3 CNRS-UMR 6037, IFRMP 23, Université de Rouen, 76821 Mont Saint Aignan Cédex, France
4 INSERM U547, Institut Pasteur de Lille, 59019 Lille, France
5 INSERM U519, IFRMP 23, Université de Rouen, 76821 Mont Saint Aignan Cédex, France
6 INSERM U413, IFRMP 23, Université de Rouen, 76821 Mont Saint Aignan Cédex, France
Received on August 27, 2002; revised on October 22, 2002; accepted on October 24, 2002
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Abstract |
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Key words: immunogenicity / N-glycans / recombinant proteins / transgenic plants
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Introduction |
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On the other hand, core xylose and core (1,3)-fucose epitopes are known to be important IgE binding carbohydrate determinants of plant allergens (Aalberse et al., 1981
; Faye and Chrispeels, 1988
; van Ree and Aalberse, 1995; Garcia-Casado et al., 1996
; Wilson and Altmann, 1998
; van Ree et al., 2000
). Furthermore, we and others have reported that immunization of goats (Kurosaka et al., 1991
) or rabbits (Faye et al., 1993
) with plant glycoproteins elicits the production of core xylose- and core
(1,3)-fucose-specific Abs. A rat monclonal Ab (YZ1/2.23) raised against elderberry abscission tissues was found to be specific for core
(1,3)-fucose (McManus et al., 1988
).
Altogether, the immunogenicity of plant N-glycans remains a major pending issue. In the context of human therapy using therapeutic proteins produced in plants, elicitation of immune responses in humans by specific plant glyco-antigens could be a major concern if people have prolonged exposure to large quantities of plant-derived glycoproteins, as may be required for certain in vivo treatments. In this article, we show that crops, such as pea, rice, and maize, that are intended for the production of therapeutic proteins, introduce plant-specific core xylose and core (1,3)-fucose on their natural or recombinant glycoproteins. Data on the immunogenicity of these plant glyco-epitopes in immunized rodents as well as in nonallergic humans are also reported.
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Results |
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To further investigate their glycosylation, N-glycans were released from natural glycoproteins or from recombinant avidin by PNGase A treatment and analysed by mass spectrometry (MS). The matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS of the pools of N-glycans isolated from pea, rice, and maize seed glycoproteins (Figure 1A, B, and C, respectively) shows a mixture of ions that were assigned to (M+Na)+ adducts of high- mannose-type N-glycans ranging from Man-5 to Man-9 (indicated by a star) as well as (1,3)-fucose- and ß(1,2)-xylose-containing N-glycans from the truncated paucimannosidic structure a to the complex N-glycan h harbouring two Lewisa epitopes (Table I). Assignments were done on the basis of the molecular masses of N-glycans by homology with previous data on plant N-glycosylation (Lerouge et al., 1998
) and considering that glycoproteins from these plants were immunodetected with antibodies specific for core xylose, core
(1,3)-fucose, and Lewisa epitopes. These assignments were confirmed by enzyme sequencing using ß-N-acetylglucosaminidase and
-mannosidase combined to MALDI-TOF MS analysis of the resulting digests, as reported in Bakker et al. (2001)
.
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Immunogenicity of core xylose and core (1,3)-fucose epitopes in rodents
We investigated the immunogenicity of core xylose and core (1,3)-fucose epitopes of plant N-glycans in two different mouse strains and in rats. To this end, BALB/c and C57BL/6 mice were immunized with horseradish peroxidase (HRP), a plant glycoprotein harboring six identical N-glycans represented in Figure 2A (referred to as c in Table I) (Kurosaka et al., 1991
). The production of anti-glycan Abs in their sera was then determined by enzyme-linked immunosorbent assay (ELISA) using two model glycoproteins having a unique common glycan epitope with HRP: honeybee venom phospholipase A2 (PLA2), an insect glycoprotein containing a core
(1,3)-fucose (Kubelka et al., 1993
), and Helix pomatia hemocyanin, a snail N-linked glycoprotein containing a core xylose (van Kuik et al., 1985
) (Figure 2A). Because no glycan-specific responses were obtained in the experiments reported by Chargelegue et al. (2000)
using 30 µg of HRP, BALB/c mice were immunized with a higher amount of HRP (100 µg) using complete Freund's adjuvant to maximize the immune response. Results are expressed as log10 Ab titers after deducing the values obtained for the pre-immune sera.
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We then studied the immunogenicity of HRP in rats. In addition to the humoral response directed against the HRP protein backbone, high titers of IgM and of IgG (isotypes IgG1 and IgG2a) against core xylose and core (1,3)-fucose were measured by ELISA using the approach developed for mouse serum analysis (Figure 3A). A comparison of the Ab reactivity with the chicken or the recombinant maize-derived avidin further demonstrates that part of the humoral response is raised against plant N-glycans introduced onto the recombinant protein.
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A proportion of human nonallergic blood donors have Abs directed against the core xylose or core (1,3)-fucose epitopes
Because humans are exposed daily to plant glycoprotein antigens in edible plant material or to other environmental glycoprotein antigens, we investigated the presence in humans of Abs raised against the core (1,3)-fucose and/or the core xylose epitopes. Sera from 53 nonallergic human blood donors were analyzed by ELISA using PLA2 and snail hemocyanin as probes. Sera were considered positive when their log of IgM or IgG1 titers were superior to 2. Surprisingly, up to 27 sera were found to react against the core xylose or core
(1,3)-fucose epitopes (Figure 4A and B). Among these sera, 23 were positive for hemocyanin and 13 for PLA2, 9 of them being both PLA2- and hemocyanin-positive. Isotype analysis indicated that Abs are almost exclusively IgM and IgG1. However, although IgG4 and IgE were undetectable, IgG2 or IgG3 were weakly detected in four sera (not shown).
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Discussion |
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The presence of nonmammalian core (1,3)-fucose and core xylose epitopes onto glycans N-linked to therapeutic glycoproteins produced in plants, in addition to the observed immunogenicity of these glyco-epitopes in some laboratory animals, raised the question of their immunogenicity in the context of a human therapy using plant-derived biopharmaceuticals. This might be investigated by the analysis of human sera following administration of a human protein bearing plant N-glycans. Indeed, the stimulation of Ab production is a concern only if the protein carrier itself turns out to be immunogenic. Immunization of rodents as described herein is far from the context of human therapy with plant-derived proteins because a plant glycoprotein, rather than a self-antigen carrying plant N-glycans, is administrated. Furthermore, to stimulate antiglycan responses, the immunization of rodents was carried out with complete Freund's adjuvants, which is a usual protocol to generate immune responses against almost any target molecules. Such adjuvants are unlikely to be used in clinical applications during human therapy.
Because humans are exposed daily to plant glycoprotein antigens through food and inhalation, the presence in humans of Abs raised against plant glyco-epitopes was questionable. As a consequence, to address this question, sera from a healthy human population were analyzed. Surprisingly, 27 out of 53 sera were found to contain IgG1 raised against plant glyco-epitopes. About 50% of sera were positive for core xylose and 25% for core (1,3)-fucose. The presence of such Abs could be related to an immune response to the core
(1,3)-fucose and/or the core xylose ubiquitously present on plant N-glycans. In the same way, other environmental antigens, such as N-glycoproteins from insects (Kubelka et al., 1993
) or parasites (Khoo et al., 1997
; van Die et al., 1999
), may also contribute to Ab production against these glyco-epitopes.
The observed human serum titers as determined by ELISA (1:100 to 1:1000) are weak but significant. Whatever the origin of glycoproteins responsible for the human immunostimulation, this demonstrates that core (1,3)-fucose and/or the core xylose, existing on plant or other glycoprotein antigens, are able to elicit immune responses in humans.
The fact that humans have Abs against plant N-glycan epitopes does not necessarily mean that there will be adverse effects when administrating plant-derived therapeutic glycoproteins. The presence of low-titer antiplant glycan Abs might be a neutralizing effect. Although the immunological significance of anti-core (1,3)-fucose and anti-core xylose Abs is too speculative at the moment, the presence of such Abs may well induce an accelerated clearance of recombinant plant glycoproteins from plasma, resulting in a therapeutic failure. In addition to this accelerated clearance, clinical effects resulting from the administration of plant-derived therapeutic glycoproteins in allergic patients are also questionable. Indeed, because these plant N-glycan epitopes appear to trigger IgE response in allergic patients (Wilson and Altmann, 1998
; van Ree et al., 2000
) and in parasite-infected mammals (van Die et al., 1999
), the presence of such glyco-epitopes on plant-derived therapeutic glycoproteins could induce clinical troubles in allergic populations. As a consequence, for a more detailed evaluation of safety concerns related to the use of plant-derived therapeutic proteins, further experiments have to be carried out in an appropriate model animal as well as in human by administering a therapeutic glycoprotein produced in a plant and analyzing immune responses to the plant glyco-epitopes in allergic and nonallergic populations.
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Materials and methods |
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N-linked glycan analysis
Protein extraction from meal of pea, rice, or maize seeds and isolation of N-glycans were carried out according to Fitchette et al. (1999). Enzyme sequencing of plant N-glycans was carried out as reported by Bakker et al. (2001)
. For permethylation, the pool of N-glycans was reduced with NaBH4 and permethylated according to Ciucanu and Kerek (1984)
.
MS analysis
MALDI-TOF mass spectra of N-glycans were acquired on a Micromass (Manchester, UK) Tof Spec E MALDI-TOF mass spectrometer equipped with a nitrogen laser. Mass spectra were performed in the reflector mode using 2,5-dihydroxybenzoic acid as matrix. Assignments to both (M+Na)+ and (M+K)+ adducts were confirmed by running an additional spectrum with CsCl. In these conditions both (M+Na)+ and (M+K)+ adducts were converted into a single (M+Cs)+ ion.
CID and MALDI-PSD experiments, selection of the precursor ion was carried out using a Bradbury-Nielsen ion gate with a m/m=100 resolution. Extraction with a delay time of 600 ns was used. CID was carried out by collision with helium at a pressure of 10-6 mbar in the collision cell. To record the full PSD spectra, reflector voltage was decreased into successive 25% steps leading to 10 spectral segments using 30 laser shots per segment. PSD was calibrated using ACTH (fragment 1839).
Immunization of mice and rats
Four inbred 67-week-old female BALB/c (H-2d) or C57BL/6 (H-2b) mice were immunized by subcutaneous injection of, respectively, 100 µg and 30 µg of HRP in complete Freund's adjuvant, followed by boosts with the same dose of immunogen in incomplete Freund's adjuvant 2, 4, and 6 weeks after the first immunization. Sera were collected 1 week after the last injection. Immunization of four 8-week-old Wistar rats was carried out according to the same protocol and using 100 µg HRP.
Western blot analysis of model glycoproteins with different sera
The glycoproteins were separated by 15% sodium dodecyl sulfatepolyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane, and blocked overnight at room temperature with 3% gelatin dissolved in Tris buffered saline buffer. The membranes were incubated with the sera of mice (1/500), rats (1/1000), and humans (1/500) for 2 h as previously described (Faye et al., 1993). The membranes were incubated with either the anti-mouse, anti-rat, anti-human IgG Abs (1/1000) conjugated with HRP or biotinylated anti-human IgG1 Abs. For detection of biotinylated anti-human IgG1 Abs, the membranes were further incubated with streptavidin conjugated with HRP. For detection of HRP activity, the membranes were revealed with 4-chloro 1-naphtol (Faye et al., 1993
). Pre-immune sera were used as negative controls. Alteration of the glycan moiety of the model glycoproteins was carried out by oxidation using sodium periodate (Fitchette-Lainé et al., 1998
).
ELISA
Ninety-six-well plates were coated overnight at 4°C with either 1 µg HRP or recombinant avidin, 3 µg PLA2, or 10 µg of hemocyanin in 50 mM sodium carbonate buffer, pH 9.6. The microplates were then washed with phosphate buffered saline (PBS) containing 0.1% Tween 20 (PBS/Tween) and blocked with PBS plus 1% bovine serum albumin for 1 h at 37°C. The plates were then incubated with serial dilutions of mice, rat, and human sera in PBS/Tween/bovine serum albumin for 2 h at 37°C. The microplates were washed with the same buffer and incubated for 2 h at 37°C with appropriate secondary Abs. Dilutions used for the secondary antibodies are: HRP-conjugated anti-mouse IgM and IgG Abs, 1/10,000; HRP-conjugated anti-rat IgM and IgG Abs, 1/1500; and biotinylated anti-human IgM and IgG1 Abs, 1/10,000.
For biotinylated conjugates, a third incubation was carried out with streptavidin conjugated to HRP (1/1500) for 1 h at 37°C. HRP activity was detected by addition of 100 µl/well of a substrate solution (15 mg of o-phenylenediamine dihydrochloride tablets in 15 ml 0.05 M citrate buffer, pH 5.6, supplemented with 15 µl H2O2). After 30 min at room temperature in the dark, the enzymatic reaction was stopped with 50 µl 2 M H2SO4, and OD was determined at 492 nM. The same plates were prepared and incubated with pre-immune sera, and the OD obtained for pre-immune sera were deduced from values obtained for immunized animals for the determination of the titers. Ab titer was determined as the highest dilution that gives an absorbance value twofold superior compared to that of background.
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Acknowledgements |
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Footnotes |
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2 To whom correspondence should be addressed; e-mail: plerouge{at}crihan.fr
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Abbreviations |
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References |
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