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
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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
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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