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*
Institute of General and Experimental Pathology, Vienna General Hospital, University of Vienna, Vienna, Austria;
Institute of Medical Physics and Biophysics, University of Münster, Münster, Germany;
European Molecular Biology Laboratory, Heidelberg, Germany;
§
Molecular Structure Division, National Institute for Medical Research, London, United Kingdom; and
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Department of Internal Medicine I, Division of Hematology, Vienna General Hospital, University of Vienna, Vienna, Austria
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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50% of allergic patients are sensitized against grass
pollen allergens. cDNAs coding for two isoforms and four fragments of a
major timothy grass (Phleum pratense) pollen allergen,
Phl p 6, were isolated by IgE immunoscreening from a pollen expression
cDNA library. Recombinant Phl p 6 (rPhl p 6), an acidic protein of 11.8
kDa, was purified to homogeneity as assessed by mass spectrometry and
exhibited almost exclusive 
-helical secondary structure as
determined by circular dichroism spectroscopy. Phl p 6 reacted with
serum IgE from 75% of grass pollen-allergic patients
(n = 171). IgE binding experiments with rPhl p 6
fragments indicated that the N terminus of the allergen is required for
IgE recognition. Purified rPhl p 6 elicited dose-dependent basophil
histamine release and immediate type skin reactions in patients
allergic to grass pollen. A rabbit antiserum raised against purified
rPhl p 6 identified it as a pollen-specific protein that, by immunogold
electron microscopy, was localized on the polysaccharide-containing
wall-precursor bodies (P-particles). The association of Phl p 6 with
P-particles may facilitate its intrusion into the deeper airways and
thus be responsible for the high prevalence of IgE recognition of Phl p
6. Recombinant native-like Phl p 6 can be used for in vitro as well as
in vivo diagnoses of grass pollen allergy, whereas N-terminal deletion
mutants with reduced IgE binding capacity may represent candidates for
immunotherapy of grass pollen allergy with a low risk of anaphylactic
side effects. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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40% of allergic individuals suffer symptoms after contact with
grass pollen, research has concentrated on the characterization of
relevant grass pollen allergens by protein and immunochemical methods
(3). Although groups of major allergens have been
identified as cross-reactive moieties that occur in most grass species
(4), nothing was known concerning their nature and
biological functions. The recent application of molecular biological techniques to allergen characterization has revealed the primary structures of allergens and facilitated the production of recombinant allergens for diagnostic and therapeutic purposes (5). Components of the plant cytoskeleton (e.g., profilin) (6) as well as calcium-binding pollen proteins (7) have been identified as relevant allergens. The fact that allergic patients exhibit immediate type reactions upon contact with various unrelated allergen sources thus can be explained by the cross-reactivity of their IgE Abs with ubiquitous allergens. Evidence that group 1 grass pollen allergens belong to a family of cell wall-loosening proteins (expansins) (8) and that grass group 5 allergens may possess RNase activity (9) has restimulated ideas that the biological function of a given protein may be related to its allergenicity. The recent findings that major grass pollen allergens can either become attached to small-sized particles (e.g., group 1 allergens to diesel exhaust, Ref. 10) or may become airborne as small pollen subcompartments (e.g., group 5 allergens in amyloplasts, Ref. 11) would provide a possible mechanism explaining how certain allergens may be able to reach the deep airways of patients and elicit allergic asthma.
We isolated cDNAs coding for a major timothy grass pollen allergen, Phl p 6. Recombinant Phl p 6 (rPhl p 6) was overexpressed in Escherichia coli and purified to homogeneity; its secondary structure content was determined by circular dichroism (CD)3 analysis. Phl p 6 IgE epitopes and the prevalence of IgE recognition by Phl p 6 among patients allergic to grass pollen were studied. The allergenic activity of purified rPhl p 6 was analyzed by basophil histamine release assays and by skin testing of allergic patients. Surprisingly, immunogold electron microscopy localized Phl p 6 predominantly on the polysaccharide-containing wall-precursor bodies (P-particles) of mature timothy grass pollen. This unique feature may possibly indicate that the high rate of sensitization of patients allergic to grass pollen against Phl p 6 is due to P-particle-linked intrusion of Phl p 6 into the deeper airways. The greatly reduced IgE binding capacity of N-terminally truncated rPhl p 6 indicates that N-terminal deletion variants of Phl p 6 may be considered as hypoallergenic candidate molecules for immunotherapy of grass pollen allergy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pollen from timothy grass (Phleum pratense), rye grass (Lolium perenne), rye (Secale cereale), Kentucky blue grass (Poa pratensis), wheat (Triticum sativum), cultivated oat (Avena sativa), and common reed (Phragmites communis) were from Allergon (Välinge, Sweden). Timothy grass seeds were purchased from Austrosaat (Vienna, Austria) and grown for 4 wk to obtain fresh leaves and roots. Patients allergic to grass pollen were characterized as described previously (4). The rabbit anti-celery profilin antiserum (RP1) has been described previously (12). A rabbit anti-rPhl p 6 antiserum was raised against purified rPhl p 6 using CFA (Charles River, Kissleg, Germany). The recombinant timothy grass pollen allergens rPhl p 1, rPhl p 2, and rPhl p 5 were purified as described previously (13). Recombinant timothy grass pollen profilin was purified by poly(L-proline) affinity chromatography (6).
Isolation and characterization of cDNAs coding for Phl p 6 isoforms/fragments
A total of 350 IgE-reactive clones were isolated from an
expression cDNA library constructed from mature timothy grass pollen in
phage 
gt 11 (14). Six cDNAs (c121, c142, c146, c171,
c223, and c233) with sequence homology to a Phl p 6-encoding cDNA
(15) were subcloned into plasmid pUC18 and sequenced
(16, 17). Sequences were analyzed using the McVector
program (Kodak, Rochester, NY). A search for Phl p 6-homologous protein
sequences was done with the FastA program (GCG package)
(18) in the SwissProt database. The sequences of Hol l 5
and Hor v 5 allergens were retrieved from the European Molecular
Biology Laboratory (EMBL) database. A multiple sequence alignment was
produced with ClustalW (19) and edited by hand. The GDE
sequence editor (S. Smith, Harvard University, Cambridge, MA) and
Colormask (J. Thompson, EMBL, Heidelberg, Germany) were used to
color conserved residues with related properties (19).
Protein secondary structure and surface accessibility predictions were
done with the PHD program on the EMBL PredictProtein server
(20).
Mapping of Phl p 6 IgE epitopes, expression, and purification of rPhl p 6
The IgE binding capacity of phage clones expressing Phl p 6 isoforms and fragments was investigated by a plaque lift assay (21). The DNA coding for the mature Phl p 6 allergen was PCR-amplified from the clone 142 DNA and subcloned into the NdeI/EcoRI site of pET-17b. rPhl p 6 was expressed in E. coli BL21 (DE3) in liquid culture. Cells were suspended in 25 mM imidazole (pH 7.4) and 0.1% Triton X-100 and lysed by the addition of lysozyme (20 µg/g cells) for 30 min at room temperature as well as by freeze-thawing cycles. DNA was digested with DNase I (0.1 mg/g cell pellet) for 20 min at room temperature. The protein extract was centrifuged for 20 min at 10,000 x g (Sorvall (Wilmington, DE) RC5C, SS34 rotor) to remove insoluble materials. rPhl p 6 was enriched in a precipitate obtained by the addition of ammonium sulfate (40–60% w/v). The precipitate was dissolved in 10 mM of Tris (pH 6) and dialyzed against this buffer; after centrifugation (20 min at 10,000 x g, Sorvall RC5C, SS34 rotor), the precipitate was applied to a DEAE cellulose-Sepharose column (Pharmacia). Unbound proteins were eluted with 10 mM Tris (pH 6) and 4% v/v isopropanol. Fractions containing >80% pure Phl p 6 were adjusted to a pH of 8 with NaOH and subjected to a second chromatography step on a DEAE-cellulose-Sepharose column. Elution of bound proteins with a 0–0.5 M NaCl gradient at a pH of 8 yielded fractions containing pure rPhl p 6; these fractions were dialyzed against double distilled H2O.
Matrix-assisted laser desorption and ionization (MALDI)-time of flight (TOF) and CD analysis of purified rPhl p 6
Laser desorption mass spectra were acquired in a linear mode
with a TOF Compact MALDI II instrument (Kratos, Manchester, U.K.)
(piCHEM, Graz, Austria). CD spectra were recorded on a Jasco (Tokyo,
Japan) J-710 spectropolarimeter fitted with a Jasco PTC-348WI Peltier
type temperature control system and interfaced with a Fisons Haake
(Paramus, NJ) GH water bath. Far-UV CD spectra were recorded at 20°C
in a 2-mm path-length quartz cuvette (Hellma, Mullheim, Baden, Germany)
at a protein concentration of 7 µM. Thermal denaturation of Phl p 6
was monitored by recording the ellipticity during temperature increase
(50°C/h) at 220 nm. The reversibility of the unfolding process was
checked by measuring the restoration of the CD signal upon cooling
(50°C/h) to the starting temperature (20°C). The fraction of folded
protein was calculated as F = 1 - U, where U =
(
220 -
N)/(U -
N). N is the
ellipticity of the protein in the native state and
U is the ellipticity of the denatured protein.
For rPhl p 6, U was assumed to be equal to
220 at 85°C and N
was assumed to be equal to 220 at
20°C.
IgE-binding capacity of rPhl p 6, and cross-reactivity with natural Phl p 6 and other timothy grass pollen allergens
The prevalence of IgE anti-rPhl p 6 reactivity was determined in sera from 171 patients allergic to grass pollen and, for control purposes, in sera from nonatopic persons by ELISA (13). The presence of cross-reactive IgE epitopes on natural Phl p 6 and rPhl p 6 was investigated by IgE immunoblot inhibition experiments (4). A possible immunological relationship between rPhl p 6 and recombinant timothy grass pollen allergens (rPhl p 1, rPhl p 2, rPhl p 5) (13) was studied by ELISA competition as described previously (4).
Histamine release experiments
Granulocytes were isolated from heparinized blood samples of
individuals allergic to grass pollen; the samples contained rPhl p
6-reactive IgE Abs by dextran sedimentation (22). Cells
were incubated with increasing concentrations of purified rPhl p 5,
rPhl p 6, and with an anti-human IgE Ab (E124.2.8 D
2,
Immunotech, Marseilles, France). Histamine released into the
supernatants was measured by RIA (Immunotech).
Skin testing
After informed consent was obtained, skin prick tests were performed on the forearms of the individuals as described previously (23). Individuals were pricked with 20-µl aliquots containing different concentrations (1 µg/ml, 10 µg/ml, and 100 µg/ml) of purified rPhl p 6, rPhl p 5, timothy grass pollen extract, histamine, and NaCl (ALK, Horsholm, Denmark).
Analysis of the presence of Phl p 6-related allergens in other grass species and tissue-specific expression of Phl p 6
Protein extracts from pollens, leaves, and roots were obtained by homogenizing the tissues in SDS sample buffer (24). Insoluble materials were removed by centrifuging the extracts (10,000 x g for 20 min; Sorvall RC5C, SS34 rotor). Protein extracts were separated by 14% SDS-PAGE (25) and blotted onto nitrocellulose membranes (26). Nitrocellulose strips were probed with a rabbit anti-celery profilin antiserum, RP1 (12), rabbit anti-rPhl p 6 antiserum, and the latter rabbit’s preimmune serum. Bound rabbit Abs were detected with a 1/1000 dilution of 125I-labeled donkey anti-rabbit Ig antiserum (Amersham, Buckinghamshire, U.K.).
In situ localization of Phl p 6 by immunogold electron microscopy
Timothy grass pollen grains were anhydrously fixed as described previously (27). Ultrathin sections were incubated with equal concentrations of either rabbit anti-rPhl p 6 Ig (Ig: protein G-purified Ig fraction) or preimmune Ig. Bound rabbit Abs were detected with goat anti-rabbit IgG Abs coupled to 10-nm-diameter colloidal gold particles (Plano, Wetzlar, Germany) (27).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six cDNA clones (c142, c223, c171, c121, c233, and c146) coding
for Phl p 6 isoforms/fragments were isolated from a timothy grass
pollen gt11 library with serum IgE from a patient allergic to grass
pollen (Fig. 1
A). The
sequences of the described clones have been deposited in the GenBank
database (accession numbers: Y16955-Y16960). The deduced amino acid
sequence of Phl p 6 (clone 142) contained a 28-aa hydrophobic leader
peptide. A molecular mass of 11.8 kDa and an isoelectric point of 5.5
was calculated for the mature Phl p 6 (clone 142) protein, which starts
with a glycine residue and shows a high content of alanine residues
(20.9%) (Fig. 1A). The computer-aided secondary structure
analysis of Phl p 6 indicates a predominant helical content; the
calculation of solvent accessibility predicts that many of the
N-terminal amino acids are solvent exposed, whereas most of the
C-terminal amino acids appeared buried (Fig. 1A). A search
for sequence motifs revealed the presence of one potential N-linked
glycosylation site (NAS: amino acids 15–17), one N-terminal
myristoylation site (GKAT: amino acids 1–4), two cAMP-dependent
protein kinase phosphorylation sites (KATT: amino acids 2–5; KYKT:
amino acids 33–36), and two peroxisomal targeting sequences (GKA:
amino acids 1–3; SKA: amino acids 54–56). The deduced Phl p 6 amino
acid sequence displayed identity with a recently submitted Phl p 6
sequence (15) and similarities with the N-terminal
portions of group 5 grass pollen allergens (Fig. 1A).
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Nitrocellulose-bound ß-galactosidase-fused complete (c223,
c142), N-terminally truncated rPhl p 6 (c171, c121, c233, and c146)
and, for control purposes, ß-galactosidase alone were exposed to
serum IgE from nine grass pollen allergic individuals and a nonallergic
person (Fig. 1
B). The results obtained showed that the two
complete Phl p 6 isoforms and a Phl p 6 fragment lacking only four of
the N-terminal amino acids strongly bound IgE from all patients tested
that were allergic to grass pollen, and that the IgE binding capacity
of the partial Phl p 6 clones decreased depending upon the number of
amino acids that were absent from the proteins’ N terminus. A partial
clone (clone 121) lacking the N-terminal 30 amino acids had almost
completely lost its IgE binding capacity (Fig. 1, A and
B).
E. coli expression and purification of rPhl p 6: IgE binding capacity of purified rPhl p 6
rPhl p 6 was overexpressed in E. coli BL21 (DE3). A
combination of several purification steps yielded pure and soluble rPhl
p 6 (5 mg protein/L E. coli culture), which was
identified by SDS-PAGE as one of the low molecular mass timothy grass
pollen allergens (Fig. 2A).
MALDI-TOF analysis of purified rPhl p 6 resulted in two mass/charge
peaks of 11,790 and 5,896 corresponding with the
MH+ and M2H2+ species of
the sample, which were in agreement with the deduced Phl p 6 molecular
mass (11,789 Da).
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rPhl p 6 folds in a stable all -helical conformation
The far-UV CD spectrum of purified rPhl p 6 (Fig. 2B)
indicates that the protein contains a considerable amount of
-helical secondary structure. The spectrum is characterized by two
broad minimums at 208 nm and 220 nm and a maximum at 191 nm. The
secondary structure prediction (Fig. 1A) is in good
agreement with the CD measurements, as it indicates predominant
-helical secondary structure content. The unfolding transition of
rPhl p 6 is monophasic and is highly cooperative, with a melting point
of 61°C. At 85°C, rPhl p 6 assumes a random coil conformation, with
a typical minimum at 200 nm. rPhl p 6 shows a high degree of folding
reversibility, evident from the cooling curve profile (Fig. 2C) and the far-UV spectrum recorded at 20°C after cooling
from 85°C (Fig. 2B).
rPhl p 6 induces dose-dependent basophil histamine release and immediate type skin reactions in patients allergic to grass pollen
Purified rPhl p 6 induced specific and dose-dependent histamine
release from basophils of a patient allergic to grass pollen (Fig. 3A). rPhl p 5, which
represents a highly active grass pollen allergen (Ref. 14 ,
R. Valenta and S. Flicker, unpublished data), induced maximal release
already at a lower concentration compared with rPhl p 6. rPhl p 6, rPhl
p 5, and timothy grass pollen extract induced immediate type skin
reactions in four patients allergic to grass pollen, but not in the
nonallergic individuals (Table I; Fig. 3B). Although no reactions to NaCl were observed, histamine
induced wheal reactions in all individuals tested (Table I; Fig. 3B).
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Although major groups of grass pollen allergens occur in pollens
of most grass species (4), group 6 allergens were reported
to occur exclusively in timothy grass (P. pratense) pollen
(15). A rabbit anti-rPhl p 6 antiserum cross-reacted
with group 5 allergens in nitrocellulose-blotted pollen extracts from
various monocots (P. pratense, L. perenne,
S. cereale, T. sativum, A. sativa,
P. communis) between 25 and 28 kDa (Fig. 4A, lane 2). Phl p
6 or Phl p 6-related allergens at 11 kDa were detected exclusively in
pollens from P. pratense and P. pratensis.
Although a putative N-glycosylation site was found in the amino acid
sequence deduced from the Phl p 6 cDNA sequence, the comparable
molecular masses observed for natural Phl p 6 and rPhl p 6 exclude
heavy glycosylation of natural Phl p 6 (Figs. 2A and
4A). Rabbit anti-rPhl p 6 Abs strongly reacted with Phl
p 6 at 11 kDa in nitrocellulose-blotted timothy grass pollen but not
with leaf or root extracts (Fig. 4B, lane 2).
Profilin was detected in all three tissues at 14 kDa (Fig. 4B, lane 1).
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Using postembedding immunogold electron microscopy, rabbit
anti-rPhl p 6 Abs bound to the numerous polysaccharide (P-)
particles, which fill much of the interior of a mature timothy grass
pollen grain (Fig. 4C). The greatest accumulation of gold
particles was observed on sectioned surfaces of the P-particles,
indicating that Phl p 6 is present on rather than in the P-particles.
Little (cytosol, exine) or no (mitochondria, intine) anti-rPhl p 6
immunoreactivity was observed in other parts of the pollen grain.
Likewise, almost no gold particles were detected in the amyloplasts.
This localization pattern, taken together with our finding that a
rabbit anti-rPhl p 5 antiserum failed to label the P-particles
(data not shown), excludes the possibility that the immunolabeling of
the P-particles resulted from the presence of cross-reactive group 5
allergens. Control experiments performed with preimmune Ig yielded only
a few nonspecifically adsorbed gold particles (Fig. 4D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The expression of Phl p 6 in E. coli yielded large amounts
of soluble and folded recombinant protein that contained an almost
exclusive -helical secondary structure. The -helical fold of Phl
p 6 is a further confirmation that there are no common structural
features that predispose a certain protein to behave as an allergen.
Although Phl p 6 is very likely an all -helical protein, Bet v 1,
the major birch pollen allergen (31) and Bet v 2, birch
profilin (32) have a mixed ß fold. As revealed by CD
spectroscopic analysis, rPhl p 6 shares with other immunologically
unrelated pollen allergens (e.g., Bet v 1 (33), Bet v 2
(6, 32)) the remarkable intrinsic tendency to refold into
a stable conformation after denaturation. Another feature that is
shared by Phl p 6 and other important plant allergens is high
expression in pollen tissue. The fact that most of the plant allergens
characterized so far are expressed predominantly in mature pollen may
therefore be interpreted as a footprint of sensitization via the
respiratory tract (34).
By immunogold electron microscopy, Phl p 6 was primarily localized on
the P-particles of mature pollen. P-particles are small
polysaccharide-containing bodies that represent 
30% of the contents
of the dormant pollen grain and, during pollen germination, transfer
material into the growing pollen-tube wall (35, 36). The
occurrence of Phl p 6 on the P-particles may be of clinical relevance,
as P-particles could act as small-sized (<2.5 µ) and therefore
respirable allergen-carriers that bring Phl p 6 in immediate contact
with the bronchial mucosa. A P-particle-linked intrusion of Phl p 6
into the deeper respiratory tract would thus explain the high
prevalence (75%) of sensitization against this allergen, although only
a few grass species (P. pratense, P. pratensis)
contained rabbit anti-rPhl p 6-reactive moieties in the low
(10–12 kDa) molecular mass range.
The E. coli-expressed purified rPhl p 6 allergen reacted with IgE Abs of the majority of patients allergic to grass pollen and induced basophil histamine release as well as immediate type skin reactions. It may therefore be used for in vitro as well as in vivo (skin test) diagnoses of grass pollen allergy. Our finding that deletion of the N-terminal portion of Phl p 6 dramatically reduced the IgE binding capacity of the allergen may lead to the construction of hypoallergenic Phl p 6 deletion mutants that may be used for specific immunotherapy of grass pollen allergy with reduced anaphylactic side effects. Recently, the bacterial expression of two hypoallergenic fragments comprising amino acids 1–74 and amino acids 75–160 of the major birch pollen allergen, Bet v 1, was reported (23). The recombinant hypoallergenic Bet v 1 fragments contained most of the Bet v 1-specific T cell epitopes (23) and, upon immunization of animals, induced Abs that blocked IgE binding to the complete Bet v 1 wild-type molecule (S. Vrtala and R. Valenta, unpublished data). Our data obtained for Phl p 6 provide good evidence that it may be possible to use a similar strategy to produce a hypoallergenic N-terminally truncated Phl p 6 version that as such or in combination with a C-terminally truncated Phl p 6 molecule could be used for immunotherapy of Phl p 6-sensitized individuals.
In conclusion, we have produced a major timothy grass pollen allergen, Phl p 6, as a recombinant molecule useful for the in vitro and in vivo diagnosis of grass pollen allergy. Hypoallergenic deletion variants of Phl p 6 may be used for specific immunotherapy of grass pollen allergy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Susanne Vrtala, Molecular Immunopathology Group, Institute of General and Experimental Pathology, Vienna General Hospital Medical School, University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria. E-mail address:
3 Abbreviations used in this paper: CD, circular dichroism; MALDI, matrix-assisted laser desorption and ionization; TOF, time of flight.
Received for publication June 25, 1999. Accepted for publication September 7, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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