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Research Communications |
1
* Department of Otorhinolaryngology,
Institute of Medical and Chemical Laboratory Diagnostics,
Institute of General and Experimental Pathology, AKH, University of Vienna, Austria;
|| European Molecular Biology Laboratory, Heidelberg, Germany; and
§ Department of Internal Medicine I, Division of Hematology, AKH, University of Vienna, Austria
1Correspondence: Molecular Immunopathology Group, Institute of General and Experimental Pathology, AKH Medical School, University of Vienna, Waehringer Guertel 18–20, A-1090 Vienna, Austria.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: Type I allergy • allergen • EF-hand protein • conformational epitopes • circular dichroism spectroscopy • cross-reactivity • pollen-specific expression
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2)
. Immediate type symptoms (allergic rhinitis,
conjunctivitis, and asthma) result from the cross-linking of effector
cell-bound IgE by allergens and the subsequent release of biological
mediators (e.g., histamine, leukotrienes) (3)
. A detailed knowledge
concerning the molecular nature of the disease eliciting antigens is
relevant for diagnosis, prevention, and therapy of Type I allergy.
Recent progress in the field of molecular allergen characterization by
cDNA cloning techniques has revealed the primary structure of many
relevant allergens, allowed to investigate their B cell and T cell
epitopes, and provided recombinant allergens for diagnostic and
therapeutic purposes (4,
5)
. Structural similarities among allergens
were found to form the molecular basis for immunological
cross-reactivity and explain why allergic patients can exhibit clinical
symptoms after contact with various unrelated allergen sources (6,
7)
.
Cross-reactive plant allergens include the family of profilins,
representing ubiquitous cytoskeletal proteins, which bind to actin (8,
9), phosphatidylinositides (e.g., PIP2) (10)
, and VASP (11)
. IgE
recognition of profilins may thus be responsible for allergic symptoms
after contact with pollens from unrelated plant species (trees,
grasses, weeds) (12)
, plant-derived food (fruits, vegetables, spices)
(13,
14)
, latex products (15)
, and perhaps for triggering and
maintaining profilin-specific antibody formation by autoimmune
mechanisms (16)
. Another group of cross-reactive plant allergens is
exemplified by Bet v 1, the major birch pollen allergen (17)
. Bet v
1-homologous proteins contain a p-loop as a typical sequence motif (18,
19)
and are thought to act as pathogenesis-related plant proteins with
RNase activity (20)
. They represent cross-reactive pollen allergens in
trees of the Fagales order (21)
and in plant-derived food
(fruits, vegetables, spices) (22)
.
Recently calcium binding plant proteins have been discovered as
relevant cross-reactive allergens (23)
. According to number and
structure of their calcium binding domains (EF-hands) as well as to
interdomain sequence similarities, calcium binding proteins can be
divided into more than 30 subfamilies (24)
. Calcium binding proteins
may simply buffer and translocate calcium or, due to calcium-induced
conformational changes, interact with ligands in a calcium-dependent
manner and thus act as cellular messengers (25)
. Calcium binding plant
allergens described so far include Bet v 3, a three EF-hand
pollen-specific birch allergen (23)
, Jun o 1, a four EF-hand cypress
pollen allergen (26)
, and a novel subfamily of two EF-hand pollen
allergens recently described for birch (Bet v 4) (27,
28)
, alder (Aln g
4) (29)
, olive (Ole e 3) (30)
, Bermuda grass (Cyn d 7) (31,
32)
, and
rape (33)
. While there is some evidence that IgE antibody recognition
of these allergens can be modulated by the presence or absence of
protein-bound calcium, the molecular mechanisms for this behavior have
been elusive.
Here we report the isolation and characterization of a cDNA coding for Phl p 7, a two EF-hand calcium binding timothy grass pollen allergen. We expressed recombinant Phl p 7 in Escherichia coli and purified the recombinant protein to homogeneity. The expression of Phl p 7 in tissues of timothy grass and its elution kinetics from hydrated pollen were studied as well as its cross-reactivity with homologous allergens in tree, grass, and weed pollens. To determine the allergenic potential of recombinant Phl p 7, basophil histamine release assays and skin prick tests were performed. The possible molecular mechanisms of the calcium-dependent modulation of IgE recognition of apo- and calcium-bound Phl p 7 were studied by circular dichroism analysis and molecular modeling.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gt 11 phage DNA was obtained from Amersham (Buckinghamshire, U.K.).
E. coli strain Y1090 was purchased from Amersham, E.
coli XL1-Blue was from Stratagene (La Jolla, Calif.), and E.
coli BL21 (DE3) was from Novagen (Madison, Wis.). Plasmid pUC19
and pET 17 b were obtained from Boehringer (Mannheim, Germany) and
Novagen, respectively. Recombinant Bet v 1, the major birch pollen
allergen, was supplied from BIOMAY (Linz, Austria). Recombinant Bet v 4
was expressed as ß-galactosidase fusion protein in E. coli
Y1089, as described (27)
.
Antibodies, sera, patients
The rabbit anti-celery root profilin antiserum RP1 is described
(13,
8)
. A rabbit antiserum was raised against purified recombinant Aln
g 4, the Phl p 7-homologous alder pollen allergen, using Freund's
adjuvans (Charles River, Kissleg, Germany) (29)
. Human sera were
collected from 102 grass pollen allergic individuals by venipuncture
and stored at -20°C until use. Patients were characterized by case
history indicative for grass pollen allergy, by a positive timothy
grass pollen RAST (radioallergosorbent test) result, and by skin prick
testing as described (34)
. Individuals with and without rPhl p
7-specific IgE antibodies were tested in basophil histamine release and
skin test experiments after informed consent was obtained.
Isolation and characterization of a Phl p 7-encoding cDNA
An expression cDNA library constructed from mature timothy grass
pollen in phage gt11 was screened with serum IgE from a grass pollen
allergic patient as described (35)
. The 350 IgE-reactive clones were
tested with antibodies/antisera with specificity for group 1, group
2/3, group 5 allergens, and profilin as well as with sera from grass
pollen allergic patients with defined IgE reactivity profile to obtain
phage clones expressing not-yet-described timothy grass pollen
allergens. cDNAs of the remaining clones were obtained from phage
liquid lysates by polymerase chain reaction (PCR) amplification using
gt11 forward and reversed primers containing internal
BamHI sites (italics): gt11 forward 5'-CGG GAT
CCC GGT TTC CAT ATG GGG ATT GGT GGC 3'; gt11 reversed 5'-CGC
GGA TCC CGT TGA CAC CAG ACC AAC TGG TAA TG 3' (MWG,
Ebersberg, Germany). The PCR products were digested with
BamHI, gel-purified, subcloned into the BamHI
site of plasmid pUC 19, and transformed in E. coli XL-1
blue. Plasmid DNA was purified using Quiagen tips (Quiagen, Hilden,
Germany) and the 5' DNA sequences of the allergen-encoding cDNAs were
determined with the gt11 forward primer using the chain termination
method (36)
, 35S-dCTP (NEN, Stevenage, U.K.), and
a T7 sequencing kit (Pharmacia, Uppsala, Sweden). All molecular
biological manipulations followed established protocols (37)
. The
obtained DNA sequences were compared with the DNA sequences deposited
in GenBank using the BLAST server. The 5' DNA sequence of clone 144
that showed significant sequence homology with cDNAs coding for calcium
binding proteins was selected for detailed characterization.
To avoid the introduction of sequence errors by PCR, original phage DNA
was isolated from clone 144 using a plate lysate method (37)
. The phage
DNA was cut with KpnI/SacI to obtain the
allergen-encoding cDNA flanked on each side by ~1000 base pairs of
gt11 DNA. The clone 144 KpnI/SacI fragment was
subcloned into the KpnI/SacI site of plasmid
pUC19 and both DNA strands were sequenced using the gt11 primers as
well as sequence specific internal primers (MWG).
Analysis of the clone 144 DNA and deduced amino acid sequence, calculations of molecular mass, pI, secondary structure, and surface accessibility were performed with the McVector Program (Kodak, Rochester, N.Y.). DNA and deduced amino acid sequence of clone 144 were compared with the sequences deposited in GenBank using the program BLASTN and BLASTP via the BLAST server.
E. coli expression and purification of rPhl p
7
The coding region of clone 144 was amplified by PCR using the
following primers: Phl p 7–5': 5' GGG AAA TTC CAT ATG GCG
GAC GAC ATG GAG AGG 3' and Phl p 7–3': 5' CCG GAA TTC ATC
AGA AGA CCT TGG CGA CGT C 3'. The primers contained a NdeI
site (italics) and an EcoRI site (italics), respectively.
The PCR product was cut with NdeI/EcoRI,
gel-purified, and subcloned into the NdeI/EcoRI
site of plasmid pET 17b. E. coli BL 21 (DE 3) colonies
expressing the rPhl p 7 allergen were identified with serum IgE from a
Phl p 7-allergic individual by colony screening. Plated E.
coli were induced to synthesize recombinant proteins by overlay
with nitrocellulose filters soaked in 10 mM IPTG. Filter-bound E.
coli were disrupted by freeze thawing and colonies expressing rPhl
p 7 were identified with serum IgE from a Phl p 7-allergic patient.
Bound IgE antibodies were detected with an alkaline phosphatase-labeled
mouse monoclonal anti-human IgE antibody (Pharmingen, San Diego,
Calif.). The DNA sequence of the IgE-reactive pET 17b clone used for
protein expression was confirmed by sequencing. Recombinant Phl p 7 was
overexpressed in E. coli BL21 (DE3). E. coli were
grown to an OD600 of 0.4 in LB-medium containing
100 mg/l ampicillin. The expression of recombinant protein was induced
by adding isopropyl-ß-thiogalactopyranoside to a final concentration
of 1 mM and culturing for an additional 4 h at 37°C. E.
coli cells from a 600 ml culture were harvested by centrifugation,
resuspended in 15 ml PBS containing 5 mM phenylmethylsulfonyl fluoride
(PMSF), and homogenized using an ultraturrax (Ika, Heidelberg,
Germany). A fraction containing soluble proteins was obtained after
centrifugation of the homogenate at 10.000 rpm (Sorval RC5C, SS34
rotor) for 30 min at 4°C. Recombinant Phl p 7 was enriched in the
soluble protein fraction after precipitation and removal of
contaminating proteins by boiling and centrifugation (10.000 rpm, 30
min, 4°C, Sorval RC5C, SS34 rotor). A similar enrichment of rPhl p 7
in the soluble protein fraction was obtained after addition of 70% w/v
ammonsulfate to the soluble E. coli fraction. The soluble
rPhl p 7-enriched protein fraction obtained after centrifugation
(18.000 rpm, Sorvall SS34, 4°C, 30') was dialyzed against water,
lyophilized, resuspended in 50 ml buffer I (25 mM Imidazole, 1 mM
ß-mercaptoethanol, pH=7.4), and applied to a DEAE anion exchange
column (Pharmacia). rPhl p 7 was eluted by a NaCl gradient (buffer I
containing 500 mM NaCl) at ~200 mM NaCl. Fractions containing pure
rPhl p 7 were pooled, dialyzed against water, and lyophilized. rPhl p 7
samples were analyzed for purity by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and protein
staining as well as by mass spectroscopy.
Protein extracts, SDS-PAGE, immunoblotting
Pollen protein extracts were prepared by homogenizing 1.5 g
pollen in 50 ml of distilled water containing 5 mM PMSF with an
ultraturrax (IKA, Heidelberg, Germany) and extraction of the homogenate
for 3 h at 4°C under continuous shaking. Homogenates were
centrifuged at 20,000 g for 30 min at 4°C to remove
insoluble particles. Supernatants were lyophilized and checked for
quantity and quality of proteins by SDS-PAGE (38)
and Coomassie blue
staining (39)
as well as by immunoblotting. The presence of Phl p 7 and
profilin in the supernatant and pellet fraction of hydrated timothy
grass pollen grains was studied by immunoblotting as described (40)
.
For the analysis of the tissue-specific expression of Phl p 7, pollen,
leaves, and roots were homogenized in SDS sample buffer with an
ultraturrax. The homogenate was boiled and insoluble materials were
removed by centrifugation (10,000 g, 20 min, RT). Total
extracts from E. coli before and after induction of protein
expression were obtained in the same way. Approximately 100 µg/cm gel
of total protein extracts and 5 µg/cm gel of purified proteins were
separated by preparative 15% SDS-PAGE and blotted onto nitrocellulose
(Schleicher & Schuell, Dassel, Germany) (41)
. Sera from allergic
patients and (for control purposes) from nonatopic individuals, were
diluted 1:10 in buffer A [50 mM Na phosphate, pH 7.5, 0.5% w/v bovine
serum albumin (BSA), 0.5% v/v Tween 20, 0.05%
NaN3] and exposed to nitrocellulose-blotted
proteins. Bound IgE was detected with
125I-labeled anti-human IgE antibodies as
described (12)
and visualized by autoradiography using KODAK X-OMAT
films and intensifying screens (Kodak, Heidelberg, Germany). Rabbit
antisera were diluted 1:2000 in buffer A and probed with
nitrocellulose-blotted proteins. Bound rabbit antibodies were detected
with 125I-labeled donkey anti-rabbit antibodies
(Amersham) and visualized by autoradiography.
MALDI-TOF (matrix-assisted laser desorption and
ionization–time-of-flight analysis) of purified rPhl p 7
Laser desorption mass spectra were acquired in a linear mode
with a time-of-flight Compact MALDI II instrument (Kratos, Manchester,
U.K.), operating at 20 kV acceleration voltage and equipped with a
nitrogen UV laser (337 nm, pulse duration 3 ns) (piCHEM, Research and
Development, Graz, Austria). The m/z values were calibrated externally.
Samples were dissolved in 10% acetonitrile (0.1% TFA). Alpha-cyano-4
hydroxy-cinnamic acid was used as a matrix dissolved in 60%
acetonitrile (0.1% TFA). For sample preparation, a 1:1 mixture of
protein solution and matrix solution was deposited onto the target and
air dried.
IgE absorption experiments
The share of IgE epitopes between natural allergens in timothy
grass (Phleum pratense), Bermuda grass (Cynodon
dactylon), birch (Betula verrucosa), and ragweed
(Ambrosia elatior) pollen extracts as well as rBet v 4 (27)
was studied by immunoabsorption experiments as described (12)
. Sera
from allergic patients containing rPhl p 7-specific IgE antibodies and
from a nonatopic individual were preabsorbed with 10 µg/ml rPhl p 7
or, for control purposes, with 10 µg/ml BSA overnight at 4°C.
Preabsorbed sera were then exposed to nitrocellulose-blotted natural
pollen extracts or rBet v 4 and bound IgE antibodies were detected with
125I-labeled anti-human IgE antibodies
(Pharmacia, Uppsala, Sweden). The reduction of IgE binding to rBet v 4
after preabsorption of the sera with rPhl p 7 was quantified by 
counting as described (21)
.
Basophil histamine release experiments
Granulocytes were isolated from heparinized blood samples
of three pollen allergic patients containing rPhl p 7-specific IgE and
(for control purposes) from a nonatopic individual by dextran
sedimentation (42,
43)
. Cells were incubated with increasing
concentrations (0.0001 µg/ml, 0.001 µg/ml, 0.01 µg/ml, 0. 1
µg/ml, 1 µg/ml, 10 µg/ml) of purified rPhl p 7 and (for control
purposes) with monoclonal anti-human IgE antibodies. Histamine released
in the supernatant was measured by radioimmunoassay (Immunotech,
Marseille, France). Total histamine was determined after freeze thawing
of the cells. Results are displayed as mean values of triplicate
determinations and represent the percentage of total histamine.
Skin prick testing
After informed consent had been obtained, three pollen allergic
patients and (for control purposes) a nonatopic individual were skin
prick tested on their forearms with 20 µl aliquots of solutions
containing different concentrations (1 µg/ml, 5 µg/ml, 10 µg/ml,
20 µg/ml) of purified rPhl p 7 and (for control purposes) with
timothy grass pollen extract, histamine, and sodium chloride (ALK,
Horsholm, Denmark), as described (44)
. The skin reactions were recorded
20 min after testing by photography and by transferring the ball point
pen-surrounded wheal area with scotch tape to paper. The mean wheal
diameters displayed in Table 1
were determined as follows: Dm = (D1 + D2)/2. D1 and D2 represent
the maximal longitudinal and transversal diameter of the wheal reaction
in mm, respectively.
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Calcium-dependent IgE binding of rPhl p 7
Equal amounts (5 µg/dot) of purified rPhl p 7 were dotted onto
nitrocellulose membranes of equal size (20 mm x 6 mm) and were exposed
to four sera from pollen-allergic patients containing rPhl p 7-reactive
IgE-antibodies in the presence (buffer A containing 0.1 mM
CaCl2.) or absence of calcium (buffer A
containing 5 mM EGTA), as described (23)
. Bound IgE antibodies were
detected with 125I-labeled anti-human
IgE-antibodies (RAST, Pharmacia). The amount of bound IgE was
quantified by counting of the nitrocellulose strips (Wizzard,
Automatic Gamma Counter, Wallac, Uppsala, Sweden) (21)
.
Circular dichroism spectroscopical analysis of purified rPhl
p 7
Circular dichroism (CD) spectra were recorded on a Jasco
J-710 spectropolarimeter fitted with a Jasco PTC-348WI peltier type
temperature control system and interfaced with a Fisons HAAKE GH water
bath. The instrument was calibrated with a 0.1% aqueous solution of
d-10-camphor-sulfonic acid. Results were expressed as the mean residue
ellipticity ([
]) at a given wavelength. Far-ultraviolet CD spectra
were recorded at 20°C, 95°, and 98°C in a 2 mm path-length quartz
cuvette (Hellma, Mullheim, Baden, Germany) at a protein concentration
of 10 µM. Spectra were recorded with 0.1 nm resolution and resulted
from averaging 10 scans. The final spectra were corrected by
subtracting the corresponding base line spectrum obtained under
identical conditions. All measurements were performed in MilliQ water,
pH 7.2. Thermal denaturation of Phl p 7 was monitored using a 2 mm
cuvette (Hellma) 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 after
cooling (50°C/h) to the starting temperature (20°C). Measurements
were performed in MilliQ water pH 7.2 at a protein concentration of 10
mM. Results were expressed as the mean residue ellipticity () at a
given wavelength. 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 that of the denatured protein. For rPhl p 7,
U was assumed to be equal to
220 at 85°C and N
to 220 at 20°C.
Molecular modeling of the open and closed states of Phl p 7
The ribbon representation of the apo- and calcium-bound form of
Phl p 7 was prepared by homology modeling using the WHAT IF program
(45)
and is displayed with MOLMOL (46)
. The coordinates of
Drosophila melanogaster calmodulin were used as a template.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). EF-hands are helix-loop-helix motifs that usually pair together to
bind to calcium through four carboxylate or carboxamide groups and a
single backbone carbonyl oxygen placed in the loop with a specific
spacing. These residues that represent the marker motif of the EF-hand
family are found in the clone 144 deduced amino acid sequence with no
diversity from the consensus. Comparison of the amino acid sequence
deduced from clone 144 with those deposited in GenBank and the known
allergen-sequences revealed best homology with a group of low molecular
weight, two EF-hand calcium binding pollen allergens recently described
for birch (Bet v 4) (27,
28)
, alder (Aln g 4) (29)
, Bermuda grass (Cyn
d 7) (31,
32)
, and rape (Bra r 1, Bra r 2, Bra n 1, Bra n 2) (33)
(Fig. 1)
. On the basis of the high sequence homology of the clone 144-encoded
timothy grass pollen allergen with the Bermuda grass pollen allergen,
Cyn d 7, and the fact that no group 7 timothy grass pollen allergen has
been identified so far, we designated the gene product Phl p 7 and
submitted it as such to GenBank (accession number: Y17835). The deduced
amino acid sequence of Phl p 7 was most closely related to that of Cyn
d 7 (93.6% sequence identity). The sequence identity of Phl p 7 with
the other two EF-hand pollen allergens was 64.1% (Aln g 4), 62.8%
(Bet v 4), 69.2% (Bra n 1, Bra r 1), and 61.5% (Bra n 2, Bra r 2).
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E. coli expression and purification of rPhl p
7
Recombinant Phl p 7 was expressed in E. coli BL21 (DE3)
using a T7 RNA polymerase-driven expression system. SDS-PAGE analysis
of E. coli extracts before (Fig. 2
A: lane 1) and after induction of protein synthesis (Fig. 2A
: lane 2) indicated expression of a band of ~6 kDa
apparent molecular mass in both samples. As observed for rAln g 4, the
Phl p 7-homologous allergen from alder pollen (29)
, rPhl p 7, showed an
apparent molecular mass of 6 kDa (Fig. 2A
), lower than
deduced from their amino acid sequences. Phl p 7 and Aln g 4 are acidic
proteins and most likely possess high electrophoretic mobility in
SDS-PAGE. A considerable amount of rPhl p 7 remained in the soluble
fraction (Fig. 2A
: lanes 3 and 4). As reported for many
other calcium binding proteins (24)
, rPhl p 7 was further enriched in
the soluble fraction after boiling and centrifugation (Fig. 2A
: lane 5). Likewise, rPhl p 7 was enriched and still
soluble after addition of 70% w/v ammonsulfate (Fig. 2A
:
lane 6). The solution obtained was desalted and passed over a DEAE
anion exchange column, and pure/soluble rPhl p 7 could be recovered
(Fig. 2A
: lane 7).
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MALDI-TOF analysis of purified rPhl p 7 yielded three mass/charge peaks
of 2855.9 Da, 4277.0 Da and 8555.6 Da corresponding to the M3H+, M2H+,
and MH+ species of the protein, respectively (Fig. 2B
). The
molecular mass determined by mass spectrometry is thus in good
agreement with the molecular mass calculated from the Phl p 7 deduced
amino acid sequence (8.6 kDa).
Purified rPhl p 7 inhibited IgE binding to a 6 kDa moiety, presumably
representing natural Phl p 7, present in nitrocellulose-blotted timothy
grass pollen extract (Fig. 2C
) and specifically bound IgE
antibodies of 10 sensitized grass pollen allergic patients, but not of
a nonallergic individual (Fig. 2D
). Preincubation of the
rPhl p 7-reactive sera with rPhl p 7 yielded according to counting
a reduction of IgE reactivity to rBet v 4 of 77.2% for one serum (Fig. 2D
: serum 1) and between 90.1%–99.2% for the other 7 Bet
v 4-reactive sera (Fig. 2D
: sera 2, 4–8, 10).
Phl p 7 represents a two EF-hand calcium binding protein that is
highly expressed in pollen
To study the expression of Phl p 7 in various tissues of timothy
grass, we used a rabbit antiserum that was raised against purified
recombinant Aln g 4, a highly homologous allergen (sequence identity of
64.1%) (GenBank accession number: Y17713). The rabbit anti-Aln g 4
antiserum (lane 2) but not the rabbits' preimmune serum (lane 1)
reacted with nitrocellulose-blotted purified rPhl p 7 (data not shown).
Comparable amounts of nitrocellulose-blotted timothy grass pollen,
leaf, and root proteins were exposed to rabbit anti-Aln g 4 antiserum
and (for control purposes) to the rabbits' preimmune serum and a
rabbit anti-profilin antiserum. Whereas anti-profilin immunoreactivity
was detected in all three tissues at 14 kDa (Fig. 3
A: 
profilin), Phl p 7 was detected in pollen but not in
root and leaf extracts (Fig. 3A
: Aln g 4). The rabbits'
preimmune serum showed no significant binding to the three extracts in
the molecular weight range of Phl p 7 (Fig. 3A
: nrs). Taken
together, these data indicate that Phl p 7 represents a protein that is
highly expressed in mature pollen and could not be detected in leaf or
root tissues.
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Rapid and complete elution of Phl p 7 after hydration of timothy
grass pollen
It has been demonstrated that many relevant pollen allergens
represent intracellular proteins that become rapidly eluted after
pollen hydration (47,
40,
48)
. The analyses of nitrocellulose-blotted
supernatant and pellet fractions obtained after hydration of timothy
grass pollen for different time intervals with a rabbit anti-Aln g 4 as
well as with a rabbit anti-profilin antiserum are displayed in Fig. 3B
. Both Phl p 7 and profilin were detected in the
supernatant fraction obtained after 1 min of pollen hydration (Fig. 3B
). The profilin content in the pollen grain-containing
pellet fraction decreased continuously until 30 min of hydration,
although substantial amounts of profilin remained detectable in the
pollen grains even after 3.5 h of hydration (Fig. 3B
).
In contrast, we found that Phl p 7 was completely eluted within the
first minutes of pollen hydration (Fig. 3B
). No Phl p 7
immunoreactivity was detected in the pellet fractions analyzed after 5
min, 10 min, 30 min, 1 h, and 3.5 h of pollen hydration. Our
results thus demonstrate that after a few minutes of hydration, Phl p 7
is completely eluted out of the pollen grains.
Phl p 7 is a cross-reactive pollen allergen
Ten out of 102 sera from grass pollen allergic patients
contained rPhl p 7-reactive IgE antibodies (Fig. 2D
). All
patients with rPhl p 7-specific IgE antibodies suffered from allergy to
pollen of other grass species, trees, and weeds. Therefore, we
investigated whether rPhl p 7 shares epitopes with allergens in pollen
of Bermuda grass (Cynodon dactylon), birch (Betula
verrucosa), and ragweed (Ambrosia elatior).
A rabbit anti-rAln g 4 antiserum (Fig. 4
a: lanes 2), but not the rabbit preimmune serum (Fig. 4a
: lanes 1), cross-reacted with two EF-hand allergens in
nitrocellulose-blotted natural pollen extracts from timothy grass
(Phleum pratense), Bermuda grass (Cynodon
dactylon), ragweed (Ambrosia elatior), and birch
(Betula verrucosa) at 6–8 kDa. These results suggest that
Phl p 7-homologous proteins are expressed in mature pollen of a variety
of higher plants, including monocotyledonic and dicotyledonic species.
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The presence of common IgE epitopes on Phl p 7 and possible homologous
pollen allergens was studied by IgE inhibition experiments.
Preabsorption of two sera from rPhl p 7-reactive patients (Fig. 4b
: A, B) with rPhl p 7 (lanes +), but not with BSA (lanes
-), led to a complete (sera A, B: Cynodon,
Ambrosia; serum A: Betula) or significant (serum
B: Betula) reduction of IgE binding to moieties of 6–8 kDa.
The reduced anti-IgE reactivity of certain high molecular weight
components (40–97 kDa) in birch (serum A) and ragweed pollen (sera A
and B) extracts could be caused by the presence of two EF-hand allergen
oligomers or independent high molecular weight allergens that share IgE
epitopes with rPhl p 7. The enhanced anti-IgE reactivity of 14–21 kDa
moieties in Bermuda grass pollen extract after preincubation of serum A
with rPhl p 7 may result from the binding of IgE-rPhl p 7
immunocomplexes to these components.
rPhl p 7 induces basophil histamine release and immediate type skin
reactions in pollen allergic patients
The biological relevance of rPhl p 7-specific IgE reactivity
was investigated by basophil histamine release experiments (Fig. 5
) and skin testing (Table 1)
. rPhl p 7 induced histamine release from
basophils of three pollen allergic patients containing rPhl p
7-reactive IgE antibodies (Fig. 5A
, data not shown), but not
from basophils of a nonallergic individual (Fig. 5B
).
Basophils from all four donors released histamine after exposure to
anti-IgE antibodies (Fig. 5A, B
; data not shown). The same
three allergic patients (A, B, C) and the nonallergic individual (N)
who were tested in basophil release experiments were skin pricked with
four concentrations of rPhl p 7 and (for control purposes) with timothy
grass pollen extract, histamine, and isotonic sodium chloride solution
(Table 1)
. rPhl p 7 and timothy grass pollen extract induced immediate
type skin reactions in all three pollen allergic patients (A-C) but not
in the nonallergic individual (N) (Table 1)
. Histamine elicited wheal
reactions in all four individuals (Table 1)
.
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Calcium-dependent IgE recognition of rPhl p 7
Exposure of purified nitrocellulose-bound rPhl p 7 under
native conditions to serum IgE antibodies of Phl p 7 allergic patients
in the presence of Ca2+ (lanes: +) or EGTA
(lanes: -) resulted in significant variations of polyclonal serum IgE
recognition of rPhl p 7 (Table 2
). Due to the polyclonal nature of the IgE response in allergic
individuals (i.e., the formation of IgE antibodies against many
epitopes), calcium-dependent modulation of IgE recognition varied. As
exemplified in Table 2
for four Phl p 7-reactive sera, certain patients
showed stronger IgE binding to the calcium-bound form of rPhl
p 7 (Table 2
; serum 1: +89%; serum 2: +112%). However, we also found
that IgE antibodies of certain patients displayed reduced IgE
reactivity to the calcium-bound-form of rPhl p 7 (Table 2
; serum 3:
-36%; serum 4: -43%). The IgE reactivity of the tested sera thus
exemplified that the polyclonal IgE antibody response in Phl p 7
allergic patients can be predominantly directed against epitopes
exposed on either the calcium-bound or apo form of the allergen.
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Circular dichroism analysis of rPhl p 7
The far-ultraviolet circular dichroism spectrum of purified
rPhl p 7 (Fig. 6
A) recorded at 20°C indicates that the protein contains a
considerable amount of -helical secondary structure. The spectrum is
characterized by a broad minimum at 208 nm, a shoulder at 222 nm, and a
maximum at 186 nm. Thermal unfolding of rPhl p 7 was monitored as
change in ellipticity at 220 nm and expressed as mean residue
ellipticity (
) (Fig. 6D
). In the presence of 5 mM EGTA,
the unfolding transition of rPhl p 7 is monophasic and highly
cooperative with a melting point of 72°C; at 95°C, calcium-free
rPhl p 7 assumes a random coil conformation, with a typical minimum at
200 nm. rPhl p 7 shows a high degree of folding reversibility evident
from the far-UV spectra recorded at 20°C, after cooling from 98°C
and 95°C (Fig. 6B, C
), respectively, in the absence or
presence of EGTA. From the thermal denaturation profiles and far-UV
spectra at 98°C (no EGTA) and 95°C (5 mM EGTA), it is immediately
evident that the thermal stability of rPhl p 7 depends on the presence
of protein-bound calcium. Calcium-bound rPhl p 7 does not unfold
completely and, even at 98°C, retains most of its native-like
conformation (Fig. 6B, D
).
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DISCUSSION |
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. Comparison
of the Phl p 7 amino acid sequence with those sequences submitted to
the databases revealed that Phl p 7 belongs to a novel subfamily of
recently described two EF-hand calcium binding pollen allergens that
are preferentially expressed in pollen of higher plants. Our contention
that Phl p 7 and the related two EF-hand allergens represent a distinct
and novel subfamily of calcium binding proteins is supported by
structural, immunological, and biological considerations. 1)
There are only a few examples of other two EF-hand calcium binding
proteins (e.g., S-100-like proteins), all of which differ from Phl p 7
and the Phl p 7-related allergens in their interdomain sequences (24,
25)
. 2) It has been demonstrated that the Phl p 7-related
two EF-hand allergens do not share epitopes with already known EF-hand
allergens (e.g., Bet v 3, parvalbumin) (23,
49)
. 3) Unlike
plant calmodulins, Phl p 7-related allergens were expressed in pollen
but not in leaves or roots.
The pollen-specific expression of Phl p 7 may be relevant for two
reasons. First, because it is well established that pollen germination
and pollen tube growth are Ca2+-dependent
processes (50)
. Pollen calcium has also been attributed to be of
importance for the reorientation of pollen tube growth (51)
and the
self-incompatibility response (52)
. Regarding pollen tube growth, it
has become apparent that the cytoplasmic Ca2+
gradient is strongly correlated with tube growth (53)
and there is a
steep, tip-directed gradient of Ca2+ (54)
;
recently it has been shown by calcium imaging that calcium pulses are
coincident with peak growth rates (55,
56)
. The importance of calcium
levels for pollen germination and pollen tube growth must therefore
require the presence of pollen proteins that tightly control and
regulate calcium metabolism. Phl p 7 and other pollen-specific calcium
binding proteins, e.g., Bet v 3, which are abundantly expressed in
plant pollens but not in other plant tissues, may therefore represent
such regulatory proteins. Within minutes of pollen hydration, Phl p 7
is completely eluted from the pollen grain, a process that likely will
influence the balance between intracellular and extracellular calcium
concentrations and thus affect pollen germination and tube growth.
Second, pollen-specific expression of Phl p 7-related allergens is of
interest regarding their role as highly cross-reactive pollen
allergens. Specific or highly up-regulated expression in pollen, but
not somatic tissues, represents a major feature of prominent plant
allergens (6)
. In the present study it is demonstrated that Phl p 7 and
related two EF-hand allergens represent highly cross-reactive allergens
in pollens of trees, grasses, and weeds. Approximately 10% of pollen
allergic individuals react with the two EF-hand pollen allergens and
therefore suffer from allergic symptoms after contact with pollens of
many unrelated plant species. Although recognized at a rather low
prevalence (10% of pollen allergic patients), we found that rPhl p 7
exhibited an extremely potent allergenic activity. rPhl p 7 induced
basophil histamine release and immediate type skin reactions at
extremely low concentrations and thus represents a biologically very
active allergen.
The allergenic activity of rPhl p 7 may be related to its proper
folding. According to CD analysis, rPhl p 7 was folded and contained
mostly -helical secondary structure elements. Proper folding
represents a prerequisite for an allergen to interact with and to
cross-link effector cell-bound IgE in solution, and thus induce the
release of biological mediators. The CD measurements also revealed that
calcium-bound rPhl p 7 had a far greater thermal stability than the
EGTA-treated apoform. Calcium-bound rPhl p 7 thus shares the property
of great thermal stability with many other calcium binding proteins
(57,
24)
, a fact that allowed us to enrich rPhl p 7 in the soluble
fraction of E. coli extracts by boiling of the homogenate.
Increased stability due to calcium binding against, for example,
proteolysis, may be of relevance to protect the protein under
physiological (e.g., pollen hydration and germination) as well as
pathological conditions, i.e., when Phl p 7 becomes eluted from the
pollen grain during contact with the mucosa of allergic patients. It
has been found that rapid elution from pollen grains after hydration as
well as high stability and refolding capacity represent major features
of prominent allergens (40,
58
; Vangelista and Valenta, unpublished
results). The latter two properties may be the reason why Phl p 7 is
able to elicit and to trigger IgE responses in allergic patients.
CD spectroscopical analysis also revealed significant structural
differences of calcium-bound and apo-rPhl p 7, a property that in the
case of calcium sensor proteins (e.g., calmodulin) allows for the
calcium regulation of interactions with various ligands (24,
25)
.
Although we have no evidence so far for physiological ligands of Phl p
7 and related two EF-hand proteins, we found that IgE antibodies of
allergic patients bound to rPhl p 7 in a calcium-dependent manner. The
ribbon representations built for the apo- and calcium-bound form of Phl
p 7 suggest that the molecule can occur in two conformations, referred
to as `closed' and `open', which correspond to the calcium-free
(apoform) and calcium-bound states (59)
(Fig. 7
). The apoform (closed form) (Fig. 7
, left) differs from the
calcium-bound form (open form) (Fig. 7
, right) by the relative
orientation of the helices. Whereas in the closed state the four
helices form a very compact structure, calcium binding would push apart
the two helices of each EF-hand motif and thus allow exposure of
hydrophobic residues that become available for interaction with target
peptides (e.g., IgE antibodies). The different IgE recognition of the
apo- and calcium-bound Phl p 7 may therefore be explained by a
preferential sensitization either to conformational epitopes present on
the calcium-bound form of rPhl p 7 or to epitopes present on the
calcium-free (apo-) form of the allergen. This hypothesis, that IgE
recognition of allergens can mirror the contact with certain allergen
conformations/states during early sensitization, is supported by data
on the IgE recognition of other prominent plant allergens. IgE epitopes
of profilin, a highly conserved actin binding protein, were mapped to
the regions involved in the binding to natural ligands (e.g., actin);
it was therefore concluded that patients were sensitized preferentially
against the ligand-free profilin (60,
61)
. Likewise, it has been
reported that IgE antibodies can distinguish between different
conformations/states of the major birch pollen allergen, Bet v 1 (58)
.
The fact that patients exhibited reduced IgE binding to certain Phl p 7
conformations may represent a basis for the generation of
`hypoallergenic' allergen variants for specific immunotherapy. The
latter concept has been used to generate recombinant fragments (44)
and
mutants (62)
of the major birch pollen allergen Bet v 1 with reduced
anaphylactic activity. Similar results may be obtained if the calcium
binding sites of Phl p 7 are destroyed by site-directed mutagenesis.
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In conclusion, Phl p 7 represents a member of a novel family of pollen-specific two EF-hand calcium binding proteins and a prominent cross-reactive pollen allergen containing calcium-modulated conformational IgE epitopes. In its recombinant form, it may be used to diagnose and treat a group of atopic patients who display allergic symptoms on contact with pollens of many unrelated plant species.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication July 31, 1998.
Revision received December 16, 1998.
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