Avtor/Urednik		Gattinger, Pia; Mittermann, Irene; Lupinek, Christian; Hofer, Gerhard; Keller, Walter; Bidovec, Urška; Korošec, Peter; Koessler, Christine; Novak, Natalija; Valenta, Rudolf
Naslov		Recombinant glycoproteins resembling carbohydrate-specific IgE epitopes from plants, venoms and mites
Tip		članek
Vol. in št.		, št. Vol. 39
Leto izdaje		2019
Obseg		str. 33-43
ISSN		2352-3964
Jezik		eng
Abstrakt		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.
Deskriptorji		Allergy and immunology Allergens Glycoproteins Molecular diagnostic techniques Alergija in imunologija Alergeni Glikoproteini Molekularne diagnostične tehnike
Proste vsebinske oznake		recombinant glycoproteins molecular allergology component-resolved diagnosis rekombinantni glikoproteini molekularna alergologija molekularna diagnostika