Glycosylation enhances allergenic activity of major bee venom allergen Api m 1 by adding IgE epitopes (2020)

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