Báo cáo khoa học: Gain of structure and IgE epitopes by eukaryotic expression of the major Timothy grass pollen allergen, Phl p 1 pdf

Gain of structure and IgE epitopes by eukaryotic expression of the major Timothy grass pollen allergen, Phl p Tanja Ball1,2, William Edstrom2, Ludwig Mauch3, Jacky Schmitt3, Bernd Leistler3, Helmut Fiebig4, Wolfgang R Sperr5, Alexander W Hauswirth5, Peter Valent5, Dietrich Kraft1, Steven C Almo2 and Rudolf Valenta1 Department of Pathophysiology, Center for Physiology and Pathophysiology, Medical University of Vienna, Austria Albert Einstein College of Medicine, Department of Biochemistry, NY, USA Pharmacia Diagnostics, Freiburg, Germany Allergopharma KG, Reinbek, Germany Division of Hematology, Department of Internal Medicine I, Medical University of Vienna, Austria Keywords allergen; allergy; epitope; eukaryotic expression; Phl p Correspondence R Valenta, Division of Immunopathology, Department of Pathophysiology, Center for Physiology and Pathophysiology, Medical University of Vienna, Waehringer Guertel 18–20, A-1090 Vienna, Austria Fax: +43 40 400 5130 Tel: +43 40 400 5108 E-mail: rudolf.valenta@meduniwien.ac.at (Received August 2004, revised 21 September 2004, accepted 22 September 2004) doi:10.1111/j.1432-1033.2004.04403.x Approximately 400 million allergic patients are sensitized against group grass pollen allergens, a family of highly cross-reactive allergens present in all grass species We report the eukaryotic expression of the group allergen from Timothy grass, Phl p 1, in baculovirus-infected insect cells Domain elucidation by limited proteolysis and mass spectrometry of the purified recombinant glycoprotein indicates that the C-terminal 40% of Phl p 1, a major IgE-reactive segment, represents a stable domain This domain also exhibits a significant sequence identity of 43% with the family of immunoglobulin domain-like group ⁄ grass pollen allergens Circular dichroism analysis demonstrates that insect cell-expressed rPhl p is a folded species with significant secondary structure This material is well ˚ behaved and is adequate for the growth of crystals that diffract to 2.9 A resolution The importance of conformational epitopes for IgE recognition of Phl p is demonstrated by the superior IgE recognition of insect-cell expressed Phl p compared to Escherichia coli-expressed Phl p Moreover, insect cell-expressed Phl p induces potent histamine release and leads to strong up-regulation of CD203c in basophils from grass pollen allergic patients Deglycosylated Phl p frequently exhibits higher IgE binding capacity than the recombinant glycoprotein suggesting that rather the intact protein structure than carbohydrate moieties themselves are important for IgE recognition of Phl p This study emphasizes the important contribution of conformational epitopes for the IgE recognition of respiratory allergens and provides a paradigmatic tool for the structural analysis of the IgE allergen interaction Type I allergy is an IgE-mediated hypersensitivity disease affecting more than 25% of the population [1,2] Grass pollen allergens belong to the group of most frequently recognized allergenic components [3] At least 40% of allergic patients are sensitized to grass pollen allergens and more than 95% of them display IgE reactivity to group I allergens [3–6] Group allergens represent a family of glycoprotein allergens of approximately 30 kDa that occur as cross-reactive antigens in almost all grasses and corn species [6,7] Abbreviations PrPhl p 1, prokaryotic recombinant Phl p 1; ErPhl p 1, eukaryotic recombinant Phl p 1; GST, glutathione S-transferase FEBS Journal 272 (2005) 217–227 ª 2004 FEBS 217 Eukaryotic expression of Phl p They are exclusively expressed in mature pollen grains where they are localized mainly in the cytoplasm [8] Using immuoelectronmicroscopy two mechanisms for the release of group allergens have been demonstrated First, contact of intact pollen grains with mucosal surfaces (e.g nasal epithelium) leads to hydration and rapid diffusion of the allergens [9] Second, it has been demonstrated that rain water induces the expulsion of respirable micron size allergen-containing particles from grass pollens [10,11] The small size of these subcellular particles allows them to reach the deeper airways and may explain the frequent occurrence of heavy asthma attacks after rainfalls [12,13] cDNAs coding for group allergens from several grasses have been isolated and showed high sequence similarity [14–20] The recombinant group allergen from Timothy grass, rPhl p 1, expressed in Escherichia coli contained many of the T cell epitopes of natural group allergens and cross-reacted with the naturally occuring isoallergens from Timothy grass and other grass species [6,21,22] However, several post-translational modifications (e.g glycosylation, occurrence of hydroxyprolines) and the formation of disulphide bonds have been described for group I allergens [23,24] These modifications not occur when proteins are expressed in prokaryotic expression systems and hence E coli-expressed group allergens exhibit impaired structural and immunological properties The importance of conformational epitopes for IgE recognition of group I allergens is highlighted by IgE competition experiments using recombinant fragments of Phl p representing continuous IgE epitopes as even a mixture of several major IgE-epitope-containing rPhl p fragments does not completely inhibit IgE binding to intact Phl p [25] To obtain properly folded rPhl p 1, the cDNA coding for the mature allergen was expressed in baculovirus-infected insect cells An expression strategy was chosen which led to the secretion of the recombinant allergen into the cell culture supernatants rPhl p was purified to homogeneity, characterized by mass spectro-metry and the presence of post-translational modification (i.e glycosylation) was investigated The secondary structure content of insect cell-expressed rPhl p was examined by circular dichroism analysis and diffraction quality crystals of the recombinant allergen were grown The IgE binding properties of insect cell-expressed Phl p were compared with those of E coli-expressed and natural Phl p by competition studies performed under native conditions and the importance of glycosylation for IgE reactivity was examined by enzymatic deglycosylation of insect cellexpressed Phl p Finally, the biological activity of 218 T Ball et al insect cell-expressed and E coli-expressed Phl p was compared in histamine release experiments and by CD203c expression in basophils from grass pollen allergic patients [26] The finding that proper folding of insect-cell expressed Phl p is related to increased IgE reactivity and allergenic activity is discussed as a general feature of respiratory allergens and has relevance for the development of allergy vaccines which are based on the reduction of allergen fold Results Comparison of natural and recombinant group grass pollen allergens Although the majority of rPhl p was detected in the insoluble pellet fraction of infected insect cells, up to 0.75 mgỈL)1 of soluble rPhl p could be purified from the culture supernatant by Ni2+-affinity chromatography under nondenaturing conditions Purified insect cell-expressed Phl p migrated at slightly higher molecular mass than the natural Phl p 1, E coliexpressed Phl p and the Phl p 1-homologous allergen from rye grass (Lol p 1) (Fig 1A) Insect cell-expressed Phl p as well as natural group allergens (nPhl p and nLol p 1) reacted with a rabbit antiserum raised against purified E coli-expressed Phl p (Fig 1B) but not with the corresponding preimmune serum (Fig 1C) A band of approximately double the molecular mass as the purified allergens, possibly representing a dimer, was detected in the bacterial and insect cell-expressed Phl p and, to a lower degree, in the nPhl p preparations Biochemical and biophysical characterization of insect-cell expressed Phl p The molecular mass of insect cell-expressed Phl p was determined by mass spectrometry to be 28 122 Dalton (data not shown) The difference of 956 Da between the calculated (27 166 Da) and the determined molecular mass is attributed to glycosylation Limited proteolysis in combination with mass spectrometry was performed to identify structural domains [27,28] Fundamental to this approach is the notion that protection against proteolysis is conferred in regions of the protein that are within a rigid structure, while proteolytic cleavage of a multiple-domain protein is biased towards solvent accessible regions (i.e exposed loops, interdomain linker chains) We identified two proteolytically stable structural domains of rPhl p by limited proteolysis, one comprising C78–K118 and a second domain spanning from FEBS Journal 272 (2005) 217–227 ª 2004 FEBS T Ball et al Eukaryotic expression of Phl p A B C Fig Coomassie staining and immunoreactivity of purified recombinant and natural group allergens (A) Coomassie stained SDS ⁄ PAGE containing natural Lol p (nLol p 1), natural Phl p (nPhl p 1), eukaryotic recombinant Phl p (ErPhl p 1) and bacterial recombinant Phl p (PrPhl p 1) B and C represent immunoblots probed with rabbit anti-(Phl p Ig) antiserum and the corresponding preimmune serum, respectively K147–K241 (data not shown) The latter corresponds to the region homologous to group allergens To confirm that the difference between the calculated and determined molecular mass is due to glycosylation of the insect cell-expressed Phl p 1, glycan detection was performed (Fig 2A) Nitrocellulose-blotted insect cell-expressed Phl p 1, but not E coli-expressed Phl p showed a positive staining for glycan moieties (blue color) Also, a nonglycosylated control protein, creatinase, and the marker proteins gave negative reaction in the glycan staining and appear brown (Fig 2A) Finally, enzymatic deglycosylation with PNGase F resulted in a reduction of molecular mass of insect cell-expressed Phl p as visualized by SDS ⁄ PAGE (Fig 2B) Insect cell-expressed Phl p represents a folded protein with considerable b-sheet structure that crystallizes as thin plates Insect cell- and E coli-expressed Phl p were analyzed by circular dichroism (CD) spectroscopy to determine their secondary structural content (Fig 3) The CD spectrum of insect cell-expressed, eukaryotic Phl p (ErPhl p 1) suggested the presence of substantial antiparallel b-sheet, while the CD spectrum for the Phl p expressed in bacteria (prokaryotic: PrPhl p 1) indicated a considerable amount of unordered structure The characteristics of the CD spectrum of insect cellexpressed Phl p indicates structural similarity with Phl p 2, an almost exclusively b-sheet containing allergen with 43% sequence identity to the C-terminal third of Phl p [29,30] Thermal denaturation of insect cellexpressed Phl p was monitored by far-UV CD in the range of 20 °C to 90° and showed an irreversible unfolding transition, with a melting point of  42 °C FEBS Journal 272 (2005) 217–227 ª 2004 FEBS (data not shown) Only the properly folded insect cell-expressed but not the E coli-expressed Phl p afforded crystals These crystals belonged to the orthorhombic space group P212121 and diffracted X-rays to ˚ a resolution of 2.9 A (Fig 4) Phl p and Phl p belong to different clusters of proteins as determined by phylogenetic analysis Amino acid sequences of seven group pollen allergens and four group ⁄ allergens were subjected to phylogenetic analysis using the phylip 3.6a2 package (http://evolution.genetics.washington.edu/phylip.html) (Fig 5) The allergens formed three main clusters, one comprising Zea m and Cyn d 1, a second consisting of Lol p 3, Dag g 3, Lol p 1, Phl p 1, Ory s and Tri a and a third cluster including Lol p 2, Hol l 1, Phl p and Pha a Although Phl p and Phl p are derived from Phleum pratense and share high sequence identity, the phylogenetic analysis shows that they are less related to each other than group and group ⁄ allergens from different species Insect cell-expressed Phl p contains the IgE epitopes of natural Phl p A comparison of the IgE binding capacity of E coliand insect cell-expressed Phl p under nondenaturing conditions in a dot-blot assay showed that insect cellexpressed Phl p was more potent than the E coliderived allergen (Table 1) IgE competition studies performed under native conditions confirmed this result (Fig 6A) Preincubation of sera from four grass pollen allergic patients with E coli-expressed Phl p completely inhibited IgE binding to the very same protein, but not to the insect cell-expressed Phl p An 219 Eukaryotic expression of Phl p almost complete reduction of IgE binding to insect cell-expressed Phl p was only observed for serum of patient 2, whereas considerable IgE reactivity of sera 1, and to insect cell-expressed Phl p was observed A T Ball et al despite preincubation with an excess of E coliexpressed Phl p Whether insect cell-expressed Phl p contains the IgE epitopes of a natural Phl p preparation was investigated by IgE competition experiments (Fig 6B) Preincubation of sera from grass pollen allergic patients with insect cell-expressed Phl p led to a strong or complete inhibition of IgE reactivity to natural Phl p (Fig 6B) Next we studied the influence of glycosylation on the IgE binding capacity of insect cell-expressed Phl p (Fig 6C) Five out of 10 patients showed stronger IgE reactivity to deglycosylated insect cellexpressed Phl p than to the untreated protein (Fig 6C, 1, 2, 3, 5, 7) Three patients exhibited comparable IgE reactivity to both protein forms (Fig 6C, #4, 9, 10) and two sera reacted stronger with the glycosylated allergen version (Fig 6C, 6, 8) Finally, we studied the possible presence of cross-reactive IgE epitopes between Phl p and Phl p Preincubation of sera from pollen allergic patients with insect cellexpressed, E coli-expressed Phl p or an unrelated control allergen (birch pollen allergen, rBet v 1) had no effect on IgE binding to rPhl p (data not shown) Allergenic activity of insect cell-expressed Phl p B The allergenic activity of insect cell-expressed Phl p was analyzed by basophil histamine release (Fig 7) and CD203c expression (data not shown) Basophils from two grass pollen allergic patients were exposed to different concentrations of E coli- or insect cellexpressed Phl p (Fig 7A,B) In both patients, insect cell-expressed Phl p was more potent, inducing histamine release at lower concentrations (10)3 lgỈmL)1) than E coli-expressed Phl p (10)2 lgỈmL)1) Measurement of CD203c expression on blood basophils of three Phl p allergic patients confirmed these results Incubation with insect cell-expressed Phl p always led to stronger upregulation of CD203c than incubation with E coli-expressed Phl p (data not shown) Fig Biochemical and biophysical characterization of insect cellexpressed Phl p (A) Glycan detection Nitrocellulose blotted insect cell-expressed rPhl p (ErPhl p 1), rPhl p expressed in bacteria (PrPhl p 1), Creatinase and marker proteins (M) were simultaneously stained for sugar moieties (blue) and reactive amino groups (fluorescent) Molecular masses are indicated on the left margin (B) SDS ⁄ PAGE containing insect cell-expressed Phl p before (ErPhl p 1-) and after (ErPhl p +) enzymatic deglycosylation Lane M: Molecular mass marker 220 FEBS Journal 272 (2005) 217–227 ª 2004 FEBS T Ball et al Eukaryotic expression of Phl p Fig Comparison of E coli- and insect cell-expressed Phl p by circular dichroism spectroscopy Far-UV CD spectra of E.coli- (grey) and insect cell-expressed Phl p (black), expressed as mean residue ellipticity (y-axis), were recorded at 20 °C in the wave length range displayed on the x-axis Fig Analysis of the sequence and phylogenetic relationship among group and group ⁄ allergens from various grass species A phylogenetic tree was reconstructed on the basis of aminoacid sequences of group (Zea m 1: Zea mays, Cyn d 1: Cynodon dactylon, Pha a 1: Phalaris aquatica, Hol l 1: Holcus lanatus, Ory s 1: Oryza sativa, Lol p 1: Lolium perenne, Phl p 1: Phleum pratense) and group ⁄ allergens (Lol p 3: Lolium perenne, Dag g 3: Dactylis glomerata, Lol p 2: Lolium perenne, Tri a 3: Triticus aestivum, Phl p 2: Phleum pratense) using the PROTDIST and KITSCH program of the PHYLIP package Fig Crystal growth of insect cell-expressed Phl p 1.Phl p crystallizes as thin plates of 0.35 · 0.35 · 0.15 mm Discussion Phl p represents one of the most important respiratory allergens known to date As Phl p is a glycoprotein containing seven cysteines, we expressed the allergen in eukaryotic insect cells to obtain a post-translationally modified and folded protein As demonstrated by mass spectrometry, glycan detection and deglycosylation experiments, insect cell-expressed Phl p was obtained as a glycoprotein The seemingly correct folding of insect cell-expressed Phl p is demonstrated by the following experiments: insect cell-expressed Phl p but not E coli-expressed Phl p exhibited a secondary structure consisting mainly of b-sheets when analyzed by CD spectroscopy Furthermore, only insect cell-expressed Phl p grew diffraction quality crystals FEBS Journal 272 (2005) 217–227 ª 2004 FEBS and thus will yield the three-dimensional structure of this allergen Phl p belongs to the group family of highly crossreactive grass pollen allergens The C-terminal domains of these allergens display sequence similarity to group Table Comparison of the IgE binding capacity of E coli- and insect cell-expressed Phl p IgE reactivity to recombinant Phl p 1, expressed in E coli (PrPhl p 1) and baculovirus-infected insect cells (ErPhl p 1) PrPhl p ErPhl p Patient number IgE binding (c.p.m.) IgE binding (c.p.m.) 158 4544 724 1010 963 1193 865 6370 2309 2980 3918 3889 221 Eukaryotic expression of Phl p T Ball et al A B C Fig (A) Superior IgE binding capacity of insect cell- vs E coli-expressed rPhl p Sera from four grass pollen allergic patients were preincubated with bacterially expressed rPhl p and exposed to dot-blotted bacterial recombinant (PrPhl p 1+) or eukaryotic recombinant Phl p (ErPhl p 1+) PrPhl p 1- and ErPhl p 1- show the IgE binding without preadsorption of sera (B) Inhibition of IgE binding to natural Phl p (nPhl p 1) by insect cell-expressed Phl p Sera from three grass pollen allergic patients (1–3) were tested for IgE reactivity to nitrocellulosedotted eukaryotic recombinant Phl p (ErPhl p 1) and natural Phl p (nPhl p 1) Sera were preadsorbed with BSA (A), natural Phl p (B), or insect cell-expressed Phl p (C) (C) IgE binding capacity of deglycosylated insect cell-expressed Phl p 1.IgE reactivity of 10 sera from grass pollen allergic patients (1–10) to untreated (–) and deglycosylated (+) Phl p is shown ⁄ grass pollen allergens, another family of major grass pollen allergens that exhibit an immunoglobulin-like fold composed almost exclusively of b-sheet structure [29,30] As shown by circular dichroism spectroscopy, Phl p showed also almost exclusively b-sheet secondary structure The results from limited proteolysis combined with mass spectrometry indicated a two domain organization of the protein with a C-terminal portion homologous to group allergens Despite these findings, Phl p and Phl p appear to represent immunologically independent allergens because significant crossreactivity of IgE antibodies was not observed and both proteins belonged to different phylogenetic clusters The analysis of Phl p IgE epitopes using recombinant allergen fragments had indicated the presence 222 of several continuous IgE epitopes, of which the most prominent could be allocated to the C-terminal portion of Phl p [25] We have identified this portion as an intact domain by the limited proteolysis experiment suggesting that intact and folded Phl p domains represent the primary targets for patients’ IgE antibodies The latter assumption is also supported by the fact that only an incomplete inhibition of IgE reactivity to the Phl p allergen could be obtained after preincubation of patients’ sera with small recombinant protein fragments suggesting the importance of conformational IgE epitopes [25] Therefore, we further tested the importance of structural integrity on IgE binding capacity and allergenic activity of Phl p by comparing insect cell-expressed FEBS Journal 272 (2005) 217–227 ª 2004 FEBS T Ball et al Fig Induction of basophil histamine release with recombinant Phl p preparations Granulocytes from patients (A, B) allergic to grass pollen were incubated with various concentrations (x-axis) of bacterial rPhl p (PrPhl p 1) and eukaryotic rPhl p The percentage of histamine released into the supernatant is displayed on the y-axis Phl p with E coli-expressed Phl p This comparison revealed a higher IgE-binding capacity and more pronounced allergenic activity of insect cell-expressed rPhl p compared to E coli-expressed rPhl p 1, as determined by basophil activation assays Deglycosylation experiments demonstrate that the higher IgEbinding capacity and increased allergenic activity of insect cell-expressed Phl p is due to intact structural integrity rather than to IgE recognition of carbohydrate moieties In fact, we found that deglycosylation rather increased the IgE binding capacity of Phl p This may be due to the exposure of protein epitopes by removing carbohydrates from a potentially hyperglycosylated insect cell-expressed Phl p On the other hand, it is unlikely that the authentic, plant-derived carbohydrates represent per se important targets for patients’ IgE antibodies because insect cell-expressed Phl p showed almost identical IgE reactivity as natural Phl p FEBS Journal 272 (2005) 217–227 ª 2004 FEBS Eukaryotic expression of Phl p The importance of native tertiary structure for the IgE recognition of Phl p seems to be a general principle applicable to the most common respiratory allergens For example, it has been demonstrated that disruption of native structure by fragmentation has led to a strong reduction of the IgE binding capacity and allergenic activity of the major birch pollen allergen, Bet v [31], the cross-reactive calcium-binding allergens Aln g [32] and Phl p [33], the major mite allergen Der p [34], and the major bovine allergen, Bos d [35] We consider the possibility that respiratory allergens may predominantely contain conformational IgE epitopes as important for at least three reasons First, it indicates that respiratory sensitization occurs preferentially against intact and folded protein antigens which elute from respirable particles (e.g pollen, mite faeces, animal dander) Second, our study emphasizes that it is important to choose an optimal expression strategy for obtaining native properly folded recombinant allergens which closely mimic the immunological properties of the natural counterparts for diagnostic purposes Finally, and perhaps most importantly, IgE recognition of mainly conformational epitopes has important implications for the design of safe allergy vaccines with reduced allergenic activity Disruption of the native structure of respiratory allergens allows for the maintenance of important T cell epitopes of a given allergen and simultaneously preserves sequences relevant for the induction of protective antibody responses [36] Controlled reduction of the fold of respiratory allergens by recombinant DNA technology or synthetic peptide chemistry thus seems to be a generally applicable strategy for the generation of recombinant allergy vaccines with reduced allergenic activity [37] Experimental procedures Materials, patients’ sera and antibodies The Sf9 cell line was purchased from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) After informed consent was obtained, sera were collected from Phl p allergic patients, following the Helsinki guidelines Allergenic patients were characterized by case history, skin prick test, and the demonstration of allergen-specific serum IgE antibodies by RAST (Pharmacia Diagnostics, Uppsala, Sweden) Natural group grass pollen allergens from Timothy grass (nPhl p 1) and rye grass (nLol p 1) were purified as described [38] Purified E coli-expressed rPhl p 1, rPhl p and rBet v were obtained from BIOMAY (Vienna, Austria) A rabbit antiPhl p antiserum was obtained by immunizing rabbits with purified rPhl p using complete Freunds’ adjuvant (Charles 223 Eukaryotic expression of Phl p River, Kissleg, Germany) Alkaline phosphatase-conjugated goat anti-(rabbit Ig) and rabbit anti-(mouse Ig) serum was purchased from JacksonImmunoResearch Laboratories (West Grove, PA, USA), a mouse monoclonal anti-Hexahistidine antibody was obtained from Dianova (Hamburg, Germany) The 125I-labeled anti-human IgE immunoglobulins were purchased from Pharmacia Diagnostics Construction of recombinant baculovirus The Phl p 1-encoding cDNA [16] was PCR amplified and cloned into the BamHI and KpnI restriction sites of the pBacPAK8 vector (Clontech Inc., Palo Alto, CA, USA), containing the baculovirus-derived ecdysteroid UDPglucosyltransferase signal peptide [39] for enhanced secretion of the recombinant protein into the culture supernatant and a C-terminal His6-tag The pBacPAK8 construct was confirmed by DNA sequencing and cotransfected with the linearized pBacPAK6 viral DNA (Clontech Inc., Palo Alto, CA, USA) into Sf9 insect cells The clones with the highest level of protein secretion were chosen by Western blotting for virus amplification Expression and purification of rPhl p from baculovirus-infected insect cells The expression of rPhl p in insect cells was optimized by infecting Sf9 cells with different amounts of virus and by expression for various periods Aliquots of the culture supernatants and cell pellets were analyzed by SDS ⁄ PAGE and immunoblotting with a rabbit anti-Phl p antiserum and a monoclonal anti-hexahistidine antibody Rabbit antirPhl p Igs were detected with an alkaline phosphatase (AP)-labeled goat anti-(rabbit Ig) antiserum Bound antihexahistidine Igs were detected with AP-labeled rabbit antimouse Igs Optimal expression of Phl p was achieved by infection of · 106 Sf9 cells per mL with recombinant baculovirus at a multiplicity of infection (MOI) of with culturing in L spinner ⁄ flasks in Insect-Xpress medium (BioWhittaker Inc., Walkersville, MD, USA) containing 2% fetal bovine serum At day two postinfection, supernatants were separated by centrifugation (8000 g, °C, 30 min) and dialyzed against start buffer [50 mm sodium phosphate (pH 8.0), 300 mm NaCl] at °C overnight Insect cell-expressed rPhl p was purified using Ni-nitrilotriacetic acid superflow matrix (Qiagen, Hilden, Germany) under nondenaturing conditions by stepwise elution with increasing (20–250 mm) imidazole concentrations The eluted samples were dialyzed against 10 mm Tris HCl (pH 8.0), 100 mm NaCl and concentrated by Centricon ultrafiltration (Millipore, Bedford, MA, USA) Protein concentrations of purified samples were estimated using BCA reagent (Pierce Chemicals, Rockford, IL, USA) and UV absorption at 280 nm The molar extinc- 224 T Ball et al tion coefficient of the protein was calculated from the tyrosine and tryptophan content [40] Mass spectrometry Purified baculovirus-expressed Phl p was analyzed by LC-MS (Liquid Chromatography-Mass Spectrometry) using a VYDAC (Hesperia, CA, USA) C4 column on a Waters HPLC 2690 (Waters Corp., Milford, MA, USA) which fed into an electrospray Thermo Finnigan LCQ quadrupole ion-trap mass spectrometer (ThermoQuest Inc., San Jose, CA, USA) Limited proteolysis followed by LC-MS Purified baculovirus-expressed Phl p was subjected to limited proteolysis by trypsin, Arg-C, Lys-C, Asp-N and Glu-C Ten microliter aliquots containing 18 lm rPhl p were digested with protease in the following ratios : 5, : 15, : 50, : 150 and : 500 (protease:rPhl p 1; w ⁄ w) for h at room temperature Proteolysis was halted by freezing at )70 °C Aliquots were analyzed by SDS ⁄ PAGE Samples showing multiple bands, indicative for successful partial digest were then selected for further investigation by LC-MS A Vydac C18 column was used on a Waters HPLC 2690 (Waters Corp.) followed by electrospray into a Thermo Finnigan LCQ Ion Trap Mass Spectrometer (ThermoQuest Inc.) The spectra were deconvoluted using Thermo Finnigan’s xcalibur software and the spectra were also verified by hand calculations of charge states The proteolytic fragments were identified using the paws software program (version 8.1.1, for Macintosh; Genomic SolutionsTM , Ann Arbor, MI, USA; http://bioinformatics.genomicsolutions.com/paws.html) Detection of glycoproteins and deglycosylation treatment Purified E coli- and insect cell-expressed Phl p proteins were separated by SDS ⁄ PAGE and transferred to nitrocellulose followed by detection of sugars using a DIG glycan ⁄ protein double labeling kit (Boehringer Mannheim GmbH, Mannheim, Germany) Briefly, glycans were oxidized to produce aldehyde groups allowing the covalent attachment of the steroid hapten digoxigenin (DIG) The latter was then detected using horseradish peroxidase-conjugated anti-digoxigenin Igs yielding a blue color reaction Creatinase and bacterial rPhl p were used as nonglycosylated controls which were stained by labeling of amino groups with fluorescein and detection with alkaline phosphatase-conjugated anti-fluorescein Igs (Boehringer) giving a brown color reaction Enzymatic deglycosylation was performed with glutathione S-transferase (GST)–PNGase F (Hampton Research, CA, USA) by using reaction ratios of GST–PNGase F:glycoprotein of : Deglycosylation was carried out for 15 h at FEBS Journal 272 (2005) 217–227 ª 2004 FEBS T Ball et al room temperature in 10 mm Tris (pH ¼ 8.0), 100 mm NaCl The GST–PNGase F was removed from the target protein using glutathione-Sepharose Circular dichroism (CD) measurements All recombinant proteins were subjected to CD analysis to access stability and secondary structure composition Far UV-CD spectra were collected on a Jasco-J720 spectropolarimeter (Jasco, Tokyo, Japan) at room temperature, at final protein concentrations of 10–25 lm in either 0.5 or 0.01 mm path-length quartz cuvettes The molar ellipticity was calculated according to [h] ¼ ⁄ 10cl, where h is the ellipticity, l is the cuvette path-length in cm and c is the protein concentration in molỈL)1 Three independent measurements were recorded and averaged for each spectral point in all experiments Thermal denaturation was monitored in the range of 20 °C to 90 °C The reversibility of the unfolding process was checked by measuring the CD signal upon cooling to the starting temperature Phylogenetic analysis of the relationships among group and group 2/3 allergens from various grass species Multiple alignment of sequences homologous to Phl p as identified by a BLAST search [41] was generated by using clustalx [42] A distance matrix among sequences was constructed using the protdist program of the phylip 3.6a2 [43,44] package The distance matrix was used as input to the KITSCH program from the phylip package for the construction of a phylogenetic tree This program implements the Fitch–Margoliash least-squares methods with the assumption of an evolutionary clock SDS/PAGE analysis and immunoblotting Samples were resolved on 12.5% polyacrylamide gels under reducing conditions Proteins were stained with Coomassie blue or transferred to nitrocellulose membranes [45] Blotted proteins were probed with sera from Phl p allergic patients¢, anti-His Igs, rabbit anti-Phl p antiserum and the corresponding preimmune serum Patients’ bound IgE antibodies were detected with 125I-labeled anti-human IgE [46], anti-His Igs with AP-conjugated rabbit anti-(mouse Ig), and bound rabbit Igs with an AP-conjugated goat anti(rabbit IgG) serum [47] IgE-binding capacity and cross-reactivity of allergens as determined by nondenaturing dot-blot experiments The IgE reactivity of the recombinant Phl p molecules was determined by dot-blot under conditions of antigen FEBS Journal 272 (2005) 217–227 ª 2004 FEBS Eukaryotic expression of Phl p excess [7] Three micrograms of the purified recombinant proteins were dotted onto nitrocellulose strips and incubated with sera from Phl p allergic patients Bound IgE antibodies were detected with 125I-labeled anti-(human IgE) Igs (Pharmacia) and quantified by c-counting (Wallac, LKB, Turku, Finland) [25] IgE inhibition experiments under conditions of antigen excess were performed as described [7] Patients’ sera were incubated with lgỈmL)1 of each allergen (or the same amount of BSA for control purposes) overnight at °C The next day, preincubated sera were exposed to lg of nitrocellulose-dotted natural Phl p 1, rPhl p 2, E coli and baculovirus expressed Phl p Bound serum IgE was detected as described for the IgE immunoblotting and quantified by c-counting [25] Basophil activation experiments Granulocytes were isolated from heparinized blood samples of individuals allergic to Phl p by dextran sedimentation The capacity of E coli- and insect cell-expressed Phl p to induce basophil degranulation was tested by incubation of granulocytes with various concentrations of the purified proteins and by measuring histamine released into the cell-free supernatant by radioimmunoassay (Immunotech, Marseille, France) Histamine release was measured in triplicates and expressed as a percentage of total histamine determined after cell lysis, as described [48] Up-regulation of CD203c expression on basophils after allergen exposure was measured as described [26] Acknowledgements We acknowledge the skillful technical assistance of Miriam Gulotta regarding the circular dichroism experiments This study was supported by grants Y078GEN, F01801, F01809, J1835 and J2122 of the Austrian Science Fund, by the CeMM Project of the Austrian Academy of Sciences, and by a research grant from BIOMAY, Vienna, Austria References Wills-Karp M, Santeliz J & Karp CL (2001) The germless theory of allergic disease: revisiting the hygiene hypothesis Nat Rev Immunol 1, 69–75 Wuthrich B, Schindler C, Leuenberger P & Ackermannă Liebrich P (1995) Prevalence of atopy and pollinosis in the adult population of Switzerland (SAPALDIA study) Swiss Study on Air Pollution and 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without preadsorption... p (nLol p 1) , natural Phl p (nPhl p 1) , eukaryotic recombinant Phl p (ErPhl p 1) and bacterial recombinant Phl p (PrPhl p 1) B and C represent immunoblots probed with rabbit anti- (Phl p Ig) antiserum... coli (PrPhl p 1) and baculovirus-infected insect cells (ErPhl p 1) PrPhl p ErPhl p Patient number IgE binding (c .p. m.) IgE binding (c .p. m.) 15 8 4544 724 10 10 963 11 93 865 6370 2309 2980 3 918 3889