Editorial
Asparagine (N)-linked glycosylation is a common modification of
secretory and membrane proteins in eukaryotes. However, plants, insects
and helminths produce N-glycosylated proteins that carry modifications
not found on mammalian glycoproteins. α1,3-linked core fucose residues
are present on plant and insect glycans and, in addition, β1,2-linked
xylose is found on plant- and helminth-derived glycans. Up to
35% of sera of allergic patients contain IgE antibodies specific for
glycan epitopes, also termed glycotopes.1 Initially,
these oligosaccharide epitopes were designated as cross-reactive
carbohydrate determinants (CCDs) based on the broad in vitrocross-reactivity of glycoproteins of plant foods, pollen and hymenoptera
venoms.2 Today, these classical CCDs are listed as
group A among 5 groups of oligosaccharides in the official allergen
nomenclature website at http://allergen.org/.3Although CCDs are probably the most frequent IgE epitopes, their
clinical significance is low. This is likely not based on the low
affinity of IgE to CCDs, but rather on the affinity of anti-CCD IgGs
which function as blocking antibodies.4 The effect of
anti-CCD IgEs are eliminated and false-positive in vitro test
results are significantly reduced by inhibition of sera with a
semisynthetic CCD blocker.1
The low clinical relevance of CCDs is in contrast to the effects caused
by another glycan, the non-reducing terminal disaccharide
galactose-α-1,3-galactose (α-Gal). Anti-α-Gal IgE causes delayed
allergic reactions to red meat and immediate anaphylactic reactions to
the humanized mouse anti-cancer antibody cetuximab.5This reaction is based on the presence of α-Gal on glycoproteins and
glycolipids of all mammals, except humans, Old World monkeys and apes.
α-Gal belongs to group B of the oligosaccharides included in the
official allergen nomenclature website.3 α-Gal
containing glycolipids seem to cause the delay of allergic symptoms
after consumption of mammalian meat, since digestion and absorption of
lipids is much slower than that of proteins.6
In their study in this issue, Plum and colleagues aimed to investigate
the interaction of antibodies and CCDs by structural and functional
analyses. As CCD-specific IgE producing cells are rare and the
establishment of IgE-derived immune repertoires is highly demanding, the
authors immunized rabbits with horseradish peroxidase (HRP), a model
protein carrying CCDs. They amplified IgG variable regions from the
heavy and light chain repertoire from the rabbit’s spleen and bone
marrow cDNAs and generated a scFv phage display library. Biopanning with
CCD-carrying glycoproteins allowed isolating CCD-specific scFv antibody
fragments. The use of HRP, a CCD with 3 terminal mannose residues, or
HSA carrying MUXF, a CCD with 2 terminal mannose residues, for
biopanning resulted in isolation of two different scFv clones (H1
selected with HRP, M5 selected with HSA-MUXF). Both clones were
converted into chimeric human/rabbit IgEs and IgGs by fusion of the scFv
fragments with human IgE and IgG Fc domains. Previous studies showed
that rabbits produce IgG antibodies that bind plant N-glycans with high
affinity.7 However, examples from allergenic proteins,
for instance the major grass pollen allergen Phl p 1, indicated that
mouse and human IgEs may recognize different
epitopes.8 Therefore, caution needs to be taken when
animal-derived antibodies are used as models for interactions between
human IgEs and allergens.
The characterization of the chimeric antibodies by ELISA revealed
specific binding of the IgE and IgG antibody constructs to the different
CCDs and interactions with soluble Fcε and Fcγ receptors. The
specificity and sensitivity of the recombinant (r) IgEs were analyzed by
ImmunoCAP using CCD-harboring molecules (HRP, HSA-MUXF, and the plant
allergen nPhl p 4), extracts from insect venoms (bee, yellow jacket, andPolistes ), and pollen (rape), as well as extracts from helminths
(Ascaris , Echinococcus spp. ) and a nematode
(Anisakis ). The recombinant wasp allergens rVes v 1 and rVes v 5
were included as allergens lacking CCDs. Comparable to human IgEs
directed against CCDs, the rIgE antibodies only recognized the
CCD-carrying glycoproteins HRP, HSA-MUXF and nPhl p 4 and the
CCD-containing extracts from bee
and yellow jacket venom and rape pollen. Polistes venom, which is
devoid of CCDs, was not recognized by the rIgEs.
Surface plasmon resonance analysis revealed high binding affinities
between CCDs and rIgEs with KD values between
8x10-7 and 8x10-9, almost in the
range of human polyclonal anti-CCD IgE responses with KDvalues of 1x10-10.4 The authors were
aware that due to the presence of several CCDs on one protein, such
affinity measurements had their limitations. For more accurate
measurements it would be important to use glycoproteins with precisely
defined carbohydrate moieties.
Basophil activation tests are regarded as reliable diagnostic tools for
predicting the clinical relevance of allergens. However, in case of
CCDs, the use of these tests as evidence for clinical relevance is
questioned, since, for instance, CCD-glycosylated human lactoferrin
expressed in rice activated basophils in vitro but did not induce
symptoms in oral challenges.9 Plum and colleagues
loaded rat basophilic leukemia SX38 cells expressing the human FcεRI
receptor with the rIgEs and showed that HRP and the MUXF conjugate could
trigger a CCD-dependent ß-hexosaminidase release. Furthermore, binding
of rIgE/antigen complexes to CD23 were blocked by CCD-specific IgG from
insect venom-allergic individuals. This clearly indicates that human
anti-CCD IgEs may in vivo play a role in triggering allergic
responses.
This study also obtained a crystal structure of a complex between an M5
IgG Fab and the epitope surrogate disaccharide FucαGlcNAcα1-OMe. The
disaccharide is recognized entirely by the VH CDRs, specifically, all
three hydroxyl groups on the fucose are recognized through hydrogen
bonds with CDR1 or CDR2. The authors conclude that the specificity and
affinity of the antibody binding is mediated by the α1,3-fucose and that
the fucose residue and the adjacent N-acetlyglucosamine residues
represent the crucial part of the glycotope. The future use of such
structural biology approaches for analysis of the interactions between
antibodies and α-Gal, could contribute to a better understanding in the
similarities and differences in the immune response to the two
glycotopes.
In summary, the data presented in this study (Figure 1) contribute
another important piece of knowledge to the role of anti-CCD IgEs in the
Th2 response to glycosylated allergens. The data also point towards
glycotopes as players in the allergic immune response.