Solution Structure of Der f 2, the Major Mite Allergen for Atopic
Diseases*
Saori
Ichikawa
§,
Hideki
Hatanaka
¶,
Toshifumi
Yuuki
,
Namiko
Iwamoto
,
Soichi
Kojima**,
Chiharu
Nishiyama
,
Kenji
Ogura
,
Yasushi
Okumura
, and
Fuyuhiko
Inagaki
From the
Department of Molecular Physiology, Tokyo
Metropolitan Institute of Medical Science, 3-18-22 Honkomagome,
Bunkyo-ku, Tokyo 113, the § Department of Material and
Biological Science, Faculty of Science, Japan Women's University,
2-8-1 Mejirodai, Bunkyo-ku, Tokyo 112, the
Bioscience Research & Development Laboratory, Asahi Breweries, Ltd., 2-13-1, Ohmori-kita,
Ohta-ku, Tokyo 143, and the ** Laboratory of Gene Technology and Safety,
Tsukuba Life Science Center, The Institute of Physical and Chemical
Research (RIKEN), Koyodai, Tsukuba, Ibaraki 305, Japan
 |
ABSTRACT |
House dust mites cause heavy atopic diseases such
as asthma and dermatitis. Among allergens from Dermatophagoides
farinae, Der f 2 shows the highest positive rate for atopic
patients, but its biological function in mites has been perfectly
unknown, as well as the functions of its homologs in human and other
animals. We have determined the tertiary structure of Der f 2 by
multidimensional nuclear magnetic resonance spectroscopy. Der f 2 was
found to be a single-domain protein of immunoglobulin fold, and its
structure was the most similar to those of the two regulatory domains
of transglutaminase. This fact, binding to the bacterial surface, and
other small pieces of information hinted that Der f 2 is related to the
innate antibacterial defense system in mites. The immunoglobulin E
epitopes are also discussed on the basis of the tertiary structure.
 |
INTRODUCTION |
Mites are the closest animals to human life and their relation is
inseparable in the modern residential environment. House dust mites
cause heavy atopic diseases such as asthma and dermatitis, which are
rapidly increasing worldwide, especially among the children in
developed countries. Dermatophagoides farinae and D. pteronyssinus are recognized as the main sources of house dust
allergens. Among their allergens, the group-2 allergen proteins, Der f
2 and Der p 2, show the highest positive rate for atopic patients (1) so they are called major allergens. Their sequences are 88% identical (2, 3), and their cross-reactivity was well confirmed (1). They are
also homologous to the major allergen from the mite Lepidoglyphus destructor (4), which is important in farming environments. These
proteins are 125-129 amino acid residues long and have three intramolecular disulfide bonds.
Although the properties of these proteins related to their
allergenicity have been well characterized, their biological function in mites is unknown. Homologous proteins were also found in human epididymis, cow milk, and moth trachea (5-7) and named HE1, EPV20 and
esr16, respectively, the first two of which are glycoproteins unlike
the group-2 mite allergens. However, there have been no clues for their
functions except for their expression patterns. This is in contrast to
the case of the other major allergen group including Der f 1 and Der p
1, because they were found to be cysteine proteases, and their activity
was suggested to be involved in the induction of allergic responses
(8). Therefore, the innate functions of Der f 2 and Der p 2 have
interested many allergy researchers.
It is generally accepted that allergic symptoms are initiated by the
specific binding of allergens to immunoglobulin E
(IgE)1 antibodies, which
cross-link the high affinity IgE receptors on mast cells and basophils
(9). So monovalent ligands to allergen-specific IgE are expected to
block IgE receptor aggregation. Structure determination of allergens
will offer the basis of design of such drugs. The tertiary structures
of some pollen allergens have been previously reported including
ragweed allergens Amb t 5 and Amb a 5, a major birch allergen Bet v 1, and a minor birch allergen profilin (10-14). However, the tertiary
structure of Der f 2 and the following drug-design processes are
exceptionally urgent considering the serious mental influences on
atopic children.
In this study, we have determined the tertiary structure of Der f 2 by
multidimensional nuclear magnetic resonance (NMR) spectroscopy. Unexpectedly we found that Der f 2 is a single-domain protein of
immunoglobulin fold. There are few single-domain proteins of this fold,
and this protein is soluble and monomeric. We feel it is of interest
also in terms of the evolution of protein folds. The structural
similarity to the two regulatory domains of transglutaminase and other
small pieces of information prompted us to suppose that Der f 2 is a
component of the innate antibacterial defense system in mites.
Furthermore, we found that Der f 2 binds to the surface of bacteria,
which is the first clue to the biological function of this class of
proteins. We also discuss previous work related to the immunoglobulin E
epitopes on the basis of the tertiary structure.
 |
EXPERIMENTAL PROCEDURES |
Sample Preparation--
Escherichia coli BL21 cells
harboring pFLT1 (pGEMEX1 derivative containing cDNA for clone 1 of
Der f 2; see Ref. 15) were cultured in M9 minimum medium in the
presence of [13C]glucose and [15N]ammonium
chloride. Labeled recombinant Der f 2 was expressed and purified as
described (16). NMR samples contained 1.5 mM of Der f 2 in
90% H2O, 10% D2O, 140 mM
N-octyl-
-D-glucoside, 0.01% NaN3
at pH 5.6.
NMR Spectroscopy--
NMR spectra were acquired at 55 °C on a
Varian Unityplus 600 NMR spectrometer equipped with a triple
resonance pulse field gradient probe. The sequential assignment of the
1H, 13C and 15N chemical shifts was
achieved mainly by through-bond heteronuclear correlations along the
backbone and side chains with the following 3D pulse sequences:
HN(CO)CA, HNCA, CBCA(CO)NH, CBCANH, HBHA(CO)NH, HN(CA)HA, C(CO)NH,
HC(C)H-TOCSY (17). Nuclear Overhauser effects (NOEs) were derived from
three-dimensional 15N-edited and 13C-edited NOE
spectroscopy spectra recorded with a mixing time of 75 ms. The pulse
sequences of HN(CO)CA, HNCA, CBCANH, HBHA(CO)NH, HN(CA)HA, C(CO)NH and
15N-edited NOE spectroscopy were modified with pulse field
gradient method and sensitivity enhancement method (17).
Structure Calculations--
Upper limits of distance constraints
were calculated as kI
1/6, where I
is the peak intensity and k is a constant adjusted in each
NOE spectroscopy spectrum and relaxed by 0.5 Å considering mobility.
Lower limits of distance constraints were all 1.8 Å. The structures
were calculated with the program X-PLOR ver. 3.1 (18). Initial
coordinates were generated using random
and
angles, whereas
peptide bonds and side-chains took extended conformations. The
macroprogram sa.inp in X-PLOR ver. 2.1 was used to carry out simulated
annealing calculation. The target function that is minimized during
simulated annealing comprises only potential terms for covalent
geometry, experimental distance restraints, and van der Waals nonbonded
repulsion. No hydrogen bonding, electrostatic, 6-12 Lennard-Jones
potential or experimental torsion angle terms were present in the
target function. The final structure calculations were based on 1086 interproton distance restraints (526 intra-, 258 sequential
(|i
j| = 1), 56 middle range (2
|i
j|
5), and 246 long range
(|i
j| > 5) NOEs). A final set of 10 converged structures was selected from 20 calculations on the basis of
agreement with the experimental data and van der Waals energy. A mean
structure was obtained by averaging the coordinates of the structures
that were superimposed in advance to the best converged structure and
then minimizing under the constraints.
In Vitro Binding Assay--
The E. coli strain C was
grown to late log phase, collected by centrifugation, and suspended in
1/10 of the original volume with 0.9% NaCl. An equal volume of 20%
acetic acid was added, and the bacteria were left at room temperature
for 5 min. 5 volumes of 1 M Tris-HCl (pH 8.2) was added,
and the bacteria were collected by centrifugation and resuspended in
one-tenth of the original culture volume in 10 mM Tris-HCl
(pH 8.2). 100 µg of Der f 2 were added to the bacterial suspension
(0.1 ml of Der f 2 to 0.1 ml of bacteria) and incubated at room
temperature for the time indicated. The suspension was centrifuged for
2 min at 10,000 × g, the pellet was washed twice in
200 µl of water and then suspended in 40 µl of 0.5 M
ammonium formate (pH 6.4). The ammonium formate eluates were
immediately adjusted to 0.1% sodium dodecyl sulfate and 10 mM dithiothreitol, heated at 70 °C for 5 min, and
subjected to electrophoresis (19).
 |
RESULTS AND DISCUSSION |
Assignments and Structure Determination--
Backbone sequential
assignments for Der f 2 (129 amino acid residues) were obtained by a
strategy using a combination of four triple resonance measurements, 3D
HN(CO)CA, HNCA, CBCA(CO)NH, CBCANH, and were complete except for
HN and N of Asp-1 and Thr-123, and C
of
Glu-53. The nitrogen chemical shifts of prolines were not assigned.
Side-chain assignments were obtained principally by 3D C(CO)NH,
HC(C)H-TOCSY and HC(C)H-COSY, and partially extended using NOE data.
All nonexchangeable resonances were assigned except for Lys-33
C
and Lys-126 H
, H
, and
H
, whose assignments were not fixed owing to heavy
overlaps. In addition, H
1 and N
1 of
Trp-92 were not detected, possibly because of fast proton solvent
exchange enhanced by interaction with added detergent molecules.
The secondary structure was determined as all
, using tertiary NOEs
between backbone protons (Fig. 1), which
had been estimated beforehand by the results of chemical shift index
method (20). Using 1086 distance constraints extracted after
assignments of 3D 15N- and 13C-edited NOE
spectroscopy spectra, we obtained 10 structures from 20 calculations. A
summary of the structural statistics for a set of the final structures
and for the mean structure is presented in Table
I. There were no violations above 0.6 Å in any of the structures, and the number of violations above 0.3 Å ranged from 4 to 13. The deviation from ideal bond lengths was 0.004 Å. These figures are relatively good, considering that our distance
constraint set was tighter than those in the typical three-level
classification. However, since the number of NOE constraints was not
large, the quality of the structure should be regarded as medium.

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Fig. 1.
Secondary structure of Der f 2. Interstrand backbone-backbone NOEs are marked by dotted
lines. The residues that project their side chains below the sheet
are shown in the shadowed box. The topology of the
-strands are depicted by arrows.
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Table I
Structural statistics
SA refers to the final set of the simulated annealing
structures and (SA)r is the mean structure. The
number of terms is given in parentheses.
|
|
Description and Evaluation of the Structure--
Fig.
2a shows the tertiary
structure determination of Der f 2. The root mean square difference
(RMSD) value of the 10 final structures from the minimized averaged
structure was 0.90 ± 0.15 Å for the backbone (N,
C
, C
) atoms of residues 1-129 and 1.44 ± 0.17 Å for the nonhydrogen atoms of the same residue range. We also calculated
local RMSD values for the backbone atoms (N, C
, C
) at
each residue after the best fit superposition of the whole backbone
(data not shown), and found that the residues of large local RMSD value
were distributed around the whole sequence. Therefore the reason the
structural convergence was not excellent is not due to local
flexibility but to the number of NOE distance constraints.

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Fig. 2.
Tertiary structure of Der f 2. a,
Best fit superpositions of the backbone atoms (N, C , C )
of the 10 final structures of Der f 2. Amino acid residue numbers are
shown every ten residues. b, Ribbon diagrams of the mean
structure of Der f 2. The start and end residue numbers of the
-strands are also shown. Both figures were produced by the program
MOLSCRIPT (44) and shown in stereo. Color changes from red (N terminus)
to blue (C terminus).
|
|
Der f 2 has an immunoglobulin fold (Fig. 2b). The topology
corresponds to s-type (21). One
-sheet consists of three strands of
residues 13-19 (a), 36-43 (b), and 85-92 (e), and the other sheet is
composed of four
-strands 62-65 (c
), 49-57 (c), 104-111 (f), and
116-127 (g), and an additional short strand of residues 5-8
(tentatively named as a
). They include three
-bulges at the
positions 14-15, 117-118, and 123-124. Der f 2 has three disulfide bonds at the positions 8-119, 21-27, and 73-78 (22), but no disulfide bridge between the
-strands b and f, which is often found
in immunoglobulin-fold domains.
The immunoglobulin fold is the most ubiquitous module and is
distributed among many protein superfamilies of different functions (21). There are, however, few single-domain proteins with the immunoglobulin fold. Der f 2 is not membrane bound like Thy-1, a
subunit in a protein complex like
2-microglobulin, nor
does it polymerize into filaments like the major sperm protein from nematode. Therefore, this simple immunoglobulin-fold protein, Der f 2, might reflect the characteristics of the most ancient immunoglobulin-like domain, and it is of special interest to compare this structure with other domains of immunoglobulin fold, mainly distributed among vertebrate immune systems, cell surface receptors, coagulation/fibrinolysis systems, and some enzymes that bind to sugar
chains.
Structural Similarity to Domains of Transglutaminase--
We
searched for structural similarities to the known protein folds using
the program Dali (23). It listed many proteins of immunoglobulin fold,
but the fourth domain of human blood coagulation factor XIII (24) was
reported to be the most structurally similar to Der f 2, where the
Z-score was 4.6. This value is not large, probably because of the
quality of our structure determination. Because the second one, the
second domain of vascular cell adhesion molecule-1 (25), had the
Z-score of 4.2, it is difficult to conclude that the domain of factor
XIII is the most similar.
Transglutaminase, including factor XIII, is composed of four domains,
and the third and fourth domains have the s-type immunoglobulin fold.
We superimposed the Der f 2 structure onto the third and fourth domains
of factor XIII using 62 C
pairs (Fig.
3a) and obtained RMSD values
2.77 Å in both cases. Then we aligned the primary sequences of these
factor XIII domains (26) to those of Der f 2 (2) and homologous
proteins (3-7) on the basis of tertiary structures (Fig.
3b). Although the sequences in each group are highly
variable, we could find identical amino acid residues shared by both
groups at many positions. The numbers of such residues were 32 both in
the third and fourth domains of factor XIII, which is much larger than
the number in the second domain of vascular cell adhesion molecule,
which is 22. Phe-41, which is located at the center of domains, is
perfectly conserved among the two groups shown in Fig. 3b.
In particular, the factor XIII fourth domain is well aligned to the
group-2 mite allergens without large insertions or deletions except for
three disulfide bond-related segments.

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Fig. 3.
Relationship between Der f 2 and factor XIII.
a, Superpositions of the mean structures of Der f 2 (black)
and human coagulation factor XIII (green; Ref. 24). The regions used
for superposition are shown in bold lines. A cluster of nine
basic amino acids in Der f 2, His-22, His-30, Arg-31, Lys-33, Lys-96, Lys-100, His-124, Lys-126, and Arg-128, is shown in blue. Domain 2 of
factor XIII is discriminated by yellowish green. The catalytic triad
residues Cys-314, His-373 and Asp-396 are shown in orange, and Tyr-372
is shown in red. b, Sequence alignments of the factor XIII
third and fourth domains (26) and Der f 2 (2) and homologous proteins
Der p 2, Lep d 1, HE1, EPV20, and esr16 (3-7). The conserved cysteines
among Der f 2 homologs are shown in cyan. Identical amino acid residues
shared by both groups are in red. -strands are
underlined. The numbers above the sequences correspond to Der f 2.
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Intriguingly, Der f 1, the other major mite allergen, is a cysteine
protease, and this protease family has been shown to be evolutionally
related to the second domain of factor XIII (27). In addition, Tyr-372
(Fig. 3a, red) of factor XIII, neighboring the
catalytic triad (orange), corresponds to the tyrosine that has been recognized as unique to the mite allergen cysteine proteases (8). The fact that Der f 2 inhibited guinea pig liver
transglutaminase2 is also
interesting as it relates to the structural similarity described
above.
Implications for Innate Functions in Mites--
Although the
biological function of Der f 2 is unknown, the above findings reminded
us of the innate immune system of invertebrates (28). In invertebrates
that do not have immunoglobulins, the coagulation system is prominent
as an antimicrobial response. Two types of coagulation mechanisms have
been reported, each of which are associated with transglutaminase and a
cascade of serine proteases, respectively. Der f 3, a minor mite
allergen, is a serine protease that has a similar substrate specificity
to blood coagulation factor XII and is reported to activate the human
serine protease cascade (29).
We found that Der f 2 binds the surface of E. coli cells
(Fig. 4) in a manner similar to hemolin,
a bacteria-binding protein from moths composed of four
immunoglobulin-like domains (30). Der f 2 has a cluster of nine basic
amino acids (Fig. 3a), which implies a negatively charged
target surface. Preliminary results show that Der f 2 does not bind to
strains K12 or BL21. All of the three types of mite allergens mentioned
are localized in the gastrointestinal tract, mouth region and feces
(31, 32), and the feces, which are suggested to cause allergic symptoms
(33), have microbial degradation activities (34). HE1, EPV20, and esr16, the Der f 2 homologs from human, cow, and moth, respectively, are included in epithelial mucosae, where antibacterial proteins are
excreted (35-37). These observations imply that Der f 2 is a component
of the antibacterial defense system in mites. A survey of recent
literature data (38-40) made us realize that Bet v 1, conalbumin and
lactoferrin, which show the highest positive rate for sera from
allergic patients of birch pollen, hen egg, and cow milk, respectively,
are components of antimicrobial host defense systems.

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Fig. 4.
In vitro binding assay. The gels were
stained with Coomassie Brilliant Blue after separation of the
following: first washing, 12 µl (lane 2); second washing,
12 µl (lane 3); ammonium formate eluate after 3 min, 10 min, and 30 min of incubation, 12 µl (lanes 4,
5, and 6, respectively); and ammonium formate eluate from untreated bacteria (lane 7). In lanes
9-11, bovine serum albumin was used instead of Der f 2 for
comparison. 1 µg of Der f 2 and bovine serum albumin were loaded in
lanes 1 and 8.
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|
IgE Epitopes--
Since the cloning of the group-2 allergens, IgE
and T-cell epitopes have been intensely studied. The experiments using
14 synthetic peptides of 15 residues in length spanning the entire sequence of Der p 2 showed that IgE antisera do not bind to most peptides; the peptide comprising residues 65-78 bound IgE, but its
activity was extremely weak (41). Therefore recognition by IgE depends
strongly on the conformation of Der p 2, and considering the intense
homology, it is probably also the case for Der f 2. Truncation of N- or
C-terminal short sequences (42) or destruction of the disulfide bond
8-119 (43) reduced IgE-binding activity severely, which corresponds to
our result that Der f 2 is composed of only one domain.
Nishiyama et al. (42) made site-directed mutants at residues
1-21, 70-81, and 114-129 of Der f 2, which were selected considering the studies mentioned above, and measured their IgE-binding activities. Using their results, we mapped the molecular surface for those substitutions that decreased IgE binding (Fig.
5). This figure suggests that two IgE
epitope areas on the surface. One epitope area includes Asp-7, Asn-10
and Lys-15, and the second one includes Cys-73, Phe-75, Lys-77, and
Cys-78. However, the borders of these areas are distorted at the
residues Asp-19 and Asn-71. Experiments that can judge whether each
decrease of IgE binding is due to global destabilization or local
effects on the allergen-antibody interface might improve epitope
definition. Although additional amino acid substitutions are desired to
clarify the borders and judge the existence of other epitope areas, our
structure suggests mosaic distribution of IgE epitopes and provides
strategies for their complete characterization.

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Fig. 5.
Partial characterization of IgE epitope
areas. The molecular surface of Der f 2 was produced by the
program GRASP (45) and colored according to the results from the
site-directed mutagenesis experiments (42). Red, residues whose
substitution decreased IgE binding; blue, residues whose substitution
did not decrease IgE binding; white, residues that were not tested. The
top figure can be related to Fig. 2 by 105° x rotation,
and the bottom can be related to the top by 180° x
rotation.
|
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The engineered Der f 2 has already begun to be applied for
immunotherapy strategies. For example, the mutated allergen in which
the disulfide bond 8-119 was disrupted retained complete activity to
stimulate T-cell proliferation (43). However, developments of
monovalent ligands to allergen-specific IgE are also desired to
suppress the symptoms by blocking IgE receptor aggregation. Since the
protein HE1 is a human homolog of Der f 2, chimeric proteins of HE1,
and Der f 2 would be candidates of monovalent IgE ligands that induce
no additional responses of antibodies. The tertiary structure of Der f
2 will provide the basis of such strategies and any other drug design
processes.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Mark B. Swindells of Helix
Research Institute, Dr. Daisuke Kohda of Biomolecular Engineering
Research Institute, Prof. Hikoichi Sakai of Japan Women's University,
Dr. Hajime Karasuyama of Tokyo Metropolitan Institute of Medical
Science, Dr. Koji Nagata of Tokyo University, and our colleagues in the
laboratories of Tokyo Metropolitan Institute of Medical Science and
Asahi Breweries, Ltd. for stimulating discussions and advice.
 |
FOOTNOTES |
*
This work was supported in part by the Tokyo Metropolitan
Government (to F. K.).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 restraints (codes 1AHK, 1AHM, R1AHKMR)
have been deposited in the Protein Data Bank, Brookhaven National
Laboratory, Upton, NY.
¶
To whom correspondence should be addressed. Tel.:
81-3-3823-2101; Fax: 81-3-3823-1247; E-mail:
hatanaka{at}rinshoken.or.jp.
1
The abbreviations used are: IgE, immunoglobulin
E; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect;
RMSD, root mean square difference.
2
S. Kojima, unpublished observation.
 |
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