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].. More-over, insect cell-expressed
Trang 1expression of the major Timothy grass pollen allergen,
Phl p 1
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
1 Department of Pathophysiology, Center for Physiology and Pathophysiology, Medical University of Vienna, Austria
2 Albert Einstein College of Medicine, Department of Biochemistry, NY, USA
3 Pharmacia Diagnostics, Freiburg, Germany
4 Allergopharma KG, Reinbek, Germany
5 Division of Hematology, Department of Internal Medicine I, Medical University of Vienna, Austria
Type I allergy is an IgE-mediated hypersensitivity
dis-ease affecting more than 25% of the population [1,2]
Grass pollen allergens belong to the group of most
fre-quently 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 1 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]
Keywords
allergen; allergy; epitope; eukaryotic
expression; Phl p 1
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 1 40 400 5130
Tel: +43 1 40 400 5108
E-mail: rudolf.valenta@meduniwien.ac.at
(Received 6 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 1 grass pollen allergens, a family of highly cross-reactive allergens present in all grass species We report the eukaryotic expression of the group 1 aller-gen 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 2⁄ 3 grass pollen allergens Circular dichroism analysis demonstrates that insect cell-expressed rPhl p 1 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 1 is demonstrated by the superior IgE recognition of insect-cell expressed Phl p 1 compared to Escherichia coli-expressed Phl p 1 More-over, insect cell-expressed Phl p 1 induces potent histamine release and leads to strong up-regulation of CD203c in basophils from grass pollen allergic patients Deglycosylated Phl p 1 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 1 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
Abbreviations
PrPhl p 1, prokaryotic recombinant Phl p 1; ErPhl p 1, eukaryotic recombinant Phl p 1; GST, glutathione S-transferase.
Trang 2They 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 1 allergens have been
demonstra-ted 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
sub-cellular particles allows them to reach the deeper
air-ways and may explain the frequent occurrence of
heavy asthma attacks after rainfalls [12,13]
cDNAs coding for group 1 allergens from several
grasses have been isolated and showed high sequence
similarity [14–20] The recombinant group 1 allergen
from Timothy grass, rPhl p 1, expressed in Escherichia
coli contained many of the T cell epitopes of natural
group 1 allergens and cross-reacted with the naturally
occuring isoallergens from Timothy grass and other
grass species [6,21,22] However, several
post-transla-tional modifications (e.g glycosylation, occurrence of
hydroxyprolines) and the formation of disulphide
bonds have been described for group I allergens
[23,24] These modifications do not occur when
pro-teins are expressed in prokaryotic expression systems
and hence E coli-expressed group 1 allergens exhibit
impaired structural and immunological properties The
importance of conformational epitopes for IgE
recog-nition of group I allergens is highlighted by IgE
com-petition experiments using recombinant fragments of
Phl p 1 representing continuous IgE epitopes as even
a mixture of several major IgE-epitope-containing
rPhl p 1 fragments does not completely inhibit IgE
binding to intact Phl p 1 [25]
To obtain properly folded rPhl p 1, the cDNA
cod-ing for the mature allergen was expressed in
baculo-virus-infected insect cells An expression strategy was
chosen which led to the secretion of the recombinant
allergen into the cell culture supernatants rPhl p 1
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 1 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 1 were compared with those
of E coli-expressed and natural Phl p 1 by competition
studies performed under native conditions and the
importance of glycosylation for IgE reactivity was
examined by enzymatic deglycosylation of insect
cell-expressed Phl p 1 Finally, the biological activity of
insect cell-expressed and E coli-expressed Phl p 1 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 1 is related to increased IgE reactivity and allergenic activity is discussed as a general feature of respiratory allergens and has rele-vance for the development of allergy vaccines which are based on the reduction of allergen fold
Results
Comparison of natural and recombinant group 1 grass pollen allergens
Although the majority of rPhl p 1 was detected in the insoluble pellet fraction of infected insect cells, up to 0.75 mgÆL)1of soluble rPhl p 1 could be purified from the culture supernatant by Ni2+-affinity chromato-graphy under nondenaturing conditions Purified insect cell-expressed Phl p 1 migrated at slightly higher molecular mass than the natural Phl p 1, E coli-expressed Phl p 1 and the Phl p 1-homologous allergen from rye grass (Lol p 1) (Fig 1A) Insect cell-expressed Phl p 1 as well as natural group 1 allergens (nPhl p 1 and nLol p 1) reacted with a rabbit antiserum raised against purified E coli-expressed Phl p 1 (Fig 1B) but not with the corresponding preimmune serum (Fig 1C) A band of approximately double the molecular mass as the purified allergens, possibly rep-resenting a dimer, was detected in the bacterial and insect cell-expressed Phl p 1 and, to a lower degree, in the nPhl p 1 preparations
Biochemical and biophysical characterization
of insect-cell expressed Phl p 1 The molecular mass of insect cell-expressed Phl p 1 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 deter-mined molecular mass is attributed to glycosylation Limited proteolysis in combination with mass spectro-metry 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 struc-ture, 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 1 by limited proteolysis, one comprising C78–K118 and a second domain spanning from
Trang 3K147–K241 (data not shown) The latter corresponds
to the region homologous to group 2 allergens To
confirm that the difference between the calculated
and determined molecular mass is due to
glycosyla-tion of the insect cell-expressed Phl p 1, glycan
detec-tion was performed (Fig 2A) Nitrocellulose-blotted
insect cell-expressed Phl p 1, but not E coli-expressed
Phl p 1 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 1 as visualized by
SDS⁄ PAGE (Fig 2B)
Insect cell-expressed Phl p 1 represents a folded
protein with considerable b-sheet structure that
crystallizes as thin plates
Insect cell- and E coli-expressed Phl p 1 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 1
(ErPhl p 1) suggested the presence of substantial
anti-parallel b-sheet, while the CD spectrum for the Phl p 1
expressed in bacteria (prokaryotic: PrPhl p 1) indicated
a considerable amount of unordered structure The
characteristics of the CD spectrum of insect
cell-expressed Phl p 1 indicates structural similarity with
Phl p 2, an almost exclusively b-sheet containing
aller-gen with 43% sequence identity to the C-terminal third
of Phl p 1 [29,30] Thermal denaturation of insect
cell-expressed Phl p 1 was monitored by far-UV CD in the
range of 20C to 90 and showed an irreversible
unfolding transition, with a melting point of 42 C
(data not shown) Only the properly folded insect cell-expressed but not the E coli-expressed Phl p 1 afforded crystals These crystals belonged to the ortho-rhombic space group P212121 and diffracted X-rays to
a resolution of 2.9 A˚ (Fig 4)
Phl p 1 and Phl p 2 belong to different clusters of proteins as determined by phylogenetic analysis Amino acid sequences of seven group 1 pollen aller-gens and four group 2⁄ 3 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 1 and Cyn d 1, a second consisting
of Lol p 3, Dag g 3, Lol p 1, Phl p 1, Ory s 1 and Tri a 3 and a third cluster including Lol p 2, Hol l 1, Phl p 2 and Pha a 1 Although Phl p 1 and Phl p 2 are derived from Phleum pratense and share high sequence identity, the phylogenetic analysis shows that they are less related to each other than group 1 and group 2⁄ 3 allergens from different species
Insect cell-expressed Phl p 1 contains the IgE epitopes of natural Phl p 1
A comparison of the IgE binding capacity of E coli-and insect cell-expressed Phl p 1 under nondenaturing conditions in a dot-blot assay showed that insect cell-expressed Phl p 1 was more potent than the E coli-derived 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 1 completely inhibited IgE binding to the very same pro-tein, but not to the insect cell-expressed Phl p 1 An
Fig 1 Coomassie staining and immunoreactivity of purified recombinant and natural group 1 allergens (A) Coomassie stained SDS ⁄ PAGE containing natural Lol p 1 (nLol p 1), natural Phl p 1 (nPhl p 1), eukaryotic recombinant Phl p 1 (ErPhl p 1) and bacterial recombinant Phl p 1 (PrPhl p 1) B and C represent immunoblots probed with rabbit anti-(Phl p 1 Ig) antiserum and the corresponding preimmune serum, respect-ively.
Trang 4almost complete reduction of IgE binding to insect
cell-expressed Phl p 1 was only observed for serum of
patient 2, whereas considerable IgE reactivity of sera
1, 3 and 4 to insect cell-expressed Phl p 1 was observed
despite preincubation with an excess of E coli-expressed Phl p 1
Whether insect cell-expressed Phl p 1 contains the IgE epitopes of a natural Phl p 1 preparation was inves-tigated by IgE competition experiments (Fig 6B) Pre-incubation of sera from grass pollen allergic patients with insect cell-expressed Phl p 1 led to a strong or complete inhibition of IgE reactivity to natural Phl p 1 (Fig 6B)
Next we studied the influence of glycosylation on the IgE binding capacity of insect cell-expressed Phl p 1 (Fig 6C) Five out of 10 patients showed stronger IgE reactivity to deglycosylated insect cell-expressed Phl p 1 than to the untreated protein (Fig 6C, 1, 2, 3, 5, 7) Three patients exhibited com-parable IgE reactivity to both protein forms (Fig 6C,
#4, 9, 10) and two sera reacted stronger with the gly-cosylated allergen version (Fig 6C, 6, 8) Finally, we studied the possible presence of cross-reactive IgE epi-topes between Phl p 1 and Phl p 2 Preincubation of sera from pollen allergic patients with insect cell-expressed, E coli-expressed Phl p 1 or an unrelated control allergen (birch pollen allergen, rBet v 1) had
no effect on IgE binding to rPhl p 2 (data not shown)
Allergenic activity of insect cell-expressed Phl p 1 The allergenic activity of insect cell-expressed Phl p 1 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 cell-expressed Phl p 1 (Fig 7A,B) In both patients, insect cell-expressed Phl p 1 was more potent, inducing hista-mine release at lower concentrations (10)3lgÆmL)1) than E coli-expressed Phl p 1 (10)2lgÆmL)1) Meas-urement of CD203c expression on blood basophils of three Phl p 1 allergic patients confirmed these results Incubation with insect cell-expressed Phl p 1 always led to stronger upregulation of CD203c than incuba-tion with E coli-expressed Phl p 1 (data not shown)
A
B
Fig 2 Biochemical and biophysical characterization of insect cell-expressed Phl p 1 (A) Glycan detection Nitrocellulose blotted insect cell-expressed rPhl p 1 (ErPhl p 1), rPhl p 1 expressed in bacteria (PrPhl p 1), Creatinase and marker proteins (M) were sim-ultaneously 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 1 before (ErPhl p 1-) and after (ErPhl p 1 +) enzymatic deglycosyla-tion Lane M: Molecular mass marker.
Trang 5Phl p 1 represents one of the most important respiratory
allergens known to date As Phl p 1 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 1 was obtained
as a glycoprotein The seemingly correct folding of
insect cell-expressed Phl p 1 is demonstrated by the
fol-lowing experiments: insect cell-expressed Phl p 1 but
not E coli-expressed Phl p 1 exhibited a secondary
structure consisting mainly of b-sheets when analyzed
by CD spectroscopy Furthermore, only insect
cell-expressed Phl p 1 grew diffraction quality crystals
and thus will yield the three-dimensional structure of this allergen
Phl p 1 belongs to the group 1 family of highly cross-reactive grass pollen allergens The C-terminal domains
of these allergens display sequence similarity to group
Fig 3 Comparison of E coli- and insect cell-expressed Phl p 1 by
circular dichroism spectroscopy Far-UV CD spectra of E.coli- (grey)
and insect cell-expressed Phl p 1 (black), expressed as mean
resi-due ellipticity (y-axis), were recorded at 20 C in the wave length
range displayed on the x-axis.
Fig 4 Crystal growth of insect cell-expressed Phl p 1.Phl p 1
crys-tallizes as thin plates of 0.35 · 0.35 · 0.15 mm.
Fig 5 Analysis of the sequence and phylogenetic relationship among group 1 and group 2 ⁄ 3 allergens from various grass spe-cies A phylogenetic tree was reconstructed on the basis of amino-acid sequences of group 1 (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 pra-tense) and group 2 ⁄ 3 allergens (Lol p 3: Lolium perenne, Dag g 3: Dactylis glomerata, Lol p 2: Lolium perenne, Tri a 3: Triticus aesti-vum, Phl p 2: Phleum pratense) using the PROTDIST and KITSCH program of the PHYLIP package.
Table 1 Comparison of the IgE binding capacity of E coli- and insect cell-expressed Phl p 1 IgE reactivity to recombinant Phl p 1, expressed in E coli (PrPhl p 1) and baculovirus-infected insect cells (ErPhl p 1).
Patient number
IgE binding (c.p.m.) IgE binding (c.p.m.)
Trang 62⁄ 3 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 1 showed also almost exclusively b-sheet
secon-dary structure The results from limited proteolysis
com-bined with mass spectrometry indicated a two domain
organization of the protein with a C-terminal portion
homologous to group 2 allergens Despite these findings,
Phl p 1 and Phl p 2 appear to represent
immunological-ly independent allergens because significant
crossreactiv-ity of IgE antibodies was not observed and both
proteins belonged to different phylogenetic clusters
The analysis of Phl p 1 IgE epitopes using
recom-binant allergen fragments had indicated the presence
of several continuous IgE epitopes, of which the most prominent could be allocated to the C-terminal por-tion of Phl p 1 [25] We have identified this porpor-tion
as an intact domain by the limited proteolysis experi-ment suggesting that intact and folded Phl p 1 domains represent the primary targets for patients’ IgE antibodies The latter assumption is also suppor-ted by the fact that only an incomplete inhibition of IgE reactivity to the Phl p 1 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 struc-tural integrity on IgE binding capacity and allergenic activity of Phl p 1 by comparing insect cell-expressed
A
B
C
Fig 6 (A) Superior IgE binding capacity of insect cell- vs E coli-expressed rPhl p 1 Sera from four grass pollen allergic patients were prein-cubated with bacterially expressed rPhl p 1 and exposed to dot-blotted bacterial recombinant (PrPhl p 1+) or eukaryotic recombinant Phl p 1 (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 1 (nPhl p 1) by insect cell-expressed Phl p 1 Sera from three grass pollen allergic patients (1–3) were tested for IgE reactivity to nitrocellulose-dotted eukaryotic recombinant Phl p 1 (ErPhl p 1) and natural Phl p 1 (nPhl p 1) Sera were preadsorbed with BSA (A), natural Phl p 1 (B), or insect cell-expressed Phl p 1 (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 1 is shown.
Trang 7Phl p 1 with E coli-expressed Phl p 1 This
compar-ison revealed a higher IgE-binding capacity and more
pronounced allergenic activity of insect cell-expressed
rPhl p 1 compared to E coli-expressed rPhl p 1, as
determined by basophil activation assays
Deglycosy-lation experiments demonstrate that the higher
IgE-binding capacity and increased allergenic activity of
insect cell-expressed Phl p 1 is due to intact structural
integrity rather than to IgE recognition of
carbo-hydrate moieties In fact, we found that
deglycosyla-tion rather increased the IgE binding capacity of
Phl p 1 This may be due to the exposure of protein
epitopes by removing carbohydrates from a
poten-tially hyperglycosylated insect cell-expressed Phl p 1
On the other hand, it is unlikely that the authentic,
plant-derived carbohydrates represent per se
import-ant targets for patients’ IgE import-antibodies because insect
cell-expressed Phl p 1 showed almost identical IgE
reactivity as natural Phl p 1
The importance of native tertiary structure for the IgE recognition of Phl p 1 seems to be a general princi-ple applicable to the most common respiratory aller-gens 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 1 [31], the cross-reactive calcium-binding allergens Aln g 4 [32] and Phl p 7 [33], the major mite allergen Der p 2 [34], and the major bovine allergen, Bos d 2 [35] We consider the possibility that respiratory aller-gens may predominantely contain conformational IgE epitopes as important for at least three reasons First, it indicates that respiratory sensitization occurs preferen-tially 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 recogni-tion of mainly conformarecogni-tional 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 main-tenance of important T cell epitopes of a given allergen and simultaneously preserves sequences relevant for the induction of protective antibody responses [36] Con-trolled reduction of the fold of respiratory allergens by recombinant DNA technology or synthetic peptide chemistry thus seems to be a generally applicable strat-egy 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 Collec-tion of Microorganisms and Cell Cultures (Braunschweig, Germany) After informed consent was obtained, sera were collected from Phl p 1 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 1 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 2 and rBet v 1 were obtained from BIOMAY (Vienna, Austria) A rabbit anti-Phl p 1 antiserum was obtained by immunizing rabbits with purified rPhl p 1 using complete Freunds’ adjuvant (Charles
Fig 7 Induction of basophil histamine release with recombinant
Phl p 1 preparations Granulocytes from patients (A, B) allergic to
grass pollen were incubated with various concentrations (x-axis) of
bacterial rPhl p 1 (PrPhl p 1) and eukaryotic rPhl p 1 The percentage
of histamine released into the supernatant is displayed on the y-axis.
Trang 8River, Kissleg, Germany) Alkaline phosphatase-conjugated
goat anti-(rabbit Ig) and rabbit anti-(mouse Ig) serum was
pur-chased from JacksonImmunoResearch Laboratories (West
Grove, PA, USA), a mouse monoclonal anti-Hexahistidine
antibody was obtained from Dianova (Hamburg, Germany)
The125I-labeled anti-human IgE immunoglobulins were
pur-chased 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
UDPgluco-syltransferase signal peptide [39] for enhanced secretion of
the recombinant protein into the culture supernatant and a
C-terminal His6-tag The pBacPAK8 construct was
con-firmed by DNA sequencing and cotransfected with the
line-arized 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 1 from
baculovirus-infected insect cells
The expression of rPhl p 1 in insect cells was optimized by
infecting Sf9 cells with different amounts of virus and by
expression for various periods Aliquots of the culture
sup-ernatants and cell pellets were analyzed by SDS⁄ PAGE and
immunoblotting with a rabbit anti-Phl p 1 antiserum and
a monoclonal hexahistidine antibody Rabbit
anti-rPhl p 1 Igs were detected with an alkaline phosphatase
(AP)-labeled goat (rabbit Ig) antiserum Bound
hexahistidine Igs were detected with AP-labeled rabbit
anti-mouse Igs
Optimal expression of Phl p 1 was achieved by infection
of 2· 106 Sf9 cells per mL with recombinant baculovirus
at a multiplicity of infection (MOI) of 5 with culturing in
3 L spinner⁄ flasks in Insect-Xpress medium (BioWhittaker
Inc., Walkersville, MD, USA) containing 2% fetal bovine
serum At day two postinfection, supernatants were
separ-ated by centrifugation (8000 g, 4C, 30 min) and dialyzed
against start buffer [50 mm sodium phosphate (pH 8.0),
300 mm NaCl] at 4C overnight Insect cell-expressed
rPhl p 1 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
con-centrated 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-tion coefficient of the protein was calculated from the tyro-sine and tryptophan content [40]
Mass spectrometry Purified baculovirus-expressed Phl p 1 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 1 was subjected to lim-ited proteolysis by trypsin, Arg-C, Lys-C, Asp-N and Glu-C Ten microliter aliquots containing 18 lm rPhl p 1 were diges-ted with protease in the following ratios 1 : 5, 1 : 15, 1 : 50,
1 : 150 and 1 : 500 (protease:rPhl p 1; w⁄ w) for 1 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 xcali-bursoftware and the spectra were also verified by hand cal-culations 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 1 proteins were separated by SDS⁄ PAGE and transferred to nitrocellu-lose followed by detection of sugars using a DIG glycan⁄ pro-tein double labeling kit (Boehringer Mannheim GmbH, Mannheim, Germany) Briefly, glycans were oxidized to pro-duce aldehyde groups allowing the covalent attachment of the steroid hapten digoxigenin (DIG) The latter was then detected using horseradish peroxidase-conjugated anti-digo-xigenin Igs yielding a blue color reaction Creatinase and bacterial rPhl p 1 were used as nonglycosylated controls which were stained by labeling of amino groups with fluo-rescein and detection with alkaline phosphatase-conjugated anti-fluorescein Igs (Boehringer) giving a brown color reac-tion Enzymatic deglycosylation was performed with gluta-thione S-transferase (GST)–PNGase F (Hampton Research,
CA, USA) by using reaction ratios of GST–PNGase F:glyco-protein of 1 : 2 Deglycosylation was carried out for 15 h at
Trang 9room temperature in 10 mm Tris (pH¼ 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
spectropola-rimeter (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
ellip-ticity, l is the cuvette path-length in cm and c is the protein
concentration in molÆL)1 Three independent measurements
were recorded and averaged for each spectral point in all
experiments Thermal denaturation was monitored in the
range of 20C 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 1 and group 2/3 allergens from various
grass species
Multiple alignment of sequences homologous to Phl p 1 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]
Blot-ted proteins were probed with sera from Phl p 1 allergic
patients¢, anti-His Igs, rabbit anti-Phl p 1 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 1 molecules
was determined by dot-blot under conditions of antigen
excess [7] Three micrograms of the purified recombinant proteins were dotted onto nitrocellulose strips and incuba-ted with sera from Phl p 1 allergic patients Bound IgE antibodies were detected with125I-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 5 lgÆmL)1 of each allergen (or the same amount of BSA for control purposes) overnight at 4C The next day, preincubated sera were exposed to 3 lg of nitrocellulose-dotted natural Phl p 1, rPhl p 2, E coli and baculovirus expressed Phl p 1 Bound serum IgE was detec-ted 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 1 by dextran sedimentation The capacity of E coli- and insect cell-expressed Phl p 1 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 trip-licates 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
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