Endolysosomal protease susceptibility of Amb a 1 as a determinant of allergenicity
Authors: Martin Wolf, Lorenz Aglas, Teresa E. Twaroch, Markus Steiner, Sara Huber, Michael Hauser, Heidi Hofer, Maria A. Parigiani, Christof Ebner, Barbara Bohle, Peter Briza, Angela Neubauer, Frank Stolz, Michael Wallner, Fatima Ferreira
Journal: Journal of Allergy and Clinical Immunology (2017)

Exposure to pollen from short ragweed (Ambrosia artemisiifolia [Amb a]) causes severe respiratory allergies worldwide. Amb a 1, a member of the pectate lyase C (pelC) family, is a highly allergenic molecule recognized by more than 90% of ragweed-sensitized individuals and accounts for more than 90% of the allergenic activity in ragweed pollen.1Wolf M. Twaroch T.E. Huber S. Reithofer M. Steiner M. Aglas L. et al.Amb a 1 isoforms: unequal siblings with distinct immunological features.Allergy. 2017; 72: 1874-1882Crossref PubMed Scopus (21) Google Scholar However, the factors/features responsible for its high allergenicity (ie, capacity to induce specific IgE antibodies) remain undefined. Efforts to understand allergenicity have lately focused on extrinsic factors normally accompanying the allergen during the initial interaction with the immune system (eg, TLR ligands and lipid mediators).2Gomez-Casado C. Diaz-Perales A. Allergen-associated immunomodulators: modifying allergy outcome.Arch Immunol Ther Exp (Warsz). 2016; 64: 339-347Crossref PubMed Scopus (13) Google Scholar However, data from our previous studies suggested that intrinsic features of Amb a 1 might be linked to its high allergenic potential.3Jahn-Schmid B. Wopfner N. Hubinger G. Asero R. Ebner C. Ferreira F. et al.The T-cell response to Amb a 1 is characterized by 3 dominant epitopes and multiple MHC restriction elements.J Allergy Clin Immunol. 2010; 126 (1071.e1-2): 1068-1071Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar Thus, we sought to investigate intrinsic molecular pattern(s) possibly involved in the allergenicity of Amb a 1. As a comparison, we used the nonallergenic pelC homologue from the phytopathogenic Dickeya chrysanthemi (previously Dickeya dadantii and Erwinia chrysanthemi),4Yoder M.D. DeChaine D.A. Jurnak F. Preliminary crystallographic analysis of the plant pathogenic factor, pectate lyase C from Erwinia chrysanthemi.J Biol Chem. 1990; 265: 11429-11431PubMed Google Scholar here termed DC-pelC. Amb a 1.01, the most allergenic isoform, was purified from ragweed pollen extracts, whereas the bacterial-derived pelC (DC-pelC) was produced as a soluble recombinant protein in Escherichia coli (see Fig E1, A, in this article's Online Repository at www.jacionline.org). After purification, their identity and purity were confirmed by mass spectrometry (Fig E1, B and C). Despite their relatively low sequence identity (24%), circular dichroism and Fourier-transform infrared experiments indicated the presence of the parallel beta-helix structural element typical for polysaccharide lyases from family 1, with a high beta-sheet content (35.6% to 40.5%) for both molecules (see Fig E2 in this article's Online Repository at www.jacionline.org). These results support the notion that the 2 proteins share some degree of structural similarity (Fig E2, D). However, because of low sequence identity, cross-reactivity is not expected. Because Dickeya species are known to cause soft root disease on many crops (eg, bell pepper, tomatoes, potatoes, and onions)4Yoder M.D. DeChaine D.A. Jurnak F. Preliminary crystallographic analysis of the plant pathogenic factor, pectate lyase C from Erwinia chrysanthemi.J Biol Chem. 1990; 265: 11429-11431PubMed Google Scholar and ornamental indoor plants, contact with this phytopathogen has been recently demonstrated by metagenomic sequencing of the microbiota of human buccal mucosa.5Zheng W. Zhang Z. Liu C. Qiao Y. Zhou D. Qu J. et al.Metagenomic sequencing reveals altered metabolic pathways in the oral microbiota of sailors during a long sea voyage.Sci Rep. 2015; 5: 9131Crossref PubMed Scopus (26) Google Scholar, 6Belda-Ferre P. Alcaraz L.D. Cabrera-Rubio R. Romero H. Simon-Soro A. Pignatelli M. et al.The oral metagenome in health and disease.ISME J. 2012; 6: 46-56Crossref PubMed Scopus (359) Google Scholar These studies unambiguously identified Dickeya dadantii as an oral commensal bacterium. Thus, we performed ELISAs to evaluate immune responses to DC-pelC. None of the 39 ragweed-allergic patients displayed IgE or IgG4 antibodies recognizing DC-pelC. However, 37.5% of the individuals showed IgG antibodies against DC-pelC, confirming exposure but no sensitization to DC-pelC (see Fig E3 in this article's Online Repository at www.jacionline.org). To further investigate the sensitization capacity of Amb a 1.01 and DC-pelC, we immunized mice in the presence and absence of aluminum hydroxide (ALUM) as adjuvant (Fig 1, A). In both immunization protocols, Amb a 1.01 behaved as a very potent sensitizer, inducing high levels of IgG and IgE antibodies (Fig 1, B). These results clearly demonstrate that the intrinsic allergenic properties of Amb a 1.01 were not significantly altered by adsorption to ALUM as adjuvant. In contrast, in the adjuvant-free model, the nonallergenic pectate lyase DC-pelC induced a moderate immune response with low levels of IgG1 and IgG2a antibodies and failed to initiate B-cell class switch for IgE production. When ALUM was coadministered with DC-pelC, a significant increase in the titers of specific IgG1 and IgG2a antibodies was observed. In addition, DC-pelC–specific IgE antibody titers increased slightly, showing mediator release capacity in only 2 out of 5 mice (Fig 1, C and E). The amount of DC-pelC needed to induce half-maximal release in basophils sensitized with sera from ALUM-immunized animals and those without ALUM did not differ significantly (Fig 1, D). Our experiments also showed that DC-pelC–induced murine antibodies did not cross-react with Amb a 1.01 and vice versa (data not shown), further supporting the notion that natural exposure of humans to Amb a 1.01 and DC-pelC or immunization of mice gives rise to antibodies of different isotypes displaying distinct patterns of epitope recognition. We next investigated resistance/susceptibility of Amb a 1.01 and DC-pelC to proteolysis. Stability to endolysosomal proteolytic processing has been proposed to correlate with the efficacy of T-cell activation and priming, and the resulting immune response in vivo.7Egger M. Jurets A. Wallner M. Briza P. Ruzek S. Hainzl S. et al.Assessing protein immunogenicity with a dendritic cell line-derived endolysosomal degradome.PLoS One. 2011; 6: e17278Crossref PubMed Scopus (43) Google Scholar, 8Machado Y. Freier R. Scheiblhofer S. Thalhamer T. Mayr M. Briza P. et al.Fold stability during endolysosomal acidification is a key factor for allergenicity and immunogenicity of the major birch pollen allergen.J Allergy Clin Immunol. 2016; 137: 1525-1534Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar We performed digestions at different pH using proteases from the endolysosomal compartment of a dendritic cell line.7Egger M. Jurets A. Wallner M. Briza P. Ruzek S. Hainzl S. et al.Assessing protein immunogenicity with a dendritic cell line-derived endolysosomal degradome.PLoS One. 2011; 6: e17278Crossref PubMed Scopus (43) Google Scholar Under the same experimental conditions, Amb a 1.01 and DC-pelC differed remarkably in their susceptibility to proteolysis in a pH-dependent manner. At pH 4.8, Amb a 1.01 and DC-pelC showed a half-life of 45 hours and 72 hours, respectively (Fig 2, A and B). In contrast, Amb a 1 at pH 4.5 showed a decreased half-life of 12 hours whereas DC-pelC remained stable. In the presence of ALUM at pH 4.5, we observed stabilizing effects on Amb a 1. Under these conditions, DC-pelC was slightly destabilized at pH 4.5 but was more susceptible to degradation at pH 4.0. Peptides generated by proteolytic degradation of Amb a 1.01 and DC-pelC differed significantly both in their sequences and in their amounts. (Fig 2, A and B). These differences in susceptibility to proteolytic degradation might be due to distinct surfaces between the proteins, which, in turn, could result in distinct accessibility and resistance/susceptibility to proteases during processing by antigen-presenting cells,8Machado Y. Freier R. Scheiblhofer S. Thalhamer T. Mayr M. Briza P. et al.Fold stability during endolysosomal acidification is a key factor for allergenicity and immunogenicity of the major birch pollen allergen.J Allergy Clin Immunol. 2016; 137: 1525-1534Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar as recently demonstrated for 2 Bet v 1 isoforms with different immunogenic/allergenic properties.11Freier R. Dall E. Brandstetter H. Protease recognition sites in Bet v 1a are cryptic, explaining its slow processing relevant to its allergenicity.Sci Rep. 2015; 5: 12707Crossref PubMed Scopus (29) Google Scholar Although the route and conditions of exposure remain unclear, the presence of specific serum IgG and no detectable IgE antibodies strongly support the notion that human exposure to DC-pelC does not induce allergic sensitization. Our findings that in an adjuvant-free mouse immunization model DC-pelC failed to induce IgE antibodies further support the view that this pectate lyase lacks intrinsic allergenic activity. It is possible that for some proteins, allergen source–derived or even extrinsic factors are important for their allergenic potential. However, here we showed that highly purified natural Amb a 1.01 is capable of inducing strong TH2 immune responses in mice in the absence of (extrinsic) adjuvants. In contrast, under the same conditions, the nonallergenic bacterial homologue induced a TH1-biased response. Our data on Amb a 1.01 and DC-pelC are in agreement with previous findings for fold-stabilized Bet v 1 mutants.8Machado Y. Freier R. Scheiblhofer S. Thalhamer T. Mayr M. Briza P. et al.Fold stability during endolysosomal acidification is a key factor for allergenicity and immunogenicity of the major birch pollen allergen.J Allergy Clin Immunol. 2016; 137: 1525-1534Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar Thus, we propose a model in which the intrinsic stability/susceptibility of a protein to proteolysis can directly influence its processing and determines the intensity and polarization of the ensuing immune response (Fig 2, C). Pollen (6 g) of Amb a (Allergon Healthcare, Ängelholm, Sweden) was extracted with 180 mL of 5 mM sodium phosphate (NaP) buffer, pH 6.8, for 16 hours at 4°C, and clarified by centrifugation at 4000g for 20 minutes. The supernatant was then treated twice with 8 g of extraction buffer–soaked polyvinylpyrrolidone (Sigma-Aldrich, St Louis, Mo), followed by a 5 minute-centrifugation step (1000g) after each addition. Before loading onto a 15-mL column packed with CHT Type I resin (Bio-Rad Laboratories Inc, Hercules, Calif), the extract was filtered through a 0.45-μm syringe filter (GE Healthcare, Chicago, Ill). The column was eluted with a 4-segment linear gradient (0 to 500 mM NaP buffer, pH 6.8, final concentration) and fractions containing Amb a 1.01 were pooled and dialyzed against 10 mM NaP, pH 8.5. After purification, proteins were stored at −20°C until further use. The bacterial pectate lyase PLYC_DICCH (Uniprot ID: P11073) from Dickeya chrysanthemi (Pectobacterium chrysanthemi) (Erwinia chrysanthemi)E1Yoder M.D. DeChaine D.A. Jurnak F. Preliminary crystallographic analysis of the plant pathogenic factor, pectate lyase C from Erwinia chrysanthemi.J Biol Chem. 1990; 265: 11429-11431PubMed Google Scholar was expressed in Escherichia coli SHuffle T7 Express (New England Biolabs, Ipswich, Mass). Bacteria were transformed via electroporation with a plasmid vector pET26b (Novagen, Merck KGaA, Darmstadt, Germany) containing a harmonized DC-pelC construct. Bacteria were cultured in baffled flasks with 2 L LB broth supplemented with 10 mL/L glycerol and 25 mg/L kanamycin at 37°C and 220 rpm. Expression was induced at an OD600 of 0.6 using 0.5 mM isopropyl β-D-1-thiogalactopyranoside for 21 hours at 16°C. Bacteria were harvested by centrifugation at 3000g for 15 minutes and dissolved in 100 mL 5 mM NaP pH 6.8/g cell pellet. Cells were disrupted by 3 freeze/thaw cycles in liquid nitrogen, followed by a sonication step. Soluble protein fractions were collected by centrifugation at 13,000g for 20 minutes and filtered with a 0.45-μm syringe filter (GE Healthcare) before loading onto a 15 mL CHT Type I column (Bio-Rad Laboratories). Proteins were eluted with a 250-mL linear gradient from 0 to 500 mM NaP buffer, pH 6.8. Fractions containing DC-pelC were pooled, dialyzed against 5 mM NaP, pH 6.8, and reloaded onto the same column. The second elution was performed with a 100-mL gradient from 0 to 1 mol NaCl in 5 mM NaP, pH 6.8. Fractions containing DC-pelC eluting at 100% B were pooled and dialyzed against 10 mM NaP, pH 8.5. Contaminating endotoxins from bacterial cell wall were removed with a Triton wash protocol, as previously described.E2Aida Y. Pabst M.J. Removal of endotoxin from protein solutions by phase separation using Triton X-114.J Immunol Methods. 1990; 132: 191-195Crossref PubMed Scopus (482) Google Scholar The endotoxin content of Amb a 1 and DC-pelC was determined using the HEK-Blue mTLR4 reporter cell assay (Invivogen, San Diego, Calif), according to manufacturer's instructions. The levels of endotoxin in both preparations were determined to be below the detection limit of 6.1 EU/mg. Amino acid analysis of the protein preparations was performed according to the PicoTag method (Waters, Milford, Mass) using an HP1100 HPLC system (Hewlett-Packard, San Jose, Calif) equipped with a 3.9 × 150 mm Novapak C18 column (Waters). The aggregation behavior and protein dispersity was assayed by dynamic light scattering using the DLS 802 system (Viscotek Corp, Houston, Tex). Data evaluation of 10 accumulated measurements was performed with the OmniSize software and displayed as mass weighted distribution of the hydrodynamic radius. For circular dichroism analysis, samples were measured at 0.1 mg/mL from 190 to 260 nm at a resolution of 1 nm with 1 nm bandwidth and a scanning speed of 1 nm/s. Spectra were recorded with a JASCO-J815 spectropolarimeter (Jasco, Tokyo, Japan) fitted with a PTC-423S Peltier-type single position cell holder in 10 mM sodium phosphate buffer, pH 8, at 20°C or 95°C, respectively. Data from 5 spectra were averaged, background corrected, and presented as mean residue molar ellipticity.E3Himly M. Nony E. Chabre H. Van Overtvelt L. Neubauer A. Van Ree R. et al.Standardization of allergen products, 1: detailed characterization of GMP-produced recombinant Bet v 1.0101 as biological reference preparation.Allergy. 2009; 64: 1038-1045Crossref PubMed Scopus (62) Google Scholar Infrared spectra were recorded at a constant temperature (25°C) using an AquaSpec transmission cell adapted to a Tensor II Confocheck Fourier transformed infrared system (Bruker Optics Inc, Billerica, Mass). Second derivatives were calculated by applying the Savitzky-Golay algorithm with 25 smoothing points. Spectra were vector-normalized and baseline corrected. Secondary structure elements were analyzed using the Quant2 method provided by Confocheck (Bruker Optics Inc). Purified proteins (1 μg) were digested with the ProteoExtract All-in-One Trypsin Digestion Kit (EMD Millipore, Billerica, Mass). After digestion, samples were desalted using C18 ZipTips (EMD Millipore) and resulting peptides separated by reverse-phase nano-HPLC (Dionex Ultimate 3000, Thermo Fisher Scientific, Bremen, Germany; column: PepSwift Monolithic Nano Column, 100 μm × 25 cm, Dionex). The column was developed with an acetonitrile gradient (solvent A: 0.1% (v/v) formic acid/0.01% (v/v) trifluoroacetic acid/5% (v/v) acetonitrile; solvent B: 0.1% (v/v) FA/0.01% (v/v) trifluoroacetic acid/90% (v/v) acetonitrile; 5%-45% B in 60 minutes) at a flow rate of 1 μL/min at 55°C). The HPLC was directly coupled via nano electrospray to a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific). Capillary voltage was 2 kV. For peptide identification, the top 12 method was used with the normalized fragmentation energy at 27%. Survey and fragment spectra were analyzed with Proteome Discoverer version 1.4 (Thermo Fisher Scientific) or Peaks Studio 7 (Bioinformatics Solutions, Waterloo, Canada), respectively. Ragweed-allergic patients were selected on the basis of typical case history, positive skin prick test results, and ImmunoCAP analysis (CAP w1 Common ragweed Ambrosia artemisiifolia, Phadia, AB, Uppsala, Sweden). Experiments using anonymized serum samples from allergic patients were approved by the ethics committee of the Medical University of Vienna (712/2010). For ELISA experiments, Maxisorp plates (Nunc, Thermo Fisher, Waltham, Mass) were coated with 100 ng protein/well in 50 μL PBS overnight at 4°C. After washing and blocking, plates were incubated either with diluted human sera (1:50 for IgG and 1:250 for IgE detection) (buffer: TBS, pH 7.4, 0.05% (v/v) Tween, 1% (w/v) BSA) or with serial dilutions of murine sera (buffer: TBS, pH 7.4, 0.05% (v/v) Tween, 1% (w/v) BSA) overnight at 4°C. Bound human IgE was detected using an AP-conjugated mouse anti-human IgE antibody (Clone G7-26) (BD Biosciences, Franklin Lakes, NJ), human IgG1 was detected by an AP-conjugated mouse anti-human IgG1-hinge antibody (Clone 4E6), and human IgG4 was detected using an AP-conjugated mouse anti-human IgG4 pFc' antibody (Clone HP6023). Murine IgG1, IgG2a, and IgE antibodies were detected using AP-conjugated rat anti-mouse IgG1, AP-conjugated rat anti-mouse IgG2a, and rat anti-mouse IgE antibodies (Clones SB77e, SB84a, and 23G3, respectively; Southern Biotech, Birmingham, Ala), respectively. Colorimetric detection with 10 mM 4-nitrophenyl phosphate (Sigma-Aldrich) was performed following incubation periods of 1 hour at 37°C and 1 hour at 4°C. All measurements were performed in duplicate and the results are presented as mean OD values. For the analysis of murine IgE antibodies, nontransfected RBL-2H3 cells (ATCC CRL2256)E4Vogel L. Luttkopf D. Hatahet L. Haustein D. Vieths S. Development of a functional in vitro assay as a novel tool for the standardization of allergen extracts in the human system.Allergy. 2005; 60: 1021-1028Crossref PubMed Scopus (124) Google Scholar were used after passive sensitization with 100 μL murine serum samples diluted 1:20 in cell culture medium. Following washing steps, serially diluted protein antigens (1 ng/mL to 100 μg/mL) were added to trigger the release of mediators. For serum dilutions, a fixed antigen concentration of 10 ng/mL was used. Antigen-dependent β-hexosaminidase release into the supernatant was measured by enzymatic cleavage of the fluorogenic substrate 4-methylumbelliferyl-N-acetyl-β-d-glucosaminide and expressed as percentage of total enzyme content of Triton X100–treated cells. Antigens were digested with a cocktail of endolysosomal proteases as previously described.E5Egger M. Jurets A. Wallner M. Briza P. Ruzek S. Hainzl S. et al.Assessing protein immunogenicity with a dendritic cell line-derived endolysosomal degradome.PLoS One. 2011; 6: e17278Crossref PubMed Scopus (58) Google Scholar In brief, microsomal fractions were harvested from JAWSII cells (ATCC CRL11904) by differential centrifugation. Five microgram of protein was digested with 7 μg of purified microsome lysates in 100 mM citrate buffer adjusted to different pH values (pH 4.0, 4.5, 4.8, 5.2, and 5.9), 2 mM dithiothreitol at 37°C. Degradation assays were also performed with proteins adsorbed to 50% Alu-Gel-S (Serva, Heidelberg, Germany). Reactions were stopped by heating to 100°C. Resulting samples were analyzed by SDS-PAGE and by nano-LC-MS/MS, as described above. Female BALB/c mice (Janvier Labs, Saint-Berthevin, France) at the age of 8 weeks were used for the immunization experiments (5 animals/group). Five microgram of antigen was either adsorbed to 50% Alu-Gel-S (Serva, Heidelberg, Germany) v/v or formulated without adjuvant in PBS, and administered as two 50-μL subcutaneous injections, bilaterally in the lumbar region at days 0, 7, 14, and 21. Sera were taken at days −2, 14, and 28. ELISA was used to analyze specific IgG1 and IgG2a antibodies, whereas mediator release assays were performed to detect IgE antibodies. All animal experiments were conducted according to national guidelines approved by the Austrian Ministry of Science (BMWF-66.012/0009-II/3b/2013).Fig E2A, Circular dichroism (CD) spectra of Amb a 1.01 and DC-pelC measured at 20°C and 95°C, respectively. B, Second derivative of FTIR spectra of Amb a 1.01 and DC-pelC in the area of α-helix (1651 cm−1) and β-sheet (1637 cm−1) signals. C, Secondary structure elements as calculated from FTIR spectra. D, Structural model of Amb a 1.01 generated by Swissmodel (template: PDB:1PXZ) and DC-pelC structure (PDB:2EWE). Lower panels, structural alignment of Amb a 1.01 model and DC-pelC structure generated with the UCSF Chimera package. FTIR, Fourier-transform infrared.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3IgE, total IgG, IgG1, and IgG4 ELISA experiments performed with sera from 39 ragweed pollen-allergic patients.View Large Image Figure ViewerDownload Hi-res image Download (PPT)