Recombinant glycoproteins resembling carbohydrate-specific IgE epitopes from plants, venoms and mites
Gattinger P., Mittermann I., Lupinek C., Hofer G., Keller W., Bidovec S. U., Korosec P., Koessler C., Novak N., Valenta R.
EBioMedicine
Vol.39, P. 33-43
Опубликовано: 2019
Тип ресурса: Статья
DOI:10.1016/j.ebiom.2018.12.002
Аннотация:
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 a
Ключевые слова:
Allergen; Allergy; Component-resolved diagnosis; Cross-reactive carbohydrate determinant; Molecular allergology; Recombinant glycoprotein
allergen; anazolene sodium; asparagine; carbohydrate; complementary DNA; epitope; glutamine; glycoprotein; hexahistidine; immunoglobulin E; immunoglobulin G1 antibody; myoglobin; recombinant protein; venom; epitope; glycoprotein; immunoglobulin E; recombinant protein; amino terminal sequence; antigen binding; Article; Baculoviridae; circular dichroism; cross reaction; enzyme linked immunosorbent assay; gel filtration; gene technology; glycosylation; human; immunoblotting; immunoglobulin G deficiency; insect cell; mite; molecular weight; nonhuman; plant; polyacrylamide gel electrophoresis; priority journal; protein expression; protein glycosylation; protein purification; sensitization; Sf9 cell line; size exclusion chromatography; animal; bee; chemistry; genetic engineering; hypersensitivity; immunology; metabolism; pollen; wasp; Allergens; Animals; Bees; Cross Reactions; Epitopes; Genetic Engineering; Glycoproteins; Humans; Hypersensitivity; Immunoglobulin E; Mites; Pollen; Recombinant
Язык текста: Английский
ISSN: 2352-3964
Gattinger P.
Mittermann I.
Lupinek C.
Hofer G.
Keller W.
Bidovec S. U. Stojkovic U.
Korosec P.
Koessler C.
Novak N.
Valenta R. Rudol`f 1963-
Гаттингер П.
Миттерманн И.
Лупинек C.
Хофер Г.
Келлер W.
Бидовеc С. У. Стойковиc У.
Коросеc П.
Коесслер C.
Новак Н.
Валента Р. Рудольф 1963-
Recombinant glycoproteins resembling carbohydrate-specific IgE epitopes from plants, venoms and mites
Текст визуальный непосредственный
EBioMedicine
Vol.39 P. 33-43
2019
Статья
Allergen Allergy Component-resolved diagnosis Cross-reactive carbohydrate determinant Molecular allergology Recombinant glycoprotein
allergen anazolene sodium asparagine carbohydrate complementary DNA epitope glutamine glycoprotein hexahistidine immunoglobulin E immunoglobulin G1 antibody myoglobin recombinant protein venom epitope glycoprotein immunoglobulin E recombinant protein amino terminal sequence antigen binding Article Baculoviridae circular dichroism cross reaction enzyme linked immunosorbent assay gel filtration gene technology glycosylation human immunoblotting immunoglobulin G deficiency insect cell mite molecular weight nonhuman plant polyacrylamide gel electrophoresis priority journal protein expression protein glycosylation protein purification sensitization Sf9 cell line size exclusion chromatography animal bee chemistry genetic engineering hypersensitivity immunology metabolism pollen wasp Allergens Animals Bees Cross Reactions Epitopes Genetic Engineering Glycoproteins Humans Hypersensitivity Immunoglobulin E Mites Pollen Recombinant
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 a