Glycosylation enhances allergenic activity of major bee venom allergen Api m 1 by adding IgE epitopes

Phospholipase A2 (Apis mellifera 1 [Api m 1]), the major bee venom allergen, is a glycoprotein that can bind IgE antibodies of patients with allergy via protein and/or carbohydrate epitopes.1Mittermann I. Zidarn M. Silar Markovic-Housley Z. Aberer W. Korosec P. et al.Recombinant allergen-based testing to distinguish and wasp allergy.J Allergy Clin Immunol. 2010; 125: 1300-1307Abstract Full Text PDF PubMed Scopus (100) Google Scholar It has been suggested epitopes Api are conformation dependant2Müller U.R. Dudler T. Schneider Crameri R. Fischer H. Skrbic D. al.Type I skin reactivity native recombinant phospholipase from honeybee similar.J 1995; 96: 395-402Abstract (75) Scholar; recently, however, IgE-reactive sequential have also described.3Zahirovic A. Koren Kopac Strukelj B. Lunder Identification characterization corresponding mimotopes.J 2019; 143: 791-794Abstract (4) Carbohydrates present on allergens plants, insects, mites, molds, parasites considered be highly cross-reactive (ie, determinants [CCDs]) low capacity induce basophil or mast cell degranulation hence allergenic activity.4Mari determinants: analysis distribution appraisal in vivo vitro reactivity.Int Arch 2002; 129: 286-295Crossref (179) Accordingly, presence CCDs may cause misleading test results. One important example grass pollen who against Phleum pratense 4 (Phl p 4)-derived showed natural CCD-containing even though they were not sensitized venom.1Mittermann To avoid such false results, nonglycosylated introduced for serologic diagnosis sensitivity.5Jakob Köhler J. Blank S. Magnusson U. Huss-Marp Spillner E. al.Comparable determinant-negative 2012; 130: 276-278Abstract (35) Conversely, galactose-α-1,3-galactose (α-GAL) trigger allergic reactions.6Platts-Mills T.A. Schuyler A.J. Tripathi Commins S.P. Anaphylaxis side chain alpha-gal.Immunol North Am. 2015; 35: 247-260Abstract (58) The influence glycosylation enzymatic activity allergen controversial issue. Certain studies suggest similar glycosylated 1,2Müller whereas seems activity.7Blank Michel Y. Seismann Plum Greunke K. Grunwald al.Evaluation different glycoforms (Api 1) produced insect cells.Protein Pept Lett. 2011; 18: 415-422Crossref (33) investigate role bee-derived structural fold, activity, 1, we compared purified (nApi (rApi expressed cells. Recombinant mutated N-glycosylation site was Spodoptera frugiperda (Sf9) cells (see Methods this article's Online Repository at www.jacionline.org); obtained Sigma-Aldrich (Vienna, Austria). Coomassie blue–stained SDS-PAGE Western blot rApi pure migrated as 15 kDa band, nApi appeared form 2 bands, prominent 16 17 14 kDa. According staining α-(1,3)-fucose–specific Aleuria aurantia lectin, band glycosylated, lacked Fig E1, A B section www.jacionline.org). Thus, E1 shows consists fraction fraction. thin corresponds version represents version. which gives single migrating Both specific by immunoblotting (Fig C); levels 1.2- 2.71-fold higher 8 12 Table By contrast, there good correlation between 1-specific determined ImmunoCAP ELISA, although differences noted certain sera E2 For both assays (ImmunoCAP ELISA), line Sf9 lacks N-linked site.5Jakob explanation observed discrepancies contains large amounts antigen immobilized its surface, much lower coated ELISA plates, allow IgG compete binding resulting signals. Finally, ImmunoCAPs CCD-specific nonspecific manner, result measurements containing IgE.8Hemmer Altmann F. Holzweber Gruber C. Wantke Wöhrl cellulose displays determinant (CCD) false-positive results high anti-CCD antibody levels.J 2018; 141: 372-381Abstract (32) Next, investigated whether regarding fold functional properties protein. However, circular dichroism proteins folded no relevant difference secondary structure contents molar ellipticities could detected Repository). Furthermore, occurred monomeric physiologic buffers, demonstrated size exclusion chromatography E3 Therefore, cannot explained structure; rather, it due attached CCDs, because most tested positive E1). m1, but control birch Betula verrucosa 1), comparable during assessment first 5 minutes 15-minute recording period see During last 10 measurement, 20% found, fact required pH conditions = 7.5; 6) storage affect B). titrated activation experiments Four (patients 6, 3, 9, 12) E1) stronger than patient 4, mounted these associated Interestingly, derivatives, differ account (for example) folded, cell– versus unfolded Escherichia coli–expressed Par j 2.9Dorofeeva Colombo Blanca Mari Khanferyan Valenta al.Expression resembling Parietaria judaica pollen.Sci Rep. 9: 15043Crossref (2) In fact, molecules, show alterations their shift dose-response curve activation; dose dependency activation, induced release ß-hexosaminidase each concentrations 2). Such qualitative usually when basophils contain more allergen-specific IgE.E4Gieras Focke-Tejkl Ball Verdino Hartl Thalhamer al.Molecular allergen-induced effector degranulation.J 2007; 119: 384-390Abstract (44) Our study thus demonstrates capacity, do depend glycosylation, increase through additional case enhances allergen. findings what described other allergens. example, Lyc e tomato bound only able histamine release.E5Foetisch Westphal Lauer Retzek Kolarich al.Biological determinants.J 2003; 111: 889-896Abstract (162) house dust mite Der had correctly, test, evidence provided found 1.E6Burtin Chabre Olagnier Didierlaurent Couret M.N. Comeau al.Production modified molecules tobacco plants.Clin Exp Allergy. 2009; 39: 760-770Crossref (17) Bla g 2, German cockroach an shown removal sugars resulted reduction human basophils.E7Li Gustchina Glesner Wünschmann Vailes L.D. Chapman M.D. al.Carbohydrates contribute interactions monoclonal antibody.J 186: 333-340Crossref (29) Scholar, E8Glesner Li Wlodawer Himly al.Mechanisms allergen-antibody interaction inhibit binding.PLoS One. 6e22223Crossref (27) E9Do D.C. Yang Yao X. Hamilton R.G. Schroeder J.T. Gao N-glycan regulates function.Immun Inflamm Dis. 2017; 5: 386-399Crossref (14) available data, one envisage possibilities how Carbohydrate mask protein-based epitopes, Phl 4.E10Zafred Nandy Pump L. Kahlert Keller Crystal immunologic 4.J 2013; 132: 696-703Abstract improve stabilize conformational would enhance activity.E11Ball Edstrom Mauch Schmitt Leistler Fiebig al.Gain eukaryotic expression Timothy 1.FEBS 2005; 272: 217-227Crossref (30) carbohydrates add addition epitopes. latter case, adds carbohydrate-specific peptide otherwise IgE-reactive, enzymatically active, activity. This mechanism operative where seem within same epitope.E7Li implications use diagnostic purposes presumably therapeutic well. effective inducing potent cell–based assay provocation tests, cell–expressed specificity avoids CCDs. We want thank Professor Dr. Natalija Novak, Bonn, Germany, providing us venom. studied using clinically well-characterized described.E1Valenta Duchene Ebner Valent Sillaber Deviller al.Profilins constitute novel family plant pan-allergens.J Med. 1992; 175: 377-385Crossref (567) Grading severity clinical reactions according Mueller 11 (Table extract (i1) quantified (Thermo Scientific, Phadia AB, Uppsala, Sweden). Carbohydrate-specific horse heart myoglobin CCD bromelain ImmunoCAPs.E2Gattinger Mittermann Lupinek Hofer G. Stojkovic U.B. glycoproteins venoms mites.EBioMedicine. 33-43Abstract (9) anonymized performed approval ethical committee Medical University Vienna (EK1641/2014). Lectin blots previously.E2Gattinger analyzed cytochrome C carbonic anhydrase molecular weight standards.E3Villazala-Merino Rodriguez-Dominguez Stanek V. Campion N.J. Gattinger al.Allergen-specific ability IgE-allergen complexes cross-link determine extent CD23-mediated T-cell activation.J 2020; 145: 958-967Abstract (6) Far-UV spectrometry previously described.E2Gattinger assessed sPLA2 Assay Kit (Cayman Chemical) (Sanova, Vienna, Austria) recommendations manufacturer. used negative control. Results time-dependent absorbance increases (mOD405nm/min). Mean values deviations less 5% duplicate shown. 1,2-dithio analog diheptanoyl phosphatidylcholine substrate following hydrolysis thioester bond sn-2 position free thiols 5,5′-dithio-bis-(2-nitrobenzoic acid). rat leukemia expressing high-affinity receptor FcεRI. loaded serum allergy. β-hexosaminidase measured described.E3Villazala-Merino ScholarFig E2Correlation sIgE (kUA/L) (OD) (Pearson coefficient R 0.7453; P < .0005).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Size (A) (B). Elution volumes marker (cytochrome C, kDa; anhydrase, 29 kDa) indicated arrows. Shown elution (x-axis) units (y-axis).View (PPT)Table E1Clinical allergyPatient IDTotal level (kU/L)∗ImmunoCAP (Phadia AB) total level.Bee (kUA/L)†ImmunoCAP AB), extract, i1, (≥0.35 kU/L).rApi (kUA/L)‡IgE i208 ImmunoCAP.nApi OD, range individual lowest highest§IgE ELISA.‖All variation 6%.nApi mean§IgE 6%.rApi highest¶IgE mean‖All 6%.¶IgE ELISA.Fold 1Mueller grade#Clinical terms grade.CCD-specific markersIgE HHM2,E8 CCD-ImmunoCAP, Bromelain-ImmunoCAP.14961007.511.70-1.731.721.37-1.411.391.22No23048.812.370.45-0.460.460.46-0.470.470.982No33931005.961.69-1.731.711.1-1.121.111.52Yes4nd44.34.571.32-1.341.331.28-1.321.301.024No54743.8316.171.89-1.881.881.89-1.891.890.992Yes6631.964.430.40-0.400.40.22-0.230.231.732Yes756654.121.931.92-1.951.941.43-1.491.461.32Yes838579.82.910.48-0.520.50.33-0.350.341.622Yes9nd10.92.630.95-0.960.950.34-0.350.352.712Yes10212177.490.89-0.930.910.45-0.500.481.93Yes11187.821.590.20-0.220.210.2-0.20.21.052No12477ndnd1.79-1.831.811.30-1.331.321.37ndYesID, Identifier; nd, done.∗ level.† kU/L).‡ ImmunoCAP.§ ELISA.‖ All 6%.¶ ELISA.# Clinical grade.∗∗ HHM2,E8Glesner Bromelain-ImmunoCAP. Open table new tab ID, done.