Pia Gattinger (Author), Irene Mittermann (Author), Christian Lupinek (Author), Gerhard Hofer (Author), Walter Keller (Author), Urška Bidovec (Author), Peter Korošec (Author), Christine Koessler (Author), Natalija Novak (Author), Rudolf Valenta (Author)

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-FiveTM 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

recombinant glycoproteins;molecular allergology;component-resolved diagnosis;

Data

Language: English
Year of publishing:
Typology: 1.01 - Original Scientific Article
Publisher: Elsevier
UDC: 616-097
COBISS: 2048486001 Link will open in a new window
ISSN: 2352-3964
Views: 923
Downloads: 634
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Other data

Secondary keywords: rekombinantni glikoproteini;molekularna alergologija;molekularna diagnostika;Allergy and immunology;Allergens;Glycoproteins;Molecular diagnostic techniques;Alergija in imunologija;Alergeni;Glikoproteini;Molekularne diagnostične tehnike;
Source comment: Soavtorji: Irene Mittermann, Christian Lupinek, Gerhard Hofer, Walter Keller, Urska Bidovec Stojkovic, Peter Korosec, Christine Koessler, Natalija Novak, Rudolf Valenta; Nasl. z nasl. zaslona; Opis vira z dne 25. 4. 2019;
Pages: str. 33-43
Issue: ǂVol. ǂ39
Chronology: Jan. 2019
DOI: 10.1016/j.ebiom.2018.12.002
ID: 12080814