20.500.12556/DiRROS-12589 Recombinant glycoproteins resembling carbohydrate-specific IgE epitopes from plants, venoms and mites 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. allergy and immunology allergens glycoproteins molecular diagnostic technique recombinant glycoproteins molecular allergology component-resolved diagnosis alergija in imunologija alergeni glikoproteini molekularne diagnostične tehnike rekombinantni glikoproteini molekularna alergologija molekularna diagnostika true false true Elsevier Angleški jezik Ni določen © 2018 The Authors Neznano 2020-10-16 12:39:22 2020-10-16 12:39:23 2022-08-18 03:38:12 0000-00-00 00:00:00 2019 0 Nizozemska 0 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; str. 33-43 Vol. 39 Jan. 2019 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 616-097 2352-3964 10.1016/j.ebiom.2018.12.002 2048486001 523063577 RAZ_Gattinger_Pia_i2019.pdf RAZ_Gattinger_Pia_i2019.pdf 1 877B01939227A3C0DCDACEDEC5C4A6F9 ca3ccb52eb0f7ba83421d38a5daffc20bdb8ea14c57e371518051ebb144736a0 88abb8fb-17b6-11ed-b6b8-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=17217 RAZ_Gattinger_Pia_i2019.zip RAZ_Gattinger_Pia_i2019.zip 1 2B96CA34BD1413ED682EF0F9160613E8 806f635bc4ddaa632c0509bd5bc6eb088f513d336d69bb5ce0ea631c1b09e8b2 88becd9e-17b6-11ed-b6b8-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=17218 https://www.ebiomedicine.com/article/S2352-3964(18)30573-5/fulltext 1 https://dirros.openscience.si/Dokument.php?lang=slv&id=15286 Univerzitetna klinika za pljučne bolezni in alergijo Golnik 0 0 0