Dissecting cross-reactivity in hymenoptera venom allergy by circumvention of α-1,3-core fucosylation
Introduction
Hymenoptera stings may cause life-threatening and sometimes fatal IgE-mediated anaphylactic reactions in humans. Although venom immunotherapy is highly effective, systemic allergic side effects to injections have been observed in 20–40% of patients. According to sting challenge tests, 10–20% of patients were not protected by honeybee venom immunotherapy and continued to develop generalised allergic symptoms (Muller et al., 1992, Rueff et al., 1996). Thus, there is considerable interest in improving diagnosis as well as design, safety and efficacy of therapy.
Although so far only a limited number of hymenoptera venom allergens are available as recombinant proteins (King and Spangfort, 2000, Muller, 2003) their use may improve existing strategies (Muller, 2002) by offering the potential for analyses on a molecular level beyond component resolution. The most prominent honeybee venom allergens include phospholipase A2 (Api m 1), hyaluronidase (Api m 2), acid phosphatase (Api m 3) (King et al., 1976) and the basic 26 amino acid peptide Melittin (Api m 4) (Arbesman et al., 1976). Major yellow jacket allergens include phospholipase A1 (Ves v 1), hyaluronidase (Ves v 2), and antigen 5 (Ves v 5) (King and Spangfort, 2000, Muller, 2002). Api m 1 and Api m 2 as well as Ves v 1, Ves v 2, and Ves v 5 could be expressed in bacteria, yeast or baculovirus-infected insect cells (Dudler et al., 1992, Gmachl and Kreil, 1993, Henriksen et al., 2001, Kuchler et al., 1989, Skov et al., 2006, Soldatova et al., 1998) and selected structures were elucidated by X-ray crystallography (Markovic-Housley et al., 2000, Scott et al., 1990). Very recently, the acid phospahatase Api m 3 and the DPPIV enzymes Api m 5 and Ves v 3 were cloned and recombinantly produced (Blank et al., 2008, Grunwald et al., 2006).
Although an increased availability of recombinant allergens will improve the dissection of individual IgE reactivities on a molecular level, allergenic cross-reactivity, a major handicap for accurate diagnosis in hymenoptera venom allergy, remains to be solved. Apart from true double sensitisation and mimicry based on the primary structure, IgE may be directed against cross-reactive carbohydrate determinants (CCDs) provided by a broad panel of proteins in food, pollen and hymenoptera venom (Aalberse et al., 2001). In general the N-glycans found on most hymenoptera venom proteins possess a number of non-mammalian features rendering them potentially immunogenic. However, the supposed hallmark of CCDs on insect venom allergens comprises carbohydrates carrying α-1,3-linked core fucose residues. IgE with specificity for such glycotopes represents the underlying principle reactive with all proteins possessing CCDs (Aalberse et al., 1981). This has even led to the estimation that IgE binding to venom proteins with pronounced glycosylation may primarily or exclusively reflect CCD reactivities (Hemmer et al., 2004), rendering their postulated allergenic character questionable. Thus, the immunoreactivity of all glycosylated allergens demands thorough re-evaluation to verify their classification as allergens in stricto sensu.
Although the clinical relevance of CCDs is still discussed (Malandain, 2005), their diagnostic relevance is beyond any controversy. Identification of the culprit hymenoptera species that a patient is sensitised to remains key for proper diagnosis and for the selection of an appropriate therapeutic strategy. Therefore, in vitro diagnosis might be markedly improved when using strategies that eliminate CCD reactivities without affecting clinically relevant IgE reactivity and allow mere cross-reactivity to be distinguished from true multiple sensitisation.
In this study, we report a molecular approach to assess the IgE reactivity to hyaluronidases Api m 2 and Ves v 2, currently recognised as the most relevant hymenoptera venom allergens displaying cross-reactivity. By exploiting both Trichoplusia ni and Spodoptera frugiperda insect cell lines, recombinant hyaluronidases and a suitable human reference protein, the high abundance serum component α-2HS-glycoprotein, could be obtained with authentic glycosylation while circumventing α-1,3-core fucose addition, which is the hallmark of CCDs. Evaluation of these proteins allowed for a detailed study of CCD-derived allergenic cross-reactivity and emphasised the potential of custom-tailored recombinant allergens for improvement of diagnostic and therapeutic strategies.
Section snippets
Materials
Whole honeybee venom was purchased from Latoxan (Valence, France) and venom of Vespula species (Euromix) was purchased from Vespa Laboratories (Spring Mills, Pennsylvania). Anti-V5 antibody was purchased from Invitrogen (Karlsruhe, Germany). Polyclonal rabbit anti-HRP serum as well as anti-rabbit-IgG-AP conjugate and anti-mouse IgG-AP conjugate was from Sigma (Taufkirchen, Germany). AlaBLOTs as well as MUXF conjugated to bovine serum albumin were obtained from Siemens Healthcare Diagnostics
Cloning of Api m 2, Ves v 2a, Ves v 2b, and α-2HS-glycoprotein
For production of the recombinant venom hyaluronidases, Api m 2 and Ves v 2a, the specific cDNA was amplified from bee and yellow jacket venom-gland cDNA. Additionally, the corresponding cDNA of Ves v 2b, a putative hyaluronidase isoform recently suggested as another component of the hyaluronidase band in V. vulgaris venom, was amplified. As a control protein to provide a non-reactive, non-allergenic protein backbone for different glycan structures we employed human fetuin, termed also
Discussion
The glycosylation of a variety of species including hymenoptera is increasingly being investigated, not only to understand the complexity of glycotypes in nature, but also to determine the clinical implications of immunogenic or allergenic CCDs in hypersensitivities. Hymenoptera venom glycoproteins display some features not found in mammals including a Lewis-type structure (Kubelka et al., 1995, Kubelka et al., 1993) that plays a detrimental role in inflammation and cancer in humans (Becker and
Acknowledgements
The contributions of Beate Heuser are gratefully acknowledged. Honeybees were provided by the bee keeper Hinrich Corleis. This work was supported in part by a grant from the German Ministery for Education and Science in the BioChancePlus-3 program (PTJ-BIO/0313817D to M.O.).
References (76)
- et al.
Immunoglobulin E antibodies that crossreact with vegetable foods, pollen, and Hymenoptera venom
J. Allergy Clin. Immunol.
(1981) - et al.
Steps in the biosynthesis of mosquito cell membrane glycoproteins and the effects of tunicamycin
Biochim. Biophys. Acta
(1981) - et al.
High-level expression in Escherichia coli and rapid purification of enzymatically active honey bee venom phospholipase A2
Biochim. Biophys. Acta
(1992) - et al.
Structures of the Erythrina corallodendron lectin and of its complexes with mono- and disaccharides
J. Mol. Biol.
(1998) - et al.
Identification of core alpha 1,3-fucosylated glycans and cloning of the requisite fucosyltransferase cDNA from Drosophila melanogaster. Potential basis of the neural anti-horseadish peroxidase epitope
J. Biol. Chem.
(2001) - et al.
Affinity purification of antibodies specific for Asn-linked glycans containing alpha 1 → 3 fucose or beta 1 → 2 xylose
Anal. Biochem.
(1993) - et al.
Characterization of the B-chain of human plasma alpha 2HS-glycoprotein. The complete amino acid sequence and primary structure of its heteroglycan
J. Biol. Chem.
(1983) - et al.
Molecular cloning and expression in insect cells of honeybee venom allergen acid phosphatase (Api m 3)
J. Allergy Clin. Immunol.
(2006) - et al.
False positive reactivity of recombinant, diagnostic, glycoproteins produced in High Five insect cells: effect of glycosylation
J. Immunol. Methods
(2008) - et al.
Characterization and cloning of GP50, a Taenia solium antigen diagnostic for cysticercosis
Mol. Biochem. Parasitol.
(2004)
Antibody binding to venom carbohydrates is a frequent cause for double positivity to honeybee and yellow jacket venom in patients with stinging-insect allergy
J. Allergy Clin. Immunol.
Sol i 1, the phospholipase allergen of imported fire ant venom
J. Allergy Clin. Immunol.
Engineering N-glycosylation pathways in the baculovirus-insect cell system
Curr. Opin. Biotechnol.
Yellow jacket venom allergens, hyaluronidase and phospholipase: sequence similarity and antigenic cross-reactivity with their hornet and wasp homologs and possible implications for clinical allergy
J. Allergy Clin. Immunol.
Allergens of honey bee venom
Arch. Biochem. Biophys.
The structure of a neural specific carbohydrate epitope of horseradish peroxidase recognized by anti-horseradish peroxidase antiserum
J. Biol. Chem.
O-glycosylation potential of lepidopteran insect cell lines
Biochim. Biophys. Acta
Crystal structure of hyaluronidase, a major allergen of bee venom
Structure
Quantitation of hyaluronidases by the Morgan–Elson reaction: comparison of the enzyme activities in the plasma of tumor patients and healthy volunteers
Cancer Lett.
Immunotherapy with honeybee venom and yellow jacket venom is different regarding efficacy and safety
J. Allergy Clin. Immunol.
A modified colorimetric method for the estimation of N-acetylamino sugars
J. Biol. Chem.
High-yield recombinant expression of the extremophile enzyme, bee hyaluronidase in Pichia pastoris
Protein Exp. Purif.
Structural and immunological characterization of the N-glycans from the major yellow jacket allergen Ves v 2: the N-glycan structures are needed for the human antibody recognition
Mol. Immunol.
Superior biologic activity of the recombinant bee venom allergen hyaluronidase expressed in baculovirus-infected insect cells as compared with Escherichia coli
J. Allergy Clin. Immunol.
The complete amino acid sequence of the A-chain of human plasma alpha 2HS-glycoprotein
J. Biol. Chem.
Cross-reactivity of IgE antibodies to allergens
Allergy
Insect cells as hosts for the expression of recombinant glycoproteins
Glycoconjugate J.
High resolution two-dimensional electrophoresis of human plasma proteins
Proc. Natl. Acad. Sci. U.S.A.
Allergenic potency of bee antigens measured by RAST inhibition
Clin. Allergy
Current Protocols in Molecular Biology
Fucose: biosynthesis and biological function in mammals
Glycobiology
Specificity of IgG and IgE antibodies against plant and insect glycoprotein glycans determined with artificial glycoforms of human transferrin
Glycobiology
Cetuximab-induced anaphylaxis and IgE specific for galactose-alpha-1,3-galactose
N. Engl. J. Med.
Baculovirus expression of alkaline phosphatase as a reporter gene for evaluation of production, glycosylation and secretion
Biotechnology (NY)
Use of mannosamine for inducing the addition of outer arm N-acetylglucosamine onto N-linked oligosaccharides of recombinant proteins in insect cells
Biotechnol. Prog.
The frequency and clinical significance of specific IgE to both wasp (Vespula) and honey-bee (Apis) venoms in the same patient
Clin. Exp. Allergy
Involvement of carbohydrate epitopes in the IgE response of celery-allergic patients
Int. Arch. Allergy Immunol.
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These authors contributed equally to this work.