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Red meat allergic patients have a selective IgE response to
the a-Gal glycan
D. Apostolovic
, T. A. T. Tran
, T. Cirkovic Velickovic
, M. Starkhammar
C. Hamsten
* & M. van Hage
Clinical Immunology and Allergy Unit, Department of Medicine Solna, Karolinska Institutet and University Hospital, Stockholm, Sweden;
Center of Excellence for Molecular Food Sciences, Faculty of Chemistry, University of Belgrade, Belgrade, Serbia;
Department of Internal
Medicine, S
odersjukhuset, Stockholm;
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.
a-Gal; cross-reactive carbohydrate determi-
nant; glycan; IgE; red meat allergy.
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
*Shared last authorship.
Accepted for publication 12 July 2015
Edited by: Reto Crameri
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
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
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
/l (range 1.3180 kU
/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
/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
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
/l; range 0.7>100 kU
/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
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
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
/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
/l). The eight patients that still had a low
IgE reactivity to deglycosylated thyroglobulin (median
0.20 kU
/l; range 0.12.8 kU
/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
/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.
The authors thank Neda Bigdeli for excellent technical assis-
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
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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Anaphylaxis is a severe allergic reaction that can be rapidly progressing and fatal, and therefore establishing its cause is pivotal to long-term risk management. Our recent work has identified a novel IgE antibody response to a mammalian oligosaccharide epitope, galactose-alpha-1,3-galactose (alpha-gal). IgE to alpha-gal has been associated with 2 distinct forms of anaphylaxis: (1) immediate-onset anaphylaxis during first exposure to intravenous cetuximab and (2) delayed-onset anaphylaxis 3 to 6 hours after ingestion of mammalian food products (eg, beef and pork). Results of our studies and those of others strongly suggest that tick bites are a cause, if not the only significant cause, of IgE antibody responses to alpha-gal in the southern, eastern, and central United States; Europe; Australia; and parts of Asia. Typical immune responses to carbohydrates are considered to be T-cell independent, whereas IgE antibody production is thought to involve sequential class-switching that requires input from T cells. Therefore, establishing the mechanism of the specific IgE antibody response to alpha-gal will be an important aspect to address as this area of research continues. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.
Virtually all cells and extracellular material are heavily decorated by various glycans, yet our understanding of the structure and function of these moieties lags behind the understanding of nucleic acids, lipids, and proteins. Recent years have seen a tremendous acceleration of knowledge in the field of glycobiology, revealing many intricacies and functional contributions that were previously poorly appreciated or even unrecognized. This review highlights several topics relevant to glycoimmunology in which mammalian and pathogen-derived glycans displayed on glycoproteins and other scaffolds are recognized by specific glycan-binding proteins (GBPs), leading to a variety of proinflammatory and anti-inflammatory cellular responses. The focus for this review is mainly on 2 families of GBPs, sialic acid-binding immunoglobulin-like lectins (siglecs) and selectins, that are involved in multiple steps of the immune response, including distinguishing pathogens from self, cell trafficking to sites of inflammation, fine-tuning of immune responses leading to activation or tolerance, and regulation of cell survival. Importantly for the clinician, accelerated rates of discovery in the field of glycoimmunology are being translated into innovative medical approaches that harness the interaction of glycans and GBPs to the benefit of the host and might soon lead to novel diagnostics and therapeutics. Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.
Background Red meat allergy presents a novel form of food allergy with severe delayed allergic reactions where IgE antibodies are directed against the carbohydrate -Gal epitope. Food preparation and processing can influence the allergenicity of proteins. The aim of this study was to characterize the proteomic profile of different beef preparations and to investigate their -Gal reactivity and potential allergenicity. Methods Extracts from raw, boiled, fried, and medium rare prepared beef were assessed by 2D PAGE for the comparison of protein profiles. IgE-binding proteins were identified using immunoblot-coupled proteomic analysis using sera from red meat-allergic patients. Presence of the -Gal epitope was verified using anti--Gal antibody and IgE inhibition immunoblot with -Gal. ResultsMultiple IgE-binding proteins were detected in the different beef preparations, many of which were also recognized by the anti--Gal antibody. Protein spots reacting with IgE in patient sera were analyzed by MS/MS, resulting in identification of 18 proteins with high identification scores. Seven of the 18 beef allergens identified using meat-allergic patient sera were also recognized by the anti--Gal monoclonal antibody, and four of them were stabile to thermal treatment. Furthermore, a dose-dependent inhibition of red meat-allergic patients' IgE to beef by -Gal was demonstrated. Conclusions We show that the -Gal epitope is commonly present in IgE-reactive beef proteins recognized by meat-allergic patients. Seven novel -Gal-containing IgE-binding proteins were identified, of which four were stable to heat treatment. Thus, the allergenicity of red meat proteins is preserved even upon different thermal cooking.
Background In 2009, we reported a novel form of delayed anaphylaxis to red meat related to serum IgE antibodies to the oligosaccharide galactose-alpha-1,3-galactose (alpha-gal). Although patients were remarkably consistent in their description of a 3- to 6-hour delay between eating mammalian meat and the appearance of symptoms, this delay has not been demonstrated under observed studies. Objectives We sought to formally document the time course of clinical symptoms after the ingestion of mammalian meat in subjects with IgE to alpha-gal and to monitor ex vivo for the appearance of markers of an allergic reaction. Methods Open food challenges were performed with mammalian meat in 12 subjects with a history of severe urticarial reactions 3 to 6 hours after eating beef, pork, or lamb, as well as in 13 control subjects. Blood samples were taken hourly during each challenge. Results Ten of 12 subjects with IgE to alpha-gal had clinical evidence of a reaction during the food challenge (vs none of the control subjects, P < .001). The reactions occurred 3 to 7 hours after the initial ingestion of mammalian meat and ranged from urticaria to anaphylaxis. Tryptase levels were positive in 3 challenges. Basophil activation, as measured by increased expression of CD63, correlated with the appearance of clinical symptoms. Conclusion The results presented provide clear evidence of an IgE-mediated food allergy that occurs several hours after ingestion of the inciting allergen. Moreover, here we report that in vivo basophil activation during a food challenge occurs in the same time frame as clinical symptoms and likely reflects the appearance of the antigen in the bloodstream.
Sensitization to the carbohydrate galactose-α-1,3-galactose (α-Gal) has been reported in patients with beef allergy. However, the proteins responsible for this allergy have not yet been identified. This study aimed to identify beef proteins that predominantly react with serum IgE in Japanese patients with beef allergy. Sera were collected from 29 patients with beef allergy who had allergic reaction(s) such as urticaria, abdominal pain, vomiting, and anaphylactic shock after ingestion of beef and pork; the sera tested positive for IgE against beef and pork. IgE-binding proteins were detected by immunoblotting sera from the patients and identified using a combination of two-dimensional gel electrophoresis and peptide mass fingerprinting techniques. The involvement of carbohydrate in the binding of IgE to allergens was examined by periodate treatment and an inhibition assay with cetuximab by immunoblotting. Specific IgE binding to cetuximab was measured using the CAP-fluorescent enzyme immunoassay. Two IgE-binding proteins (240 kDa and 140 kDa) were detected in beef extract and identified as laminin γ-1 and the collagen α-1 (VI) chain from Bos taurus, respectively. Periodate treatment or the inhibition assay resulted in the loss of IgE binding to these proteins. Immunoblotting with anti-α-Gal antibody revealed the presence of α-Gal on the 240- and 140-kDa beef proteins. The amount of IgE bound to cetuximab was significantly correlated with that to beef in the patients with beef allergy. The carbohydrate moiety (α-Gal) on laminin γ-1 and collagen α-1 (VI) chain are possibly common IgE-reactive proteins in the Japanese patients with beef allergy.
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.
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.