Format

Send to

Choose Destination
EBioMedicine. 2018 Dec 20. pii: S2352-3964(18)30573-5. doi: 10.1016/j.ebiom.2018.12.002. [Epub ahead of print]

Recombinant glycoproteins resembling carbohydrate-specific IgE epitopes from plants, venoms and mites.

Author information

1
Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.
2
Institute of Molecular Biosciences, BioTechMed Graz, University of Graz, Graz, Austria.
3
University Clinic of Respiratory and Allergic Diseases, Golnik, Slovenia.
4
Department of Dermatology and Allergy, University of Bonn, Bonn, Germany.
5
Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria; NRC Institute of Immunology FMBA of Russia, Moscow, Russia; Laboratory for Immunopathology, Department of Clinical Immunology and Allergy, Sechenov First Moscow State Medical University, Moscow, Russia. Electronic address: rudolf.valenta@meduniwien.ac.at.

Abstract

BACKGROUND:

N-linked glycans present in venoms, pollen and mites are recognized by IgE antibodies from >20% of allergic patients but have low or no allergenic activity.

OBJECTIVES:

To engineer recombinant glycoproteins resembling carbohydrate-specific IgE epitopes from venoms, pollen and mites which can discriminate carbohydrate-specific IgE from allergenic, peptide-specific IgE.

METHODS:

One or two N-glycosylation sites were engineered into the N-terminus of the non-allergenic protein horse heart myoglobin (HHM) using synthetic gene technology. HHM 1 and HHM 2 containing one or two N-glycosylation sites were expressed in baculovirus-infected High-Five™ insect cells and a non-glycosylated version (HHM 0) was obtained by mutating the glycosylation motif. Recombinant HHM proteins were analyzed regarding fold and aggregation by circular dichroism and gel filtration, respectively. IgE reactivity was assessed by ELISA, immunoblotting and quantitative ImmunoCAP measurements. IgE inhibition assays were performed to study cross-reactivity with venom, plant and mite-derived carbohydrate IgE epitopes.

RESULTS:

HHM-glycovariants were expressed and purified from insect cells as monomeric and folded proteins. The HHM-glycovariants exhibited strictly carbohydrate-specific IgE reactivity, designed to quantify carbohydrate-specific IgE and resembled IgE epitopes of pollen, venom and mite-derived carbohydrates. IgE-reactivity and inhibition experiments established a hierarchy of plant glcyoallergens (nPhl p 4 > nCyn d 1 > nPla a 2 > nJug r 2 > nCup a 1 > nCry j 1) indicating a hitherto unknown heterogeneity of carbohydrate IgE epitopes in plants which were completely represented by HHM 2.

CONCLUSION:

Defined recombinant HHM-glycoproteins resembling carbohydrate-specific IgE epitopes from plants, venoms and mites were engineered which made it possible to discriminate carbohydrate- from peptide-specific IgE reactivity.

KEYWORDS:

Allergen; Allergy; Component-resolved diagnosis; Cross-reactive carbohydrate determinant; Molecular allergology; Recombinant glycoprotein

PMID:
30581149
DOI:
10.1016/j.ebiom.2018.12.002
Free full text

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center