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Red meat allergic patients have a selective IgE response to
the a-Gal glycan
D. Apostolovic
1,2
, T. A. T. Tran
1
,S.S
anchez-Vidaurre
1
, T. Cirkovic Velickovic
2
, M. Starkhammar
3
,
C. Hamsten
1,4,
* & M. van Hage
1,
*
1
Clinical Immunology and Allergy Unit, Department of Medicine Solna, Karolinska Institutet and University Hospital, Stockholm, Sweden;
2
Center of Excellence for Molecular Food Sciences, Faculty of Chemistry, University of Belgrade, Belgrade, Serbia;
3
Department of Internal
Medicine, S
odersjukhuset, Stockholm;
4
Center for Inflammatory Diseases, Karolinska Institutet, Stockholm, Sweden
To cite this article: Apostolovic D, Tran TAT, S
anchez-Vidaurre S, Cirkovic Velickovic T, Starkhammar M, Hamsten C, van Hage M. Red meat-allergic patients
have a selective IgE response to the a-Gal glycan. Allergy 2015; 70: 14971500.
Keywords
a-Gal; cross-reactive carbohydrate determi-
nant; glycan; IgE; red meat allergy.
Correspondence
Marianne van Hage, MD, PhD, Clinical
Immunology and Allergy Unit, Department
of Medicine, Solna, Karolinska Institutet,
Karolinska University Hospital Solna L2:04,
SE 171 76 Stockholm, Sweden.
Tel.: +46-8-5177-5942
Fax: +46-8-33-57-24
E-mail: marianne.van.hage@ki.se
*Shared last authorship.
Accepted for publication 12 July 2015
DOI:10.1111/all.12695
Edited by: Reto Crameri
Abstract
Galactose-a-1,3-galactose (a-Gal) is a mammalian carbohydrate with significance
in a novel type of food allergy. Patients with IgE against a-Gal report severe
allergic symptoms 36 h after consumption of red meat. We investigated whether
IgE from red meat allergic patients recognizes other mammalian glycans than a-
Gal or glycans from the plant kingdom and insects of importance in allergy. We
found that none of the 24 red meat allergic patients investigated had an IgE anti-
body response against the other abundant mammalian glycan N-glycolylneu-
raminic acid or against cross-reactive carbohydrate determinants from plant or
venom sources (nCup a 1, nArt v 1, and MUXF3). Deglycosylation of an a-Gal-
containing protein, bovine thyroglobulin, significantly reduced the IgE response.
In conclusion, we show that red meat allergic patients have a selective IgE
response to the a-Gal glycan found in red meat. Other common glycans reactive
in allergic disease are not targets of red meat allergic patients’ IgE.
Many allergens are glycoproteins that carry one or several
carbohydrates linked to the protein structure. Not only pro-
teins but also carbohydrates can stimulate the production of
IgE antibodies and be strong inducers of Th2 responses (1).
The most common carbohydrate epitopes recognized by
human IgE are glycans with a1-3 fucose and xylose cores
that are present in plants, insects, ruminant nematode, and
trematodes (2). About 20% or more of allergic patients
generate a specific antiglycan IgE response (3). Galactose-
a-1,3-galactose (a-Gal) is a mammalian carbohydrate epitope
with recently discovered significance in a novel type of severe
food allergy (4). Patients with IgE to a-Gal report symptoms
of anaphylaxis, angioedema, or urticaria 36 h after con-
sumption of red meat (4, 5). Immunoproteomics of processed
beef has revealed that the a-Gal epitope is commonly present
in IgE-reactive beef proteins, four of which are stable to heat
treatment (6). The red meat allergy syndrome has been recog-
nized in many countries in Europe as well as in the United
States, Japan, and Australia (4, 713), highlighting a-Gal as
a clinically relevant food allergen. Furthermore, a strong
association between tick bites, anti-a-Gal IgE responses, and
red meat allergy has been reported (7, 14).
The only mammalian glycan besides a-Gal present in high
amounts in beef, lamb, pork, and cow’s milk is the sialic acid
N-glycolylneuraminic acid (Neu5Gc) (15). Neu5Gc is present
in most mammals including primates (16) but not in humans
due to an irreversible mutation in the gene encoding
the enzyme responsible for Neu5Gc synthesis (17). As a con-
sequence of dietary sources, Neu5Gc-induced immune
Abbreviations
a-Gal, Galactose-a-1,3-galactose; CCD, Cross-reactive carbohydrate
determinant; Neu5Gc, N-glycolylneuraminic acid.
Allergy 70 (2015) 1497–1500 ©2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 1497
Allergy
responses are observed in humans (18). Anti-Neu5Gc anti-
bodies have been detected in serum in as many as 85% of
the human population, in which IgG against Neu5Gc reaches
up to 0.25% of total circulating IgG in some subjects. The
findings are similar to what is known about IgG antibodies
to a-Gal, but no correlations between IgG levels to Neu5Gc
and a-Gal have been reported (15, 19).
In this study, we investigated whether IgE from red meat
allergic patients recognizes other mammalian glycans or gly-
cans from the plant kingdom and insects of importance in
allergic disease. Moreover, we scrutinized whether IgE
responses to a-Gal target the whole glycoprotein or the gly-
can structure only. Twenty-four patients with a-Gal-induced
red meat allergy and a median IgE level to a-Gal of
14.9 kU
A
/l (range 1.3180 kU
A
/l; Table 1) (ImmunoCAP
o215, bovine thyroglobulin; Thermo Fisher Scientific/Phadia
AB, Uppsala, Sweden) and three healthy controls IgE
negative to a-Gal (<0.10 kU
A
/l) were enrolled. Bovine thy-
roglobulin is a glycoprotein heavily decorated with a-Gal,
which is commonly used in the diagnosis of red meat
allergy (7, 20).
IgE reactivity against the glycans a-Gal (a-Gal-sp-biotin)
and Neu5Gca(Neu5Gca-sp-biotin) (Glycotech, Gaithers-
burg, MD, USA) was tested by streptavidin ImmunoCAP
(Thermo Fisher/Phadia AB, for details see Supporting Infor-
mation). IgE reactivity to the cross-reactive carbohydrate
determinant (CCD)-containing allergens MUXF3 (a1,3-
fucose and a1,2-xylose core), nCup a 1 (horseradish peroxi-
dase-based carbohydrates), and nArt v 1 (arabinogalactan-rich
protein) was determined by ImmunoCAP (Thermo Fisher/
Phadia AB) according to the manufacturer’s instructions with
a cutoff at 0.10 kU
A
/l.
We found that none of the red meat allergic patients had
an IgE antibody response against Neu5Gcaor against
MUXF3 or the CCD-containing proteins (nCup a 1 and
nArt v 1; Table 1). One patient had a very low IgE response
to nArt v 1, but pre-inhibition with bovine thyroglobulin did
not affect the nArt v 1 response. The result confirms that
the immune response against nArt v 1 was independent of
the a-Gal response. Thus, the only glycan target of signifi-
cance in red meat allergy is a-Gal. The IgE levels to the pure
a-Gal glycan were lower in red meat-allergic patients (median
8.9 kU
A
/l; range 0.7>100 kU
A
/l) compared to IgE levels
using ImmunoCAP o215 (bovine thyroglobulin; Table 1).
However, this was not unexpected as the relative a-Gal con-
tent on these ImmunoCAPs is uncertain. The healthy
Table 1 Analysis of selected glycans and glycoproteins on Swedish red meat allergic patients
Patient ID
Age
(year)/sex
IgE*
Total TG dTG a1,3Gal Neu5Gc MUXF3 nCup a 1 nArt v 1
1 53/M 260 10 <0.10 8.1 <0.10 <0.10 <0.10 <0.10
2 48/F 44 5.0 <0.10 0.7 <0.10 <0.10 <0.10 <0.10
3 34/M 380 180 0.4 64.4 <0.10 <0.10 <0.10 <0.10
4 67/F 123 38 <0.10 26 <0.10 <0.10 <0.10 <0.10
5 66/M 705 100 1.1 >100 <0.10 <0.10 <0.10 <0.10
6 49/F 111 11.7 0.22 8.4 <0.10 <0.10 <0.10 <0.10
7 38/F 112 32.3 0.10 16.2 <0.10 <0.10 <0.10 0.21
8 40/F 171 14.9 <0.10 9.0 <0.10 <0.10 <0.10 <0.10
9 46/F 140 13.2 <0.10 9.1 <0.10 <0.10 <0.10 <0.10
10 59/F 79 17.4 <0.10 11.4 <0.10 <0.10 <0.10 <0.10
11 23/M 127 11.2 <0.10 6.6 <0.10 <0.10 <0.10 <0.10
12 43/F 166 9.0 <0.10 5.2 <0.10 <0.10 <0.10 <0.10
13 68/F 138 27.4 0.13 11.7 <0.10 <0.10 <0.10 <0.10
14 75/M 510 140 2.8 >100 <0.10 <0.10 <0.10 <0.10
15 53/F 166 6.3 <0.10 3.9 <0.10 <0.10 <0.10 <0.10
16 78/M 210 30 <0.10 9.6 <0.10 <0.10 <0.10 <0.10
17 78/F 30 1.6 <0.10 1.1 <0.10 <0.10 <0.10 <0.10
18 20/F 189 1.3 <0.10 0.9 <0.10 <0.10 <0.10 <0.10
19 20/F 520 24 0.18 8.1 <0.10 <0.10 <0.10 <0.10
20 37/M 42 6.4 <0.10 2.2 <0.10 <0.10 <0.10 <0.10
21 67/F 110 29 <0.10 12.5 <0.10 <0.10 <0.10 <0.10
22 26/M 817 2.8 <0.10 1.4 <0.10 <0.10 <0.10 <0.10
23 47/F 67 19 <0.10 12 <0.10 <0.10 <0.10 <0.10
24 49/F 36 21 0.17 8.8 <0.10 <0.10 <0.10 <0.10
C1 41/F 12 <0.10 <0.10 <0.10 <0.10 <0.10 <0.10 <0.10
C2 44/F 2.0 <0.10 <0.10 <0.10 <0.10 <0.10 <0.10 <0.10
C3 55/M 120 <0.10 <0.10 <0.10 <0.10 <0.10 <0.10 1.1
C, control; M, male; F, female; TG, bovine thyroglobulin (ImmunoCAP o215); dTG, deglycosylated thyroglobulin; a1,3Gal, Galactose-a-1,3-
galactose; Neu5Gc, N-glycolylneuraminic acid.
*ImmunoCAP IgE results: allergen-specific IgE levels in kU
A
/l.
Allergy 70 (2015) 1497–1500 ©2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd1498
Selective IgE response to the a-Gal glycan Apostolovic et al.
controls did not have IgE reactivity against the tested com-
ponents except one patient who was sensitized to Artemisia
vulgaris (nArt v 1 IgE level 1.1 kU
A
/l, C3 in Table 1).
To enable a thorough investigation of the IgE responses to
the pure a-Gal glycan vs aa-Gal-containing glycoprotein,
bovine thyroglobulin was deglycosylated with PNGase F
(New England Biolabs, Ipswich, MA, USA). Native and
deglycosylated bovine thyroglobulin were analyzed by CD
spectroscopy and similar secondary structures were observed,
indicating that the protein structure was unaffected by the
enzymatical procedure (Fig. S1). The removal of glycans was
visualized by SDS-PAGE where the deglycosylation of thy-
roglobulin resulted in a shift of the major protein band to
lower molecular weight (below 250 kDa) compared to
untreated thyroglobulin (band above 250 kDa) (Fig. 1A).
This was further supported by immunoblot where a signifi-
cant reduction in IgE binding to deglycosylated compared to
untreated thyroglobulin was noted (Fig. 1B). Comparable
results were obtained when using a monoclonal anti-a-Gal
antibody (Enzo Life Science, Inc., Farmingdale, NY, USA)
(Fig. 1C). Next, IgE levels to deglycosylated thyroglobulin
were measured using streptavidin ImmunoCAP (for details
see Supporting Information). In two-thirds of the patients,
deglycosylation of thyroglobulin reduced the IgE levels as
much as one hundred-fold in 50% of the cases to below the
cutoff (<0.10 kU
A
/l). The eight patients that still had a low
IgE reactivity to deglycosylated thyroglobulin (median
0.20 kU
A
/l; range 0.12.8 kU
A
/l) had high IgE levels to
untreated thyroglobulin (ImmunoCAP o215; Table 1).
Hence, this probably reflects an incomplete glycan removal
by PNGase F, which could be due to steric hindrance on the
glycoprotein. This was investigated by pre-incubation with
500 lg/ml of a-Gal (a-Gal-sp-biotin) prior to the measure-
ment of deglycosylated thyroglobulin-specific IgE (described
in Supporting Information) which resulted in a reduction to
below cutoff (<0.10 kU
A
/l) in all patients (data not shown),
indicating that their IgE responses were indeed specific for
the a-Gal glycan with no binding to the protein structure.
In conclusion, we show that red meat allergic patients have
a selective IgE response to the pure a-Gal glycan that is
unrelated to the carrier protein. Common CCDs from plants
or venoms are not targets of the IgE response in these
patients. Even though the glycan Neu5Gcais present in
mammalian meat and milk alongside a-Gal, it is not recog-
nized by IgE from red meat-allergic patients. The specificity
of the a-Gal response could be due to the route of sensitiza-
tion through the skin via tick bites. We have recently shown
that a-Gal is present within ticks, thus potentially explaining
the strong association between anti-a-Gal IgE and tick bites,
with the development of red meat allergy as a secondary
phenomenon (7, 11, 14). We consider these data are of
importance as they reveal that cross-reactions to other
glycans, common in other allergic diseases, are not an issue
in the pathogenesis and diagnosis of red meat allergy.
Acknowledgments
The authors thank Neda Bigdeli for excellent technical assis-
tance.
Author contributions
The study was conceived by DA, CH, and MvH. DA
participated in all stages of the project and performed the
experiments. DA, TAT, CH, and MvH wrote the manuscript
together with SSV and TCV. MS provided the patient mate-
rial. All authors contributed to the interpretation of the data
and provided critical review of the manuscript.
Figure 1 (A) Protein profile of thyroglobulin and deglycosylated thy-
roglobulin, (B) representative IgE-binding profiles of thyroglobulin
and deglycosylated thyroglobulin in one red meat allergic patient (+)
and one healthy control (), and (C) a-Gal binding of thyroglobulin
and deglycosylated thyroglobulin using monoclonal anti-a-Gal anti-
body.All experiments were performed using bovine thyroglobulin.
Allergy 70 (2015) 1497–1500 ©2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 1499
Apostolovic et al. Selective IgE response to the a-Gal glycan
Funding
This article was supported by the Swedish Research Council,
Stockholm County Council; the Swedish Asthma and Allergy
Association’s Research Foundation; Ministry of Education,
Science and Technological Development of the Republic of
Serbia (GA No. 172024); FP7 RegPot project FCUB ERA
(GA No. 256716); the Swedish Heart-Lung Foundation; the
Center for Inflammatory Diseases, Karolinska Institutet; the
Swedish Cancer and Allergy Foundation; the Konsul Th C
Berg Foundation; the King Gustaf V 80th Birthday Founda-
tion; the Magnus Bergvall Foundation; Karolinska Institutet;
and EAACI Fellowship Award 2013.
Conflicts of interest
The authors declare that they have no conflicts of interest.
Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Figure S1. Far UV CD spectra of thyroglobulin (solid line)
and deglycosylated thyroglobulin (dash line).
Data S1. Material and methods.
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Patients with IgE antibodies against the carbohydrate epitope galactose-α-1,3-galactose (α-Gal) have reported severe allergic reactions after consumption of red meat. Investigations have revealed associations between IgE to α-Gal and tick bites. We provide the first direct evidence that α-Gal is present within ticks thus potentially explaining the relationship between tick exposure and sensitization to α-Gal, with development of red meat allergy as a secondary phenomena. Serum from Swedish patients with delayed severe reactions to red meat was included in the study. A dose-dependent inhibition of IgE responses to α-Gal by the tick Ixodes ricinus is demonstrated. Furthermore, using cryostat-cut sections of I. ricinus, we show that both a monoclonal and a polyclonal antibody against α-Gal stains the gastrointestinal tract of the tick. The same pattern is seen when staining with patient sera IgE positive to α-Gal. These results confirm that the α-Gal epitope is present in I. ricinus and imply host exposure to α-Gal during a tick bite. This provides further evidence that tick bites are associated with IgE responses to α-Gal and red meat allergy.
Article
Carbohydrate-specific IgE antibodies present on nonprimate mammalian proteins were incriminated recently in delayed meat anaphylaxis. The aim of this study was to explore whether anaphylaxis to mammalian kidney is also associated with galactose-α-1,3-galactose (αGal)-specific IgE. Fourteen patients with anaphylaxis to pork or beef kidney underwent prick tests to meat and kidney. Some patients also underwent skin tests to Erbitux(®) (cetuximab). IgE antibodies to αGal, swine urine proteins, beef and pork meat, serum albumin proteins, cat, and rFel d 1 were measured by ImmunoCAP(®). The αGal levels were estimated in meats and kidney by ELISA inhibition assay. Cross-reactivity between αGal and pork kidney was studied with the ImmunoCAP(®) inhibition assay. Among the 14 patients, 12 presented with anaphylactic shock. Reactions occurred within 2 h from exposure in 67% of patients. Associated risk factors were observed in 10 cases, and alcohol was the main cofactor. Three patients underwent an oral challenge to pork kidney, and anaphylaxis occurred after ingestion of small quantities (1-2 g). Prick tests to kidney were positive in 54% of patients. All tested patients showed positive skin tests to Erbitux(®). All patients tested positive for IgE to αGal, with levels ranging from 0.4 to 294 kU/l. IgE binding to αGal was inhibited by raw pork kidney extract (mean, 77%; range, 55-87%), which showed a high amount of αGal determinants. Pork or beef kidney anaphylaxis is related to αGal IgE. Its peculiar severity could be due to an elevated content of αGal epitopes in kidney.