same volume of human sera (1/3200) and preincubated for 1 h at 37°C. The immunodominance of each reduced O-linked oligosaccharide was evaluated by ELISA as described.


    Acknowledgements
 
We thank Maria de Fatima F. Soares for technical assistance. This work was supported by grants from Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Fundacão de Amparo a Pesquisa do Estado do Rio de Janeiro, and Programa de Apoio a Nucleos de Excelência. The QTOF mass spectrometer was purchased by the HbfG grant ( Land Nordrhein Westfalen) to J.P.-K.


    Footnotes
 
1 Deceased, June 19, 2001 Back

2 To whom correspondence should be addressed; email: eliana.bergter{at}micro.ufrj.br Back


    Abbreviations
 
COSY, correlation spectroscopy; ELISA, enzyme-linked immunosorbent assay; ESI-QTOF-MS, electrospray/quadrupole time-of-flight mass spectrometry; GC, gas-liquid chromatography; HMQC, heteronuclear multiple quantum coherence; HPLC, high-performance liquid chromatography; HPTLC, high-performance thin-layer chromatography; MS/MS, tandem electrospray/quadrupole-time-of-flight mass spectrometry; NMR, nuclear magnetic resonance; pGM, peptidogalactomannan; TOCSY, total correlation spectroscopy


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 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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