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J Biol Chem, Vol. 274, Issue 4, 2337-2343, January 22, 1999
From the The three-dimensional structure of the major
bovine allergen Bos d 2 has been determined by using x-ray diffraction
at 1.8-Å resolution. Structurally Bos d 2 is a member of the lipocalin family comprising proteins with transport functions. There is a flat
small cavity inside the Bos d 2 protein core suitable for ligand
binding, and it is possible that Glu115 and
Asn37 inside the core are able to make hydrogen bonds with
the ligand. Many allergens from different animals belong to the
lipocalin family. The amino acid residue similarities between these
lipocalins indicate putative regions for IgE binding. Comparison with
the available allergen structures from other sources suggests that these allergens are roughly the same size and that their shape is more
spherical than elliptical.
Allergy is a result of an inappropriate immune response in persons
with a genetic predisposition or atopy. At least 20% of the population
in the industrialized countries suffer from different forms of
allergies (1). The development of allergy is a multistep process, and
the mechanisms leading to sensitization, production of IgE antibodies,
and allergic diseases are complex and not fully understood. However,
allergies arise in response to certain otherwise innocuous proteins,
allergens. The list of denominated allergens contains more than 170 proteins from different species (2). These proteins are structurally
and functionally a heterogeneous group, including hydrolytic enzymes,
enzyme inhibitors, and transport molecules, but there are also a number
of proteins without a known function (3). One group of proteins, which
has been found recently to include several animal allergens, is the
lipocalin family (4).
Recognition of a protein by IgE antibodies and by receptors on the T
helper type 2 lymphocytes is a critical requirement for allergenicity.
The receptor of T helper type 2 cells recognizes allergens as processed
linear peptides in association with major histocompatibility class II
molecules on antigen-presenting cells (5). On the other hand, IgE
antibodies bind on the surface of the allergen molecules and therefore
the binding sites or B-cell epitopes are often conformational and thus
dependent on the three-dimensional structure. The allergic reaction is
triggered by the binding of an allergen to IgE molecules on mast cells.
The cross-linking of mast cell-bound IgE molecules by an allergen
postulates that the allergenic protein contains more than one B-cell epitope.
During the past few years, the primary structure of a large number of
different allergens has been resolved, and the three-dimensional structures of some allergens have also been determined by using x-ray
diffraction or NMR. To date, the coordinates of seven different allergenic proteins are available at the Brookhaven Data Bank (Table
I). Four of the proteins are from plants:
Birch pollen Bet v 1 (6) and Bet v 2 (7), mouse ear cress Ara t (8), and timothy grass pollen Phl p 2 (9). Three proteins are from animals:
house dust mite Der f 2 (10), mouse urine Mus m 1 (11), and bovine milk
Bos d 5 ( The three-dimensional structures of allergens are important in
understanding the molecular basis of allergenicity. Structural information in relation to the information on B-cell and T-cell epitopes is also necessary for a rational strategy to design effective, safe recombinant derivatives of allergenic proteins for specific immunotherapy. In this study, we have determined the three-dimensional structure of the major bovine dander allergen Bos d
21 at 1.8-Å resolution. The
structure of Bos d 2 is compared with other allergenic proteins within
the lipocalin protein family. We also make a general comparison between
currently released allergen structures in an attempt to find features
shared by these molecules.
X-ray Crystallography--
The crystals of recombinant Bos d 2 were grown at 20 °C by the hanging drop method as described (13).
Bos d 2 crystallized in a space group
P212121 with cell dimensions
a = 37.0, b = 55.6, c = 77.2 Å containing one molecule in the asymmetric unit. The native data
set was collected at room temperature with an R-AXIS IIc imaging plate
using CuK
Bos d 2 is homologous with two lipocalins in which three-dimensional
structures have been determined. Its identity to the odorant-binding
protein (PDB code 1OBP) is 26% and to the major mouse urinary protein
(1MUP) 20%. Because of the low identity, some heavy atom derivatives
were sought. One data set obtained from a crystal soaked overnight in
10 mM Hg(OAc)2 showed a single heavy atom site
in the difference Patterson map. Using one mercury site, the single
isomorphous replacement map was calculated with both hands using the
program XtalView (15). Both maps showed positions of protein molecules
in the unit cell but no other details.
The molecular replacement calculations were performed with the program
AMoRe (16). Because the odorant-binding protein is a dimer and part of
its structure has swapped, it was not used. Therefore, the major murine
urinary protein 1MUP without the ligand was used as a search model. The
rotation function was calculated between the data at 8 and 4 Å, and a
22-Å integration radius was used. This resulted in eight peaks, which
were 3.9-4.0
The building of the model was performed with the program O and the
refinement with the program X-PLOR (17). The initial refinement with
the mouse urinary protein against the Bos d 2 data resulted in the
crystallographic R factor of 39.3% and free R
factor of 54.2%. The residues of the model were then gradually replaced by the residues of Bos d 2, and after several cycles of manual
rebuilding, simulated annealing refinement and addition of water
molecules the R factor dropped to 18.4% and the free R factor to 23.2%. The statistics of the final model are
given in the Table II.
Immunologic Assays--
The capacity of nBos d 2 and rBos d 2 to
inhibit the binding of patient IgE or the binding of monoclonal
antibodies, mAb1 and mAb3 (18), to the solid-phase rBos d 2 or nBos d 2 was examined by the ELISA technique as described previously (19).
First, six 10-fold dilutions of each protein preparation (0.001-100
µg/ml) were allowed to react with a predetermined dilution of an
antibody in a test tube for 30 min at 37 °C. The inhibited antibody
as well as the uninhibited control antibody were then transferred into
the wells of a microwell plate. In IgE inhibitions, the ELISA procedure
was completed using rabbit anti-human IgE (Dako A/S, Glostrup, Denmark;
A0094), biotinylated goat anti-rabbit IgG (Dako; E0432) and Vectastain
ABC Elite Kit (Vector Laboratories). In the inhibitions of mAbs, the
bound antibody was detected with horseradish peroxidase-conjugated
rabbit anti-mouse immunoglobulins (Dako; P161). The color reaction was
developed, and optical density values were measured at 450 nm. The
values are the means of two replicate wells calculated after
subtracting the reagent blank value.
Skin prick tests were performed according to Nordic recommendations
(20) on the forearm of cow-asthmatic patients in duplicate with nBos d
2 and rBos d 2. Five 10-fold dilutions (0.02-200 µg/ml) of each
preparation were used with appropriate controls. After 15 min, the
wheals were marked and the diameters were measured.
Immunological Properties of Recombinant Bos d 2--
Recombinant
Bos d 2 (rBos d 2) was produced in Pichia pastoris and
crystallized as described earlier (13). To ascertain that the
observations made on the relationship between the allergenicity and the
structure of the rBos d 2 were valid, we characterized the
immunoreactivity of rBos d 2 by the ELISA inhibition technique and skin
prick tests. In the ELISA inhibition, the capacity of rBos d 2 or
natural Bos d 2 (nBos d 2) to inhibit the binding of serum IgE of four
allergic patients to the allergens was tested as described under
"Experimental Procedures." All sera gave identical results, showing
that rBos d 2 was as effective an inhibitor per weight unit as nBos d 2 (Fig. 1). The complete inhibition of the binding of the two monoclonal antibodies revealed that the two different epitopes recognized by these antibodies were intact.
The relevance of rBos d 2 as an allergen was further confirmed with
in vivo skin prick tests in ten patients. Again, the results showed (Fig. 2) that rBos d 2 was as
effective as nBos d 2, indicating that rBos d 2 was recognized by IgE
antibodies on the effector cells leading to cross-linking, release of
mediators, and to positive skin prick tests. In preliminary experiments
with two Bos d 2-specific T-cell clones (19), rBos d 2 also induced
stimulation identical to that caused by nBos d 2 (results not
shown).
The results described above showed that structural characteristics
responsible for the clinically relevant, conformational B-cell epitopes
were present in the rBos d 2. One of the aims when modified allergens
are designed for immunotherapy is to reduce allergic side effects by
minimizing the IgE binding capacity. Once the structural sites in the
molecule critical for B-cell epitopes have been localized, this can be
achieved by recombinant DNA technology.
The Structure of Bos d 2--
The three-dimensional structure of
rBos d 2 was determined by using x-ray diffraction at 1.8-Å resolution
and then refined to a crystallographic R factor of 18.4%.
The structure was solved with a molecular replacement technique by
using the mouse urinary protein 1MUP as a search model. The refined
model contains 150 residues (7-156) and 76 water molecules. A short
N-terminal region of the protein was disordered and was not included in
the model. The structure is monomeric with no glycosylation or other observable post-translational modifications. Previous molecular weight
determinations by ion trap mass spectrometry suggested that the
N-terminal residue appears either as glutamine or as pyrrolidone
carboxylic acid. Because the first six N-terminal residues were not
visible, the existence of pyrrolidone carboxylic acid could not be
verified by the crystal structure analysis. The final electron density
was of good quality except in the two loops B-C and C-D, which
indicated disorder in the crystal and resulted in high B factors.
The side chains for two residues, Arg17 and
Leu117, were built using two conformations.
Bos d 2 is clearly a member of the lipocalin protein family sharing all
the structural elements characteristic of other lipocalins (4). The
central
The electron density unambiguously showed the existence of two
disulfide bridges. Cys44-Cys48 is located in
the loop between strands B and C. Cys63-Cys154
is located between the N terminus of the
Of lipocalins with known three-dimensional structures, 1MUP (11) and
odorant-binding protein (1OBP) (21) have the highest sequence
identities with Bos d 2, 20 and 26%, respectively. The structures of
the three proteins were compared with the program O (22). We obtained
an root mean square deviation of 1.645 Å for 138 C
The biological function of Bos d 2 is currently unknown. There is only
indirect evidence of the biological function of Bos d 2 (25). However,
most proteins in the lipocalin family are ligand-binding proteins
capable of binding and transporting small hydrophobic molecules. The
VOIDOO program was used to calculate the molecular surface of the
protein and detect possible cavities (26). A flat cavity was found
inside the protein, and its size would approximately correspond two
small fused rings, as in indan. Because a recombinant protein was used
in the study, the ligand binding cavity was only occupied by two water
molecules. The ligand binding pocket is usually formed by a number of
hydrophobic residues. However, the striking feature of Bos d 2 is the
existence of hydrophilic residues in the pocket (Fig.
4). An important residue is Glu115, in
which the first carboxylate oxygen makes hydrogen bonds with Tyr39 and Thr100. The second carboxylate oxygen
is hydrogen-bonded with two water molecules. The second important
hydrophilic residue in the pocket is Asn37 with the side
chain amino group hydrogen-bonded to the main chain carbonyl of
Pro34 and the side chain oxygen hydrogen-bonded to the same
water molecule as Glu115. These two hydrogen bonds from
Glu115 and Asn37 to the water molecule are well
oriented geometrically. Because both residues are hydrogen bond
acceptors, it is plausible to suggest that the endogenous ligand of Bos
d 2 contains a heteroatom(s), which can donate two hydrogen bonds. One
such group could be the charged nitrogen with two hydrogen atoms.
Allergenic Lipocalins--
Many recently characterized animal
allergens have been found to be members of the lipocalin family.
Examples of such allergic proteins are mouse Mus m 1 (27), bovine Bos d
5 (
The main unifying features of allergenic lipocalins are the
three-dimensional structure and the transport function. Therefore, the
relationship between the structure and B-cell epitopes could reveal
valuable information on the determinants of allergenicity of
lipocalins. Three different methods have been used to map B-cell epitopes: the use of polypeptide fragments of allergenic proteins, mutagenesis of allergenic proteins, and investigation of surfaces of
protein models, e.g. by mapping homologous residues.
Generally, much less is known about B-cell (conformational) epitopes
than T-cell (sequential) epitopes. We have reported earlier that the C-terminal part of Bos d 2 is important for IgE binding (19). Among the
other lipocalin allergens, B-cell epitopes have been characterized only
for
Because the knowledge of B-cell epitopes of lipocalin allergens is very
limited, we have used another method to probe the properties of
these molecules. First, a multiple amino acid sequence alignment was
carried out for seven proteins with the program GeneWorks (Fig. 5).
Despite the probable structural similarity, the identity between the
sequences was very low, showing only the two identical residues,
Gly14 and Trp16 (Bos d 2 numbering), of the
main conserved segment of the lipocalin family. The computer program
used also estimates a penalty or a "cost," a number from 0 to 9 for
alignment; 0 represents strongest similarity and 9 weakest. These
values are shown in the Fig. 5 for each residue and are also mapped on
the surface of Bos d 2 (Figs. 3, B and C). A
resemblance between the molecules can be observed in several areas of
the surface. These areas include the Comparison with Allergens of Other Families--
In the past few
years, the three-dimensional structures of allergens of other families
have also been determined. The coordinates for four plant and one mite
allergen are available at the Brookhaven Data Bank (Table I).
The major birch pollen allergen Bet v 1 contains a seven-stranded
anti-parallel
Knowledge of the B-cell epitopes of these allergens is also very
limited. Peptides have been used to study the IgE binding of Bet v 2. In particular, it has been suggested that N- and C-terminal fragments
are important for binding (7). Nishiyama et al. (32) have
studied Der f 2 by using site-directed mutagenesis. They have found
that two small areas on the opposite faces of the protein are important
for IgE binding. One area is composed of polar or charged residues and
the other of more hydrophobic residues.
When comparing the available allergen structures, it is easy to note
that they are composed mainly of *
This work was supported by the Academy of Finland, Maud
Kuistila Foundation, and Kuopio University Hospital (Grant 5035).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The atomic coordinates and structure factor (code 1BJ7) have been
deposited in the Protein Data Bank, Brookhaven Laboratory, Upton,
NY.
§
To whom correspondence should be addressed. Tel.: 358-13-2513318;
Fax: 358-13-2513390; E-mail: juha.rouvinen{at}joensuu.fi.
The abbreviations used are:
Bos d 2, bovine
lipocalin allergen Bos d 2; rBos d 2, recombinant Bos d 2; 1MUP, major
mouse urinary protein; ELISA, enzyme-linked immunosorbent assay; mAb, monoclonal antibody.
Probing the Molecular Basis of Allergy
THREE-DIMENSIONAL STRUCTURE OF THE BOVINE LIPOCALIN ALLERGEN Bos
d 2*
§,
, and Department of Chemistry, University of
Joensuu, POB 111, FIN-80101 Joensuu, Finland, the ¶ Department of
Clinical Microbiology, University of Kuopio, POB 1627, FIN-70211
Kuopio, Finland, and the Department of Pulmonary Diseases,
Kuopio University Hospital, POB 1777, FIN-70211 Kuopio, Finland
ABSTRACT
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Abstract
Introduction
References
INTRODUCTION
Top
Abstract
Introduction
References
-lactoglobulin) (12).
Structural features of some common allergens
-lactoglobulin are calculated for a monomer.
EXPERIMENTAL PROCEDURES
radiation and processed with the DENZO and SCALEPACK
programs (14). The native data set consisted of 14,230 unique
reflections (redundancy 2.9) up to 1.8-Å resolution with an
R-merge of 6.6%.
above the mean. The first 20 peaks from the rotation
function were used in the translation search at 4-10-Å resolution.
The fifth and sixth solution from the rotation function produced peaks
18.1 and 15.1 above the mean, respectively. The rigid body
refinement resulted in an R factor of 52.1% for both
solutions. The solutions are partially overlapping so that one part of
the -sheet of the search model is common to the solutions and the
rest are not. When comparing the solutions with the both hand single
isomorphous replacement maps we found that the first solution
overlapped with one of the single isomorphous replacement maps. In
consequence, this model was used as a starting model in the structure determination.
Structure refinement
RESULTS AND DISCUSSION
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Fig. 1.
B-cell epitopes in the natural and
recombinant Bos d 2. ELISA inhibition was used to demonstrate the
IgE antibody binding capacity of rBos d 2 and the presence of the two
B-cell epitopes recognized by monoclonal antibodies mAb1 and mAb3.
Microtiter plates were coated with natural or recombinant Bos d 2, and
the bound antibodies were detected by the ELISA technique. The
inhibition with one patient's serum and with the mAb3 by natural or
recombinant Bos d 2 is shown in the figure. Sera of all four patients
tested and the mAb 1 also gave the same results regardless of the
coating antigen.
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Fig. 2.
In vivo reactivity of recombinant Bos d
2. Skin prick tests were used to compare the efficacy of natural
and recombinant Bos d 2 in provoking a positive skin reaction in
patients allergic to cow dander. The diameters of the skin lesions
induced by 200 or 20 µg/ml of the allergens are shown. Each
dot-line-dot indicates the reactivity to the two allergens
in an individual patient.
-barrel is composed of eight antiparallel -strands
(A-H) with (+1)8 topology forming a continuous
hydrogen-bonded flattened barrel. The structure also contains a short
-strand (I), a 310-helix between strands A and B, an
-helix between strands H and I, and a C-terminal
310-helix (Fig.
3A).
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Fig. 3.
A, schematic representation of Bos d 2. The -strands are in green and 310 and
-helices in blue. The positions of disulfide bridges are in
yellow. A small red stick model shows
the position of the putative ligand binding pocket inside the protein
core. The picture has been created with the program Setor (34).
B, the molecular surface of Bos d 2. The surface is mapped
according to the "cost" from the sequence alignment of allergenic
lipocalins (Table II). The white indicates residues of
highest similarity and smallest cost. The dark blue shows
the areas of weakest similarity. C, as in B but
rotated 180° along the y axis. The surface pictures were
made with the program GRASP (35) and rendered with the PhotoShop.
-strand D and C terminus of
the protein, a structure shared by other proteins in the lipocalin protein family, for example, 1MUP.
atoms of 1MUP
and 1.623 Å for 129 C atoms of 1OBP. These values are close to
those reported between the insect biliverdin-binding protein and the
human retinol-binding protein of the lipocalin protein family, namely
1.65 Å for 97 C atoms (23). For comparison, the root mean square
deviation of 1.557 Å for 144 C atoms is obtained when the crystal
structure of Bos d 2 and the model based on comparative modeling (24)
are compared.
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Fig. 4.
The representative electron density map from
the final refined model of Bos d 2. The picture shows the
"empty" ligand binding pocket inside the protein. The hydrogen bond
network around the Glu115 is shown.
-lactoglobulin) (28), cockroach Bla g 4 (29), dog Can f 1 and Can
f 2 (30), and horse Equ c 1 (31). Three-dimensional structures are only available for Mus m 1 and Bos d 5, but their allergenic properties have
not been discussed in terms of structure. The best known allergens of
the lipocalin family exist as monomers. Mus m 1 was reported to be a
dimer in the crystal but when observing the packing of the molecules it
is difficult to believe that it is a true dimer. On the other hand, the
dimeric structure of -lactoglobulin is well characterized. In this
protein, the longer -strand I forms an important part of the dimer
interface containing a number of hydrogen bonds. The protein exists as
a dimer at neutral pH but as a monomer at acidic pH. Four of the
allergens, Bla g 4, Can f 1, Can f 2, and Equ c 1, have putative
N-glycosylation sites (Fig.
5). These sites are located on the
protein surface between -strands A and B, and between -strands B
and C, and in -strand D.
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Fig. 5.
The sequence alignment of allergenic
lipocalins. Putative N-glycosylation sites are shown in
boldface. The first row shows the secondary structure elements of Bos d
2: ~, electron density weak or missing; 
, -strand; =,
310 or -helix. Cost is the estimation of similarity
according to the GeneWorks program (0 means absolute identity).
-lactoglobulin (Bos d 5) by using peptide fragments. Ball
et al. (28) determined that polypeptide 97-108 inhibited
the antibody binding of native -lactoglobulin. This fragment is
composed mainly of -strand G and the preceding loop. Many residues
of this fragment would be inside the core in the native protein
structure, and it is questionable whether this kind of short fragment
would have a native extended -strand conformation.
-helix, N- and C-terminal ends
of -strand A, and the loop between -strands C and D. Some well
conserved charged residues can be found in these areas, especially on
the surface of the -helix (Glu126 and
Glu129). It would be interesting to continue the analysis
to other, nonallergenic lipocalins, but problems would arise, because
we do not know if these molecules are potential allergens or not. However, we can conclude that surprisingly many characterized allergens
belong to the lipocalin family. These allergens are predominantly
monomeric molecules. Some, but not all, are glycosylated, which would
indicate that glycosylation is not a common critical determinant of
allergenicity in lipocalins. We have identified surface residues of
high similarity between lipocalin allergens. These regions would be
suitable targets for mutational studies in the development of new
preparations for allergen immunotherapy.
-sheet, which wraps around a long -helix. The
structure also contains two shorter -helices. The biological function of Bet v 1 is unknown (6). The second birch pollen allergen,
Bet v 2, is a profilin and partly resembles Bet v 1, since it also has
a seven-stranded antiparallel -sheet, but it has a slightly
different strand order and helix positions (7). Mouse ear cress
profilin Ara t has a similar structure to birch pollen profilin Bet v 2 (8). Major mite allergen Der f 2 is a single domain -protein with an
immunoglobulin fold, and it has been suggested that it is related to
the antibacterial defense system (10). Only preliminary structural data
have been published for timothy grass pollen Phl p 2 (9). It is
composed of an eight-stranded anti-parallel -barrel, thus resembling
lipocalins, but the connection between the -strands is clearly different.
-structures. Der f 2 and Phl p 2 contain only -structures, whereas lipocalins, Bet v 1, and profilins
also have some helices. Because the experimental knowledge of B-cell
epitopes is very limited, it is not possible to suggest any structural
motif or sequence pattern common to all allergenic proteins. Therefore
we have compared dimensions and shapes of available allergenic
molecules. Since many molecules seemed to be a bit flat, we calculated
ellipticity for molecules (Fig. 6, Table
I). The shortest dimension for these molecules is on average 34 Å, the
middle dimension 37 Å, and the longest dimension 44 Å. When looking
at the picture, it is evident that these allergens appear to have
similar dimensions. Islam et al. (33) defined the
ellipticity to be the ratio between the longest ellipsoidal axis and
the shortest. These allergens are only slighly elliptical (ratio 1.3).
Islam et al. (33) have calculated the ellipticity for most
single domain proteins to range from 1.25 to 2.25, which would suggest
that these allergens should be considered more spherical than
elliptical.
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Fig. 6.
The molecular surfaces of eight
allergens. The molecules were positioned so that the longest axis
is in the x direction and the shortest axis in the
y direction (upper row). The upper and
lower rows are related by the rotation of 90° along the
x axis. The molecules are drawn in the same scale and
color-coded according to the electrostatic potential: blue
representing positive areas and red negative. The figure was
created with the program GRASP (35).
FOOTNOTES
REFERENCES
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Abstract
Introduction
References
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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