2Institut für Chemie der Universität für Bodenkultur, Muthgasse 18, A-1190 Wien, Austria, and 3Bijvoet Center for Biomolecular Research, Department of Bio-Organic Chemistry, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
Received on July 12, 2000; revised on November 28, 2000; accepted on December 1, 2000.
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Abstract |
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These results are not only compatible with the hypothesis that the carbohydrate structures are another potential source of immunological cross-reaction between different plant allergens, but they also demonstrate that the Lea-type structure is very widespread among plants.
Key words: allergy/MALDI-TOF-MS/Lewis a/plant glycoproteins
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Introduction |
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Food glycans are of interest because the IgE cross-reactions that have been reported between pollen and vegetable foods are at least partly due to carbohydrate components (Aalberse and van Ree, 1997; van Ree et al., 2000
); indeed it has been claimed that major food allergens are typically water-soluble glycoproteins (Bernhisel-Broadbent, 1995
). The roles of proteins themselves in cross-reactivity should, however, not be understated because many clinically relevant cross-reactions are between proteins (Breiteneder, 1998
), such as analogues of Bet v I (Schöning et al., 1995
), profilins (e.g., Bet v II) (Valenta et al., 1992
), isoflavone reductases (Vieths et al., 1998
), and hevein/type I chitinases (Salcedo et al., 1999
).
Carbohydrate-dependent cross-reactions have been probed by crude means, such as examining loss of binding of IgE to periodate-oxidized or chemically deglycosylated food extracts. More specifically, neoglycoconjugates consisting of glycopeptides carrying core 1,3-linked fucose coupled to bovine serum albumin have been found, as judged by direct enzyme-linked immunoassay (ELISA) and/or inhibition ELISA, to bind IgE from either pollinosis patients who displayed also food hypersensitivity or patients allergic to celery (Petersen et al., 1997
; Fötisch et al., 1999
). In addition, studies in this laboratory have shown that IgG antibodies recognizing core
1,3-linked fucose will bind a range of pollen and food extracts (Wilson et al., 1998
); indeed a structural survey of glycans verified the presence of this epitope in 10 pollens (Wilson and Altmann, 1998
). Whether such carbohydrate-mediated cross-reactions are clinically significant is controversial (Van der Veen et al., 1997
), although core
1,3-fucosylated glycoconjugates have been shown to elicit the release of histamine from mast cells (Batanero et al., 1999
; Fötisch et al., 1999
), suggesting that glycans may indeed be biologically active in allergy. However, it is obvious that their cross-reactivity can complicate allergy diagnosis, and this assumption is corroborated in several reports (van Ree and Aalberse, 1993
, 1999; Aalberse, 1997
; Aalberse and van Ree, 1997
; Mari et al., 1999
; van Ree et al., 2000
).
Among cross-reactions of interest in the present study, an allergy to latex is often associated with hypersensitivity to avocado, banana, and kiwi (M'Raihi et al., 1991; Blanco et al., 1994
; Ahlroth et al., 1995
; Lavaud et al., 1995
; Müller et al., 1998
); in two cases, cross-reactivity between latex and buckwheat was also reported (de Maat-Bleeker and Stapel, 1998
). On the basis of comparing IgE blots with Western blotting with an anticarbohydrate antibody, these cross-reactions to latex have recently been assigned to chitinases, rather than to N-linked oligosaccharides (Diaz-Perales et al., 1999
). On the other hand, possibly complicating the picture also is the finding that kiwi allergy can also be associated with birch allergy (Gall et al., 1994
); one study suggested that this cross-reaction is mainly due to carbohydrate (Fahlbusch et al., 1998
). A similar conclusion was made about the cross-reaction between latex and grass pollen (Fuchs et al., 1997
). Indeed, both birch and grass pollens carry N-glycans with xylose and core
1,3-linked fucose (Wilson and Altmann, 1998
). In yet another example of overlapping allergy syndromes, sensitivity to birch or mugwort pollen can also be associated with allergy to apple and other Rosaceae fruits, carrot, celery, and potato (Calkhoven et al., 1987
; Ebner et al., 1996
; Heiss et al., 1996
; Fernández-Rivas et al., 1997
). Of specific interest with regard to the present study, RAST of apple- and peach-allergic patients can be inhibited up to 85% by proteinase Kdigested pollen extract, suggestive of the importance of nonprotein (possibly carbohydrate) components in the cross-reactivity (van Ree et al., 1995
); in another study, a number of celery-allergic patients displayed carbohydrate cross-reactivity as judged by periodate oxidation and by binding to a neoglycoconjugate containing the MUXF3 structure (Jankiewicz et al., 1998
).
Hazelnut allergy has also been described in tree pollenallergic patients (Hirschwehr et al., 1992). Indeed, nuts, whether legume peanut or true tree nuts, are a major source of allergy (Burks et al., 1998
; Sicherer et al., 1999
); a rare allergy to coconut was also found to be associated with allergy to other nuts, especially walnut, but also almond (Teuber and Peterson, 1999
). Among other foods examined in the present study, allergic reactions to asparagus (Eng et al., 1996
; Escribano et al., 1998
), cauliflower (van Ketel, 1975
), pignoli (pine nut) (de las Marinas et al., 1998
), onion (Valdivieso et al., 1994
), pistachio (Parra et al., 1993
; Fernandez et al., 1995
; Liccardi et al., 1996
), strawberry (Grattan and Harman, 1985
), and tomato (Petersen et al., 1997
) have also been reported.
As part of ongoing studies in this laboratory to verify the structural basis for carbohydrate-mediated IgE cross-reactivity, the glycan structures from twenty-seven food extracts have been examined. By means of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), supplemented by size-fractionation and reverse-phase high-performance liquid chromatography (RP-HPLC) as well as nuclear magnetic resonance (NMR) spectroscopy, it was found that all plant foods contain N-glycans carrying xylose and core 1,3-linked fucose, and many also contain Lea epitopes; thus, not even considering O-glycans or cell wall polysaccharides, it is obvious that N-glycans are major candidates for explaining carbohydrate cross-reactivity between different plant species.
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Results |
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MALDI-TOF-MS profiling of N-linked oligosaccharides from plant foods
The Asn-linked glycans from various foodstuffs, covering large sections of the phylogenetic tree of plants, were analyzed by MALDI-TOF-MS of underivatized oligosaccharides prepared by immediate proteolytic digestion of these foods. This procedure has been devised to avoid two possible errors inherent to the analysis of soluble extracts as has been performed previously (Wilson and Altmann, 1998). First, the immediate pepsin digestion of proteins at pH 2 is considered to prevent any enzymatic degradation of glycans that might occur during extraction. Second, N-glycans from insoluble, e.g., membrane-associated, glycoproteins will likewise be included by this technique. Due to the different masses of hexoses, GlcNAc, xylose, and fucose, MS allows a fairly detailed insight into the structural species present in a sample. Assuming a conserved biosynthetic pathway of N-glycans in plants (Lerouge et al., 1998
), masses can be translated to primary structures as depicted in Figure 1. Several examples of N-glycan profiles obtained by this procedure are shown in Figure 2, and Table I gives a comprehensive overview of the results of our study. As will be shown below, in several cases the conclusions drawn from mass data were proven valid by other methods. The purification procedure relies on the affinity of peptides and glycopeptides to cation exchange resin under fairly acidic conditions. Even under these conditions, a small percentage of very acidic peptides or peptides with very acidic glycan moieties may not bind. The latter, however, are not supposed to occur in plant glycoproteins (Lerouge et al., 1998
). After release of the (neutral) oligosaccharide from peptide, the glycans will no longer bind to the resin. Additional purification steps, such as gel filtration and reversed-phase chromatography, facilitated the preparation of N-glycans from whole foodstuffs in sufficient purity to be analysed by MALDI-TOF-MS.
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Variation of the quantitative pattern can also arise from the samples themselves as the N-glycan profile may depend on variety, ripening status, and storage conditions. For instance, in sequential preparations of peanut glycans (GF)(GF)XF3 was detected (albeit as a minor component) in very fresh peanuts, whereas this structure was no longer detectable after 10 weeks of storage at room temperature. Generally, however, the results from those foods from which two or more preparations had been performed (almond, apple, celery, coconut, pea, peanut, pear, potato, tomato, and strawberry) clearly showed that the overall profile remains conserved: the major species in one preparation are the major species in a duplicate preparation.
Artifactual variation can be introduced by inappropiate methodology, for instance, by lengthy extraction procedures. For example, when analyzed by the direct proteolysis method MMX accounted for only 6% of hazelnut glycans, whereas in the soluble extract this was a main compound and the relative amount of larger glycans was reduced. Likewise, tomato N-glycans when analyzed from extracts of acetone powder (Zeleny et al., 1999) contained relatively much more MUXF3 and other very small structures than the directly proteolyzed sample (compare Table IV in Zeleny et al., 1999
; and Table I herein). Therefore, we believe that the direct proteolysis method used in the current study results in a less distorted and more accurate overview of a samples N-glycan profile than any other recently applied procedure.
HPLC analysis of glycans
To demonstrate the validity of the mass spectrometric analyses, structures of the major N-glycans in some of the samples were elucidated by additional methods. In an approach similar to our previous study on pollens (Wilson and Altmann, 1998) and tomato (Zeleny et al., 1999
), pyridylaminated N-linked oligosaccharides from proteolyzed slurries of apple, banana, celery, kiwi, and strawberry and from soluble extracts of almond, avocado, coconut, pea, pistachio, and soya were fractionated according to size as shown for celery in Figure 3. Each nonvoid peak collected from size fractionation HPLC was rechromatographed by RP-HPLC; in addition, the full spectrum of N-glycans was also analysed by RP-HPLC of pyridylaminated N-glycans (data not shown). The effects of exoglycosidase digestion on retention times were compared with the properties of standard oligosaccharides MMXF3, MUXF3, MMX, and MM to assign the structure. In a number of cases, fractions were subject to MALDI-TOF-MS analysis. For kiwi and banana N-glycans identity was inferred by comparison of their three-dimensional properties to those of corresponding celery glycans, but additional exoglycosidase digests were not performed. In the following account of experimental evidence results are presented in groups of related structures. As a detailed rationale for the structural assignments of N-glycans of the oligomannosidic and of the truncated type (up to GnGnXF3) has been presented in previous articles (Kubelka et al., 1994
; Wilson and Altmann, 1998
), the respective results for food N-glycans will only be outlined here.
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Truncated structures with both xylose and fucose (UUXF3, MUXF3, MMXF3, GnMXF3, MGnXF3, GnGnXF3).
UUXF3 was identified in apple and celery ([M+Na]+ = 967.2) based on its RP-HPLC retention time (5.2 g.u.-RP) being identical to the final mannosidase digest product of MUXF3 and MMXF3. According to "three-dimensional" properties, MUXF3 was found in apple, avocado, celery ([M+Na]+ = 1128.3), and soybean extract and MMXF3 in almond ([M+Na]+ = 1290.4), apple, avocado, celery ([M+H]+ = 1268.6), coconut ([M+Na]+ = 1290.7), pea, pistachio ([M+Na]+ = 1291.6), soybean, and strawberry ([M+H]+ = 1268.2). -Mannosidase digestions of these glycans resulted in the expected shift to UUXF3 (5.2 g.u.-RP). In the case of MMXF3 an intermediate digestion product of 6.0 g.u. on reverse-phase (the MUXF3 structure) was apparent because the
1,6-mannose is more resistant to
-mannosidase treatment. On further incubation with
-mannosidase, this intermediate product was converted completely or almost completely to UUXF3 in all cases.
GnMXF3 and MGnXF3, as present in almond, apple, avocado, celery ([M+Na]+ = 1492.6), pea, and strawberry ([M+Na]+ = 1493.1), co-elute on Micropak (7.3 g.u.) but separate on RP-HPLC (5.8 and 4.7 g.u.). Similar to the previously reported results with pollen N-glycans (Wilson and Altmann, 1998), GnMXF3 (from almond and pea) was sensitive to
-mannosidase as shown by its conversion to GnUXF3 eluting at 8.0 g.u.-RP. On the other hand, MGnXF3 is apparently insensitive to this treatment, compatible with the known resistance of
1,6-linked mannose (Oku et al., 1990
). GnUXF3 itself was found in celery due to its retention time of 7.8 g.u.-RP and its mass ([M+Na]+ = 1330.1).
GnGnXF3 (8.2 g.u.-MP, 5.4 g.u.-RP) was found in apple, celery ([M+Na]+ = 1696.5), and strawberry ([M+Na]+ = 1698.9). ß-Hexosaminidase treatment of GnMXF3, MGnXF3, or GnGnXF3 resulted in a single peak of 4.8 g.u.-RP, which is the retention expected for MMXF3. Subsequent -mannosidase digestion, when performed, of hexosaminidase-treated fractions showed the same pattern of digestion indicative of the MMXF3 structure.
In addition to these experiments, celery fractions assigned as MMXF3 and GnGnXF3 were subject to monosaccharide analysis by gas liquid chromatographymass spectrometry (GLC-MS) and found to contain besides fucose, mannose and GlcNAc only xylose but no arabinose.
Solely fucosylated structures (MMF3).
MMF3 was identified in almond ([M+Na]+ = 1158.2) due to its -mannosidase sensitivity (shift from 5.0 to 4.5 g.u.-RP) being similar to that of MMF3 from bee venom phospholipase (Kubelka et al., 1993
).
Galactosylated structures with xylose and core 1,3-linked fucose.
Two structures only resolvable by RP-HPLC but consistent in terms of mass with being GnGXF3 and GGnXF3 were found to be present in celery fraction 12 (12c and 12d). Treatment of 12c with ß-hexosaminidase resulted in a shift in RP-HPLC elution time from 5.2 to 4.7 g.u, whereas treatment of 12d with ß-hexosaminidase resulted in a shift from 5.4 to 6.0 g.u.-RP. This product was purified and then subjected to a combined mannosidase and galactosidase treatment, resulting in a peak of 7.8 g.u.-RP. Thus, it is concluded that fraction 12d contains GGnXF3 because the combined enzyme digestions resulted in an increase in elution time to the same as the elution time of GnUXF3, whereas the decrease in elution time on hexosaminidase treatment of 12c would suggest that the galactose was on the 3-arm (i.e., a structure of GnGXF3).
HPLC analysis of structures containing the Lea epitope.
Oligosaccharides with a long retention time on the sizing column (812 g.u.-MP) were found in apple, banana, celery, kiwi, and strawberry, but which by RP-HPLC had similar retention times in the range 4.85.8. g.u., thus making them and their exoglycosidase digestion products difficult to distinguish by the latter HPLC method. However, on the basis of mass, the compositions of these glycans are compatible with the presence of terminal galactose and fucose residues, in addition to core xylose and 1,3-linked fucose. Bearing in mind the recent findings on the presence of Lea epitopes in plants, putative Lea-containing structures from celery and strawberry were analyzed by HPLC in conjunction with exoglycosidase digests as follows.
The largest N-glycan, e.g., from celery (peak 17 in Figure 3), had a mass of [M+Na]+ = 2313.2, which is consistent with the complete secreted-type structure (Fitchette-Lainé et al., 1997; Melo et al., 1997
). Its HPLC elution position (12 g.u.-MP, 5.2 g.u.-RP) was identical with the largest structure from sycamore cell laccase, which was previously shown to contain two Lea-determinants (Fitchette-Lainé et al., 1997
). The putative (GF)(GF)XF3 [or (G3F4)(G3F4)XF3 if linkages are to be fully assigned] from celery as well as strawberry was sensitive to almond
-fucosidase, which removed two residues as judged by MicroPak and MALDI-TOF-MS. Partial fucosidase digest resulted in a mixture of monofucosylated products with elution times of 11.0 g.u.-MP and 5.0 and 5.2 g.u.-RP; full digestion gave a product of 10 g.u.-MP and 5.1 g.u.-RP. Because the core
1,3-fucose is not sensitive to almond
-fucosidase (Kubelka et al., 1993
), these data show that two outer-arm fucosyl residues had been removed. The defucosylated glycan now was sensitive to bovine kidney ß-galactosidase but resistant to Aspergillus oryzae ß-galactosidase. This was in contrast to the sensitivity of the ß1,4-linked galactose residues of bovine fibrin glycans to the Aspergillus enzyme (data not shown). Since the fungal enzyme has a strong preference for ß1,4-linkages (Zeleny et al., 1997
), the two glycans from celery and strawberry apparently contained ß1,3-linked galactose residues. The structural analysis of strawberry (GF)(GF)XF3 was completed by NMR spectroscopy (see NMR spectrometric analysis of Lea-containing structures).
In addition to the complete vacuolar type, smaller species were found to be present in apple, celery, and strawberry when analysed by HPLC.
(GF)GXF3/G(GF)XF3 (celery 16; 11 g.u.-MP; 5.15.2 g.u.-RP; [M+Na]+ = 2167) appeared to be a mixture of two isomers that had the chromatographic properties of a partial -fucosidase digest of 17, which also results in two isomers (11.0 g.u.-MP; 5.1 and 5.2 g.u.-RP).
A small fraction of celery peak 16 behaved differently, having a RP-retention time of 5.9 g.u. and a mass of [M+Na]+ = 2151. With -fucosidase and ß-hexosaminidase, there was no change in RP elution time of the presumed (GF)FXF3, but the expected digestion product would also have 5.9 g.u.-RP (GMXF3, cf. digestion of 12d). Subsequent digestion though with
-mannosidase resulted in a retention time of 7.0 g.u.-RP (presumably GUXF3). Thus, we conclude this trace compound to have the structure (GF)FXF3.
(GF)GnXF3/Gn(GF)XF3 (celery 14d; 10 g.u.-MP; 5.2 g.u.-RP; [M+Na]+ = 2005) was insensitive to ß-galactosidase but converted to a peak of 5.4 g.u. (RP-HPLC) on digestion with both almond -fucosidase and ß-galactosidase. ß-hexosaminidase digestion resulted in the almost complete conversion of the doublet to two peaks (4.7 and 5.8 g.u.-RP), which correspond in retention time to M(GF)XF3 and (GF)MXF3, respectively, suggesting that celery peak 14d is a mixture of both possible isomers. The corresponding peak from strawberry was converted by almond
-fucosidase to a peak of 9.4 g.u.-MP and by combined fucosidase/galactosidase digestion to a peak of 8.2 g.u.-MP, suggestive of the respective removal of one and two residues. In theory, the Gal and Fuc residues might be located on different GlcNAc residues, but structures GFXF3/FGXF3 were present in only minute amounts, if at all, as judged from the ß-hexosaminidase digest of celery 14d and from NMR in the case of the respective strawberry structure.
Celery peak 14 also contained a small amount of an additional structure (10.0 g.u.-MP; 4.8 g.u.-RP; [M+Na]+ = 1989), tentatively assigned as FFXF3 by means of its mass.
M(GF)XF3/(GF)MXF3 (celery 12b and 12e; 9.2 g.u.-MP; 4.8 and 5.8 g.u.-RP) were both insensitive to bovine testes ß-galactosidase, but also a combined -fucosidase and ß-galactosidase digest of 12e resulted in no obvious change in elution time by RP-HPLC; previous studies have shown that the GnMXF3 product of such a digest would have a retention time of 5.8. g.u.-RP anyway. However, a subsequent
-mannosidase digest resulted in GnUXF3 as the probable final product (7.8 g.u.-RP), thus suggesting that 12e is indeed (GF)MXF3, whereas the predominant isomer is presumably M(GF)XF3.
For other samples, extensive fractionation and analysis of individual glycan species were not performed. However, the PA-glycans from apple, asparagus, buckwheat, carrot, cauliflower, hazelnut, onion, and pear were subject to RP-HPLC. The profiles obtained agreed with the assumption that these samples contained the same N-glycans as celery and strawberry.
NMR spectrometric analysis of Lea-containing structures
Since the linkage of the fucose to the celery and strawberry putative Lea-containing structures cannot be specified by the results of almond -fucosidase digestion, peaks 9 and 11 from strawberry (corresponding to peaks 14 and 17 in celery, see Figure 3) were analyzed by 1D-1H-NMR as pyridylaminated N-glycans.
For peak 11, putative (GF)(GF)XF3, 1D-1H-NMR spectroscopy showed the H-1, H-5, and CH3 signals for three -L-Fuc residues, the H-1 signals for two ß-D-Gal residues, and the H-1 signals for two antennary ß-D-GlcNAc residues (Table II). The values of the structural-reporter-group signals matched those reported by Takahashi et al. for laccase (Takahashi et al., 1986
) and miraculin (Takahashi et al., 1990
), and by Fitchette-Lainé et al. for laccase (Fitchette-Lainé et al., 1997
). The originally reported terminal sequence in laccase diantennary Xyl-containing N-glycans with
1,3-linked Fuc at the Asn-bound GlcNAc residue, Galß4(Fuc
6)GlcNAc (Takahashi et al., 1986
, 1990) was revised to be the Lewisa epitope Galß3(Fuc
4)GlcNAc (Fitchette-Lainé et al., 1997
). Unfortunately, however, in a recent paper on peanut peroxidase glycans Shaw et al. followed the original interpretations of Takahashi et al. (Shaw et al., 2000
).
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Additionally, two 2D-1H-NMR experiments were carried out, a total correlation spectroscopy (TOCSY) measurement for the assignment of the relevant skeleton protons and a nuclear Overhauser effect spectroscopy (NOESY) measurement for sequence/glycosidic linkage information. These data confirmed the presence of a ß1,3-linkage between Gal and GlcNAc in both antennae. The chemical shifts of H-2, H-3, and H-4 of the 1,4-linked Fuc residues match those of an O-glycan Lea-determinant (Strecker et al., 1992
). The H-1 signal of the antennary Fuc residues showed NOEs with the H-4, H-6a, and H-6b signals of the antennary GlcNAc residues, as shown to be the case for the synthetic Lea-trisaccharide (Kogelberg and Rutherford, 1994
). These findings thus constitute an illustration that caution should be applied when using NOEs as the sole basis for a structural assignment.
For strawberry peak 9, putative (GF)GnXF3/Gn(GF)XF3, the 1D-1H-NMR also showed the presence of a Lea-determinant (Fuc H-1, 5.008; Fuc H-5,
4.870; Fuc CH3,
1.177). In view of the different structural-reporter-group signals for the antennary Gal and GlcNAc residues (Table II), this peak was determined to be a mixture of two isomers, with either a Lea-determinant on the 6-branch and terminal GlcNAc on the 3-branch or vice versa (estimated ratio 3:1).
Oligomannosidic oligosaccharides
In initial studies, endoglycosidase H treatment of complete N-glycan pools from soya and pea extracts suggested that these extracts had a number of oligomannosidic oligosaccharides. Percentages of endoglycosidase Hsensitive glycans, as judged by the integration of the GlcNAc peak of 3.5 g.u. on reverse-phase were 78% and 63% for the extracts from soya and pea, respectively, whereas for other foods the percentage of oligomannose structures was far lower. Verification of the nature of the presumed oligomannosidic oligosaccharides was by means of (1) size estimation on Micropak; (2) comparison with experimentally determined retention times of the oligomannosidic series of soybean 7S glycoprotein (Man6Man8), and Man5 from A. oryzae -amylase; (3) demonstration that
-mannosidase treatment resulted in a final digestion product with retention time of 6.8 glucose units; and (4) by MALDI-TOF-MS.
In the case of three large pea peaks, the determined [M+Na]+ masses were 1500.9, 1661.9, and 1824.4, consistent with the respective HPLC-determined designations of Man6 (Man6GlcNAc2), Man7, and Man8.
The oligomannosidic glycans from apple, celery, pea, soya, and strawberry were analyzed by 2D HPLC. For Man5 to Man8, a number of isomers can theoretically exist, although in the present study only multiple isomers of Man7 were found. For strawberry all three isomers of this structure were detected (5.2, 5.6, and 7.6 g.u.-RP). Based on the compatibility of the reverse-phase retention times with those given by Kubelka et al. (1994) and Tomiya et al. (1988)
, which fortunately agree with each other to within ± 0.1 g.u., the isomers were, in the order of theír elution, assigned to be Man7(1), Man7(3), and Man7(2) (Altmann et al., 1999
) occuring in a ratio of 0.8 to 1.0 to 0.1 in strawberry.
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Discussion |
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Utility of glycome analysis of foods
The results presented in this article were primarily obtained by a novel method for preparation and purification of N-glycans from whole foodstuffs. This strategy is believed to avoid two problems inherent to the currently applied procedures of glycoprotein analysis: (1) enzymatic degradation of N-glycans during extraction and purification of glycoproteins and (2) preferential detection of N-glycan species present on abundant, soluble glycoproteins. The data presented here for a wide variety of edible parts of food crops derived from various dicotyledons and monocotyledons as well as one gymnospermic plant therefore may well reflect the general glycosylation capacity of this tissue at a given time. By analogy to its older "cousins" genome and proteome analysis we thus would like to call the herein presented strategy glycome analysis, in which the glycome is represented by a mass profile obtained by MALDI-TOF-MS. Knowledge of the structures and the biosynthesis of N-glycans in plants is utilized to translate mass data into compositions and structure (Lerouge et al., 1998). We have tested the suitability of these tentative assignments by separating and analyzing the glycans from a number of samples by additional methods, such as HPLC of pyridylaminated glycans and 1H-NMR. Generally, the quantitative and qualitative results obtained by MALDI-TOF-MS and by HPLC agreed, notwithstanding the ability of RP-HPLC to resolve isomeric structures such as MGnXF3/GnMXF3 or M(GF)XF3/(GF)MXF3. Only in the case of trace amounts of (GF)FXF3 and FFXF3 from celery, which were detected by MALDI-TOF-MS in fractions purified by HPLC but not in the whole mixture, did the results deviate. As the masses of these compounds ([M+Na]+ = 2072.7 and 1901.7) are close to that of more abundant glycans, detection of these trace compounds was beyond the limits of MALDI-TOF-MS with a linear flight path. As the presented method for glycome analysis is very new, the results cannot be directly compared with published data. Only in the case of tomato, a recent publication in general validates the structural assignments from the glycan masses. However, it also reveals the discrepancies between results obtained with different methods of N-glycan preparation (see Results). Another similar study has been performed on soluble extracts from allergenic pollens (Wilson and Altmann, 1998
). In this case, no structures larger GnGnXF3 were found, and thus it is possible to ask whether the complete lack of such larger structures was likewise an artifact of allergen extraction rather than a reflection of the pollens authentic N-glycan profile.
A remarkable situation is found with soybeans, where the N-glycans obtained from the entire beans qualitatively and quantitatively essentially resemble to those on the 7S major storage protein (Neeser et al., 1985). In some samples, oligomannosidic N-glycans carrying 10 hexose residues were found. Considering the biosynthesis of N-glycans and a recent paper on Glc-containing Man9 in jack bean
-mannosidase (Kimura et al., 1999
), the 10th hexose is presumed to be glucose. For papaya leaves, the occurence of the unusual structures Man5XF3 and Man5GnXF3 has been reported (Makino et al., 2000
). These N-glycans have also been found as major constituents of papaya fruit (Table I). In the present study, further elaborations of the 3-arm leading to the novel structures Man5GXF3 and Man5(GF)XF3 could be shown for a small fraction of papaya fruit N-glycans.
A severe limitation of MALDI-TOF-MS is that isobaric structures and monosaccharides, such as arabinose and xylose, cannot be distinguished. One study suggested the presence of arabinose in free tomato N-glycans (Priem et al., 1993). HPLC fractionation did not yield fractions with an elution behavior deviating from the Xyl-containing standards. However, Ara-containing glycans might comigrate; but compositional analysis of MMXF3 and GnGnXF3 from celery revealed the presence of xylose only. Xylose-containing glycans that carry additional rhamnose and/or arabinose residues have been suggested to be present on secreted carrot glycoproteins (Sturm, 1991
); in the present study, however, masses indicative of such structures were not found during the analysis of carrot N-glycans.
Glycome analysis is, of course, only a first step in the determination of which glycans have a role in allergy and IgE cross-reactivity. One limitation of most studies in this area, including the present one, is that O-glycans have been generally ignored, although there is data suggesting a role for O-glycans as IgE epitopes on the major allergen of Parthenium pollen (Gupta et al., 1996). Unfortunately, methods developed for the analysis of mammalian O-glycans are inapplicable to plant O-glycans due to the entirely different linkages of sugar to protein (Klis, 1995
). Studies are being initiated to investigate these glycans in a range of foods, which may prove to be another source of cross-reactivity.
Presence of Lea
The literature on plant glycosylation has until recently given the impression that plant N-linked oligosaccharides are either of the truncated "vacuolar" type, with core 1,3-linked fucose and/or ß1,2-linked xylose, or are oligomannosidic. Until 1997, only three instances of larger structures had been reported. These glycans, suggested to carry the nonreducing terminal Galß4(Fuc
6)GlcNAc sequence were found in highly "exotic" sources, that is, sycamore cell laccase, the taste-modifying protein miraculin, and Japanese cedar pollen allergen Cry j I (Takahashi et al., 1986
, 1990; Ogawa et al., 1996
). However, more recently it has become apparent that these terminal fucosylated sequences take the form Galß3(Fuc
4)GlcNAc, the same as and cross-reacting with antibodies against mammalian Lea structures (Fitchette-Lainé et al., 1997
; Melo et al., 1997
). The combined HPLC and NMR data from the present study are consistent with the revisionist interpretation of the data on plant glycans with branches containing galactose and fucose residues.
Antibody-binding studies have shown that the Lea epitope is present in a range of plant tissues, such as onion and tomato roots, sycamore, gingko, spruce, crocus, walnut, chive, celery, and columbine leaves, while being completely or nearly absent from Arabidopsis, canola, radish, and cauliflower (Fitchette-Lainé et al., 1997; Fitchette et al., 1999
). In keeping with these results, no Lea-containing glycans could be found in cauliflower by MALDI-TOF-MS (Table I). However, papaya (which, like Arabidopsis and cauliflower, belongs to the Brassicaceae family) did exhibit Lea determinants albeit as part of a unique type of hybrid N-glycans (Table I, Figure 1). Mass spectrometry data also indicate the presence of the Lea epitope in tobacco (Fitchette et al., 1999
), an important plant with respect to expression of recombinant proteins. As shown for the first time by direct structural analysis, the N-glycan repertoire of gymnospermic plants (e.g., conifers) resembles that from angiospermic plants, including the presence of smaller xylosylated and core-
1,3-fucosylated as well as of larger Lea-containing N-glycans. In contrast to structural analyses by others (Kimura and Matsuo, 2000
) but in agreement with the aforementioned antibody-binding data (Fitchette-Lainé et al., 1997
), our recent data (Kolarich, D., unpublished observations) indicate the presence of the Lea structure in gingko, another old phylogenetic neighbor of the angiospermic plants.
Our results constitute direct structural confirmation that the Lea epitope is widespread and that members of a wide spectrum of taxonomic orders are capable of synthesizing this determinant. Lea-carrying glycans were major components primarily in "fleshy" foods, that is, in apple, asparagus, banana, carrot, celery, kiwi, onion, orange, pear, and strawberry. Morever, pignoli (from a coniferous plant), avocado, hazelnut, and walnut contained appreciable amounts of these glycans, whereas tomato and potato (Solanaceae) and legume seeds generally contained only small amounts of Lea-carrying glycans. Furthermore, our data indicating the presence of the Lea-epitope in kiwi is compatible with the interaction of glycoproteins from this fruit with Aleuria aurantia lectin (Fahlbusch et al., 1998), which binds a number of fucose-containing oligosaccharides including some carrying Lea (Kochibe and Furukawa, 1982
). The presence of this epitope raises questions as to its effect on animals exposed to plant material expressing these structures.
Comparison with antibody data
The presence of xylose and core 1,3-linked fucose in all tested foods is consistent with our previous findings (Wilson et al., 1998
) on the binding of anticarbohydrate antibodies to a range of soluble vegetable, fruit, and nut extracts. In the present study, direct comparison can be made between the antibody data (using antihorseradish peroxidase and the core
1,3-linked fucose-specific monoclonal YZ1/2.23) and the structural data for pea, soya, avocado, pear, strawberry, almond, coconut, and pistachio. All these extracts showed high binding to antihorseradish peroxidase, in general inhibitable by the addition of a conjugate of bovine serum albumin with a glycopeptide carrying MUXF3 but not inhibitable by the defucosylated analogue conjugate. In addition all of these samples, except pea, showed significant binding (also inhibitable) to YZ1/2.23. Although pea and coconut both carry very little MMXF3, there is a comparably high binding of YZ1/2.23 to coconut where glycans with xylose only predominate. Thus, although nonfucosylated glycopeptide-albumin conjugates were poor inhibitors for the binding of YZ1/2.23 to MUXF3 structures (Wilson et al., 1998
), solely xylosylated N-glycans like MMX nevertheless might have affinity to this monoclonal. Another explanation would be that MMXF3 determinants are presented more properly by the extract from coconut than that from pea. In the literature there are other reports that polyclonal antibodies against carrot fructosidase (Faye and Chrispeels, 1988
) and peanut peroxidase (Wan and van Huystee, 1994
) are also predominantly "anticarbohydrate"; the former antibodys binding to apple and carrot is consistent with the present data.
Knowledge of the glycan structures, rather than just antibody-binding data, present in the complete mix means that further research can be better directed. Approaches with promise include direct analysis of glycans on allergens separated by SDSpolyacrylamide gel electrophoresis (Kolarich and Altmann, 2000), as well as the use of specific glycoconjugates as inhibitors of IgE binding or RAST assays or as elicitors of histamine release.
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Materials and methods |
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Preparation of N-glycans
Approximately 100 g of fleshy fruit or vegetable (e.g., apple or carrot) or 40 g of dry foods (e.g., nuts and beans) were chopped or ground and suspended in 200 ml of water. After mixing with a kitchen blender, the slurries were made up to 300 ml with a final concentration of 5% (v/v) formic acid and about 0.1 mg/ml pepsin (and 0.1% 2-mercaptoethanol in the case of potato). After prewarming in a water bath, the slurry was incubated at 37°C for 20 h with occasional stirring. Insoluble material was then removed by centrifugation (20 min, 15°C, 10,000 rpm, Sorvall-DuPont GSA rotor). The supernatant was mixed for 15 min with 100 ml Dowex 50W x2 (prewashed with 500 ml 0.4 M ammonium acetate, pH 6.0, and 1 L 2% [v/v] acetic acid). The resin was then washed in a funnel with 300 ml 2% (v/v) acetic acid, poured into a column, washed with one column volume of 2% (v/v) acetic acid and then with 0.4 M ammonium acetate, pH 6.0. Forty fractions of 400 drops were collected, and fractions that gave a positive orcinol test were pooled and the volume reduced by rotary evaporation. This glycopeptide fraction was subject to gel filtration on Sephadex G25 (coarse; 2.5 x 120 cm) and eluted with 1% (v/v) acetic acid. The orcinol-positive fractions were pooled, lyophilized, and dissolved in approximately 1 ml citrate-phosphate buffer, pH 5.0. After incubation at 95°C for 10 min, the N-glycans were released by incubation with 0.7 U of peptide:N-glycosidase A, 37°C, 24 h. An equal volume of 10% (v/v) acetic acid was added to acidify the sample, which was then passed over Dowex 50W (45 ml) to separate released N-glycans from residual glycopeptides (including possible O-linked glycans). The column was washed with 2% (v/v) acetic acid; the unretained orcinol positive fractions were concentrated and subject to gel filtration (Sephadex G15, elution with 1% [v/v] acetic acid). The orcinol positive fractions were pooled, concentrated, and applied to a small reversed-phase column (200 µl; Lichroprep RP 18, 2540 µm prewashed with 2 ml of 60% [v/v] propan-2-ol in water containing 5% [v/v] acetic acid) and then 5 ml 5% [v/v] acetic acid). The N-glycans were eluted with 23 ml 5% (v/v) acetic acid, concentrated with a rotary evaporator, lyophilized, and finally dissolved in 50 µl water for MALDI-TOF-MS analysis.
In some cases, the glycans were then subject to derivatization by reductive pyridylamination (Hase et al., 1984; Kubelka et al., 1993
; Wilson and Altmann, 1998
). N-glycans from extracted soluble glycoproteins were prepared as described for pollen extracts (Wilson and Altmann, 1998
).
MALDI-TOF-MS
Aliquots of 0.8 µl of underivatized or pyridylaminated N-glycans were applied to a flat sample platen. To the dry sample, 0.8 µl of matrix (2% 2,5-dihydroxybenzoic acid in 30% [v/v] acetontrile) were added and and dried immediately under mild vacuum. MALDI-TOF-MS spectra were acquired on a DYNAMO (Thermo BioAnalysis, Santa Fe, NM) linear time-of-flight mass spectrometer capable of dynamic extraction, a synonym for delayed extraction. The instrument was operated with a dynamic extraction setting of 0.1. External mass calibration was performed with pyridylaminated N-glycans or with a partial dextran hydrolysate.
HPLC analysis and glycosidase digestions
Pyridylaminated oligosaccharides were fractionated by a "two-dimensional" mapping technique starting with separation according to size on a Micropak AX-5 column (0.4 x 30 cm) (Tomiya et al., 1988). Peaks were collected and dried thoroughly prior to subfractionation in the second dimension by reverse-phase chromatography on an Hypersil ODS column (0.4 x 25 cm) (Kubelka et al., 1993
; Wilson and Altmann, 1998
). Columns were calibrated daily in terms of glucose units with a pyridylaminated partial dextran hydrolysate (310 glucose units). Peaks from either size fractionation or reverse-phase chromatographies were subject to exo- or endoglycosidase digestions as follows: Canavalia ensiformis (jack bean)
-mannosidase ("medium" dose; 25 mU in 20 µl 50 mM sodium acetate, 0.1 mM zinc chloride, pH 4.2); C. ensiformis ß-hexosaminidase (25 mU in 20 µl 0.1 M sodium citrate, pH 5.0); endoglycosidase H (2 mU in 20 µl, 0.1 M citrate-phosphate, pH 5.0); almond
-fucosidase 1 µl (0.1 µU in 20 µl, 0.1 M citrate-phosphate, pH 5.0); bovine testes ß-galactosidase 2 µl (1.6 mU in 20 µl, 0.1 M citrate-phosphate, pH 5.0).
Monosaccharide analysis
Sugars were analyzed as alditol acetates by GLC-MS as described (Kubelka et al., 1993).
1H-NMR spectroscopy
The oligosaccharide samples were exchanged twice with 2H2O (99.9 atom% 2H, Cambridge Isotope Ltd.) with intermediate lyophilisation, then dissolved in 2H2O (99.96 atom% 2H, Isotec Inc.). 1D-1H-NMR spectra were recorded at 600 MHz on a Bruker AMX 600 instrument (Bijvoet Center, Department of NMR Spectroscopy, Utrecht University) at probe temperatures of 300 K. Chemical shifts () are expressed in p.p.m. by reference to internal acetone (
2.225). A 2D TOCSY spectrum at 600 MHz was recorded using Bruker software with a MLEV-17 spin-lock pulse sequence (100 ms) preceded by a 2.5-ms trim pulse, and a 2D NOESY spectrum was recorded using a mixing time of 400 ms. Data matrices of 512 x 1024 points were collected representing a spectral width of 4800 Hz in each dimension. The residual water signal was suppressed by presaturation for 1 s during the relaxation delay. Phase-sensitive handling of the data was performed by the TPPI method inplemented in the Bruker software. The time domain data were zero-filled to data matrices of 1024 x 1024 points, prior to multiplication with a phase-shifted square bell function.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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