From The Austin Research Institute, Austin Hospital,
Studley Road, Heidelberg, Victoria 3084, Australia and ¶ The
Centre for Drug Design and Development, University of Queensland,
Brisbane, Queensland 4072, Australia
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ABSTRACT |
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The high affinity receptor for IgE
(Fc Fc The binding of IgE by Fc Studies from our group and others using chimeric receptors
together with the epitope mapping of anti-Fc Despite the localization of multiple binding regions in Fc Generation of Chimeric Fc Chimeric Fc Chimeric and mutant receptor cDNA expression constructs were
produced by subcloning the cDNAs into the eukaryotic expression vector pKC3 (17). Each cDNA was engineered in the PCRs to have an
EcoRI site at their 5'-end (the 5'-flanking oligonucleotide primer containing an EcoRI recognition site) and a
SalI site at their 3' end (the 3'-flanking oligonucleotide
primer containing a SalI recognition site), which enabled
the cDNAs to be cloned into the EcoRI and
SalI sites of pKC3. The nucleotide sequence integrities of
the chimeric cDNAs were determined by dideoxynucleotide chain
termination sequencing (18) using SequenaseTM (U.S.
Biochemical Corp.) as described (19).
Monoclonal Antibodies and Ig Reagents
The anti-Fc Transfection
COS-7 cells (30-50% confluent per 5-cm2 Petri
dish) were transiently transfected with FcR cDNA expression
constructs by the DEAE-dextran method (22). Cells were incubated with a
transfection mixture (1 ml/5-cm2 dish) consisting of 5-10
mg/ml DNA, 0.4 mg/ml DEAE-dextran (Amersham Pharmacia Biotech), and 1 mM chloroquine (Sigma) in Dulbecco's modified Eagle's
medium (Flow Laboratories, Australia) containing 10% (v/v) Nuserum
(Flow Laboratories, Australia), for 4 h. The transfection mixture
was then removed, and the cells were treated with 10% (v/v) dimethyl
sulfoxide in phosphate-buffered saline (7.6 mM
Na2HPO4, 3.25 mM
NaH2PO4, 145 mM NaCl), pH 7.4, for
2 min, washed, and returned to fully supplemented culture medium for
48-72 h before use in assays. COS-7 cells were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% (v/v) heat-inactivated fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine (Commonwealth Serum
Laboratories, Melbourne, Australia), and 0.05 mM
2-mercaptoethanol (Koch Light Ltd., Birmingham, United Kingdom).
Ig Binding Assays
The binding of Ig by COS-7 cells following transfection with
chimeric or mutant receptor cDNAs was determined using two approaches.
Erythrocyte-Antibody Rosetting--
COS-7 cell monolayers
transfected with FcR expression constructs were incubated with EA
complexes, prepared by coating sheep red blood cells with
trinitrobenzene sulfonate (Fluka Chemika, Switzerland) and then
sensitizing these cells with mouse IgE or IgG1
anti-2,4,6-trinitrophenyl mAb (23). Two ml of 2% EAs (v/v) were added
per 5-cm2 dish of transfected cells and incubated for 5 min
at 37 °C. Plates were then centrifuged at 500 × g
for 3 min and placed on ice for 30 min. Unbound EA were removed by
washing with L-15 medium modified with glutamine (Flow Laboratories,
Melbourne, Australia) and containing 0.5% bovine serum albumin.
Direct Binding of Monomeric Human IgE--
COS-7 cells
transfected with FcR expression constructs were harvested; washed in
phosphate-buffered saline, 0.5% bovine serum albumin; and resuspended
at 107 cells/ml in L-15 medium, 0.5% bovine serum albumin.
50 µl of cells were incubated with 50-µl serial dilutions of
125I-hIgE for 120 min at 4 °C. 125I-hIgE was
prepared by the chloramine-T method as described (24) and shown to
compete equally with unlabeled hIgE in binding to Fc receptor
expressing COS-7 cells. Cell-bound 125I-hIgE was determined
following centrifugation of cells through a 3:2 (v/v) mixture of
dibutylphthalate and dioctylphthalate oils (Fluka Chemika, Buchs,
Switzerland), and cell bound 125I-hIgE was determined.
Nonspecific hIgE binding was determined by assaying on mock-transfected
cells and subtracted from total binding to give specific hIgE bound.
Levels of COS-7 cell surface expression of the mutant Fc Generation of Fc Molecular modeling of domain 2 (D2) of human Fc Molecular Modeling of the Extracellular Domains of
Fc Chimeric Receptors Identify Multiple Regions of Fc
As described above, the segment of Fc Fine Structure Analysis of the Fc
First, the individual alanine substitution of residues
Lys154-Glu161 in the F-G loop indicated that
each mutant retained hIgE binding, with the striking exception of the
Val155-Ala mutant, where binding of monomeric hIgE was
almost totally abolished, this receptor exhibiting only 3.2 ± 2.1% (mean ± S.D.) binding relative to the wild-type receptor
(Fig. 3, A and D). The loss of hIgE binding by
this mutant receptor was not due to decreased cell surface expression
as demonstrated by its expression on the cell surface in levels
comparable with that of wild-type Fc
As observed for residues Lys154-Glu161 of the
F-G loop, alanine substitution of residues
Tyr129-His134 of the C'-E loop was found to
result in loss or enhancement of hIgE binding. Substitution of
Tyr131 and Glu132 substantially decreased hIgE
binding to 30.3 ± 4.4 and 61.4 ± 3.9% that of wild-type
Fc
Although the chimeric receptor strategy failed to reveal a direct
binding role for the B-C loop (residues
Gly109-Tyr116), mutagenesis of residues
Trp113, Val115, and Tyr116 within
this loop suggests that it may also contribute to IgE binding by
Fc Two approaches have been used to identify and analyze the IgE
binding site of Fc The molecular model of Fc Interestingly, a recent study examining the IgE inhibitory capacity of
synthetic peptides designed to mimic regions of Fc The alanine scanning mutagenesis of the F-G, C'-E, and B-C loops of
Fc Studies examining the binding regions on the Fc portion of IgE for
Fc The findings described herein for FcRI) plays an integral role in triggering IgE-mediated
hypersensitivity reactions. The IgE-interactive site of human Fc
RI
has previously been broadly mapped to several large regions in the
second extracellular domain (D2) of the
-subunit (Fc
RI
). In
this study, the IgE binding site of human Fc
RI
has been further
localized to subregions of D2, and key residues putatively involved in
the interaction with IgE have been identified. Chimeric receptors
generated between Fc
RI
and the functionally distinct but
structurally homologous low affinity receptor for IgG (Fc
RIIa) have
been used to localize two IgE binding regions of Fc
RI
to amino
acid segments Tyr129-His134 and
Lys154-Glu161. Both regions were capable of
independently binding IgE upon placement into Fc
RIIa. Molecular
modeling of the three-dimensional structure of Fc
RI
-D2 has
suggested that these binding regions correspond to the "exposed"
C'-E and F-G loop regions at the membrane distal portion of the domain.
A systematic site-directed mutagenesis strategy, whereby each residue
in the Tyr129-His134 and
Lys154-Glu161 regions of Fc
RI
was
replaced with alanine, has identified key residues putatively involved
in the interaction with IgE. Substitution of Tyr131,
Glu132, Val155, and Asp159
decreased the binding of IgE, whereas substitution of
Trp130, Trp156, Tyr160, and
Glu161 increased binding. In addition, mutagenesis of
residues Trp113, Val115, and Tyr116
in the B-C loop region, which lies adjacent to the C'-E and F-G loops,
has suggested Trp113 also contributes to IgE binding, since
the substitution of this residue with alanine dramatically reduces
binding. This information should prove valuable in the design of
strategies to intervene in the Fc
RI
-IgE interaction for the
possible treatment of IgE-mediated allergic disease.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RI binds monomeric IgE with high affinity
(Ka = 1010 M
1) and is
expressed on mast cells, basophils (1, 2), Langerhans cells (3, 4),
peripheral blood dendritic cells (5), eosinophils (6), and monocytes
(7). The receptor can exist in two distinct multimeric forms, a
tetrameric complex comprising an
-subunit, a
-subunit, and a
disulfide-bonded homodimer of two
-subunits (1, 2) or a trimeric
complex (
2), which lacks the
-subunit (5, 7). The
-subunit of Fc
RI (Fc
RI
) is the IgE binding chain and is
structurally related to the Ig binding chains of the leukocyte
receptors for IgG (Fc
R) and IgA (Fc
R), containing an
extracellular region of two Ig-like domains (1). The associated
-
and
-subunits plays crucial roles in both cell surface expression and signal transduction of the receptor (8).
RI on mast cells and basophils is a
fundamental step in the cascade of events that lead to allergic disease. The interaction of multivalent allergen with Fc
RI-bound IgE
results in cross-linking of the receptor, which triggers a range of
biological sequelae that ultimately leads to the release of
inflammatory mediators and the onset of the type I hypersensitivity response (1, 2). Approaches that intervene in the binding of IgE by
Fc
RI may prove useful in the treatment of allergic disease. Clearly,
understanding the molecular basis of the interaction of Fc
RI with
IgE would provide valuable information for such a therapeutic strategy.
RI
monoclonal
antibodies have identified the second extracellular domain of
Fc
RI
as the principle IgE interactive domain (9-12). The first
extracellular domain has not been demonstrated to have a direct IgE
binding role; however, it does appear to make an important structural contribution in the maintenance of the high affinity IgE binding of the
receptor (9, 11). Multiple regions of Fc
RI
-D2 have been
implicated in the binding of IgE. In a series of "gain of function"
experiments using chimeric Fc
RI
/Fc
RIIa receptors, we
identified three relatively large regions of Fc
RI
-D2, each capable of independently binding IgE (9). The Fc
RI
regions encompassed by residues Trp87-Lys128,
Tyr129-Asp145, or
Ser146-Val169 when inserted into Fc
RIIa
were each able to impart IgE binding to the receptor. Mallamaci
et al. (10) have used a similar approach with chimeric
Fc
RI
/Fc
RIII receptors, however, in "loss of function" experiments and identified four regions of Fc
RI
-D2 that
putatively contribute to IgE binding. The replacement of each of the
Fc
RI
regions encompassed by residues
Ser93-Phe104,
Arg111-Glu125,
Asp123-Ser137, and
Lys154-Ile167 with the corresponding regions
of Fc
RIII, was found to result in reduced IgE binding. In addition,
a recent study by McDonnell et al. (13) has demonstrated
that residues Ile119-Tyr129 of Fc
RI
-D2,
when synthesized as a conformationally constrained peptide, can inhibit
the binding of IgE to Fc
RI.
RI
-D2,
the interaction of Fc
RI with IgE at the level of individual residues
has not been defined. In this study, we have identified small IgE
binding subregions of Fc
RI
-D2, which have been analyzed by
site-directed mutagenesis, and residues putatively involved in the
interaction with IgE have been determined. These findings have enabled
the development of a model of how Fc
RI
binds IgE and contribute
to our understanding of the interaction of the leukocyte FcR family
with their Ig ligands.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RI
/Fc
RIIa and Mutant Fc
RI
Receptor cDNA Expression Constructs
RI
/Fc
RIIa or mutant Fc
RI
cDNAs
were constructed by splice overlap extension
PCR1 (14) using an
expressible form of the Fc
RI
chain (15) or Fc
RIIaNR cDNA (16) as templates. The expressible
form of the Fc
RI
chain consists of the extracellular region of
Fc
RI
linked to the transmembrane and cytoplasmic tails of
Fc
RIIa and is expressed on the cell surface and binds monomeric hIgE
with an affinity comparable with that of the wild-type Fc
RI
chain, as described previously (15). Splice overlap extension PCR was
performed as follows. Two PCR reactions were used to amplify the
Fc
RI
-Fc
RIIa or Fc
RI
fragments to be spliced together.
The reactions were performed on 100 ng of the Fc
RI
cDNA in
the presence of 500 ng of each oligonucleotide primer, 1.25 mM dNTPs, 50 mM KCl, 10 mM Tris-Cl,
pH 8.3, 1.25 mM dNTPs, 1.5 mM MgCl2
using 2.5 units of Taq polymerase (Amplitaq; Cetus) for 25 amplification cycles. A third PCR was performed to splice the two
fragments and amplify the spliced product. 100 ng of each purified
fragment was used with the appropriate oligonucleotide primers under
the above PCR conditions.
RI
mAb 3B4 and the anti-Fc
RIIa mAb 8.2 were
produced in this laboratory (20). The anti-Fc
RI
mAb 15A5 was a
gift of Dr. J. Kochan (12). The mouse IgE anti-2,4,6-trinitrophenyl mAb
(TIB142) was produced from a hybridoma cell line obtained from the
American Type Culture Collection (Rockville, MD); the mouse
IgG1 anti-2,4,6-trinitrophenyl mAb (A3) was produced from a
hybridoma cell line that was a gift of Dr. A. Lopez (21). Human IgE
myeloma protein was purified from the serum of a myeloma patient. IgE
was precipitated with NH4SO4, and then IgG was
removed by chromatography on protein A, and IgE was purified by size
fractionation chromatography on Sephacryl S-300 HR ( Amersham Pharmacia
Biotech). Purified IgE was analyzed by SDS-polyacrylamide gel
electrophoresis and by enzyme-linked immunosorbent assay, and
contaminating IgG was estimated at <1%.
RI
receptors were determined by assessing the binding of the
anti-Fc
RI
mAb 22E7 (shown to bind distantly to the binding site;
see Ref. 12) at 2 µg/ml in a direct binding assay as described for
the binding of hIgE. Any variation in cell surface receptor expression
between the mutant Fc
RI
and wild-type Fc
RI
COS-7 cell
transfectants (levels ranged from 80 to 120% of wild-type Fc
RI
)
was then normalized, and the binding of hIgE by the mutant Fc
RI
receptors was corrected using the following formula: (mutant
mock IgE binding) × ((wild type
mock 22E7 binding)/(mutant
mock 22E7 binding)).
RI
Domain 2 Model Structure
RI
was
performed using the Homology and Discover modules of the InsightII environment of Molecular Simulations Inc. on a Silicon Graphics Indigo
workstation. The model of Fc
RI
-D2 was constructed by mutation of
our previously described model of human Fc
RIIa-D2 (25), which was
based on the crystal structure of domain 2 of CD4 (protein data base
file pbd2cd4.ent; Brookhaven National Laboratory, Upton, NY) (26, 27).
Briefly, using a sequence alignment of Fc
RI
-D2 with Fc
RIIa-D2
and CD4-2 (28), regions of Fc
RIIa-D2 aligned with the
-sheet
residues of CD4-2 were designated as structurally conserved residues,
with other residues designated as loops. The coordinates for the atoms
of the structurally conserved residues of Fc
RIIa were assigned from
those of the equivalent residues in the pbdcd4 file, with the
coordinates of the side chain atoms assigned through mutation of the
pbdcd4 side chains. Using the Homology Loop Search function, segments
of Protein Data Bank files with the correct number of residues and
appropriate gap distance were obtained. Incorporation of the loops was
then followed by elimination of severe atomic overlaps ("bumps") by altering the torsion angles of side chain C
-C
bonds. The
structure was then minimized using the Discover module to a maximum
root-mean-square derivative of 0.0001. The Fc
RIIa-D2 model was then
converted to a Fc
RI
-D2 model by mutation of the residues followed
by further minimization of the structure using the above protocol.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RI
--
The three-dimensional structure of Fc
RI
has not
yet been solved. To aid in the localization of putative IgE binding
regions of Fc
RI
, we have generated a three-dimensional model of
the second extracellular domain (D2) of Fc
RI
based on the known structure of a related domain, CD4-2. CD4-2 belongs to the C2 set of Ig
superfamily members, and sequence alignment of Fc
RI
-D2 with CD4-2
suggests that this domain will adopt a similar folding pattern (25,
28). The structure of the Fc
RI
-D2 model is characteristic of the
C2 Ig-fold, comprising seven
-strands (A, B, C, C', E, F, G) that
form two antiparallel
-sheets of ABE and CC'FG (Fig.
1A).
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Fig. 1.
Molecular modeling of the extracellular
region of human Fc RI
domain 2 and location of residues putatively involved in the
interaction with IgE. A, Fc
RI
domain 2 model
structure displayed as a ribbon diagram. The
model is orientated such that it adjoins domain 1 at the top
and the transmembrane region at the bottom. The three
regions of domain 2 putatively involved in IgE binding (B-C, C'-E, and
F-G loops) are shown in white. An additional region (C'-C
loop) postulated to also be involved in IgE binding (13) is shown in
magenta, with the remainder of the domain shown in
green. B, location of residues putatively
involved in the interaction of Fc
RI
domain 2 with IgE. The side
chains of amino acids implicated in IgE binding as described under
"Results" are indicated. Those side chains that when substituted
result in decreased or increased binding are shown in red or
yellow, respectively. The computer model of Fc
RI
domain 2 was generated by molecular modeling based on the structure of
the related CD4 domain 2 as described under "Experimental
Procedures."
RI
Involved
in IgE Binding--
Based on the location of the previously described
IgE binding regions (9-13) on our three-dimensional molecular model of
Fc
RI
-D2 and by analogy with mapping studies of the homologous
interaction of the Fc
R with IgG (25, 28-31), we targeted the B-C,
C'-E, and F-G loop regions of Fc
RI
-D2 as likely to be involved in the binding of IgE. In order to assess the contribution these three
loops made to the binding of IgE by Fc
RI
, chimeric receptors were
generated, whereby Fc
RIIa was used as a scaffold to accept each of
these Fc
RI
loop regions. The three resultant chimeric receptors
consisted of Fc
RIIa containing the following regions of
Fc
RI
-D2: (i) the B-C loop, residues
Gly109-Tyr116; (ii) C'-E loop, residues
Tyr129-His134; and (iii) F-G loop, residues
Lys154-Glu161, designated the
109-116
,
129-134
, and
154-161
chimeric receptors, respectively.
COS-7 cells were transfected with expression constructs of these
chimeric receptors and tested for their capacity to bind mouse IgE (or
IgG1) immune complexes by EA rosetting. Cells transfected
with the
154-161
chimeric receptor bound IgE-EA (Fig.
2A, Table
I), and the binding was specific, since
mock-transfected cells or cells transfected with Fc
RIIa did not bind
IgE-EA (Table I). These data indicate that the Lys154 to
Glu161 region of Fc
RI
can direct the binding of IgE.
As expected, this chimeric receptor was unable to bind
IgG1, since the previously described IgG binding region,
residues Asn154-Ser161 (25), has been replaced
with the homologous Fc
RI
sequence (Table I). Similar experiments
demonstrated that the
129-134
chimeric receptor could also
specifically bind IgE-EA (Fig. 2B, Table I), indicating that
the Tyr129-His134 region also contains an IgE
binding site. As expected, this chimeric receptor was able to bind
IgG1-EA (Table I) due to the presence of the Fc
RIIa
Asn154-Ser161 IgG binding sequence.
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Fig. 2.
IgE complex binding of chimeric Fc
receptors. COS-7 cell monolayers were transfected with the
following chimeric cDNA constructs: 154-161
(A),
129-134
(B), and
109-116
(C). The
binding of IgE immune complexes was assessed directly on monolayers by
EA rosetting using mouse IgE-sensitized erythrocytes. The transfections
were performed in a transient expression system, resulting typically in
30-50% of cells expressing the chimeric FcR. Cells were considered to
be expressing functional receptors if >10 red blood cells were bound
per cell.
Chimeric FcR composition and Ig complex binding
RI
chain, and unshaded regions are derived from Fc
RIIa. The relative
positions of the putative
-strands are shown above as labeled solid
lines. The binding of mouse IgE and IgG1 (mIgE and mIgG) was
assessed by EA rosetting as described under "Experimental
Procedures." +, >10% of cells rosetting;
, no cells
rosetting.
RI
-D2 encompassed by
residues 87-128 had previously been shown to contain an IgE binding site, which we predicted to be the B-C loop (28). However, when transfected into cells, the
109-116
chimeric receptor containing the Fc
RI
B-C loop did not bind IgE-EA (Fig. 2C). Since
the receptor was clearly expressed on the cell surface, demonstrated by
its ability to bind IgG-EA (Table I), these results suggest that the
Gly109-Tyr116 region is insufficient to bind
IgE in its own right and therefore that the IgE binding region in the
87-128 segment is either not the B-C loop or requires the B-C loop in
combination with additional surrounding region(s). This was further
investigated by site-directed mutagenesis (see below).
RI
IgE Binding Site--
To
identify the key residues of the Fc
RI
binding regions (C'-E loop,
residues Tyr129-His134; F-G loop, residues
Lys154-Glu161) involved in the interaction
with IgE, site-directed mutagenesis was used to replace each residue in
these regions with alanine. In addition, residues Trp113,
Val115, and Tyr116 in the B-C loop were also
substituted with alanine, since the Fc
RI
-D2 model predicts this
region is likely to be adjacent to the F-G and C'-E loops and may
therefore contribute to IgE binding. The alanine substitution mutants
of Fc
RI
were expressed in COS-7 cells, and the binding of
monomeric human IgE was examined in direct binding assays by titration
of 125I-labeled hIgE (Fig.
3). The levels of cell surface expression of the Fc
RI
mutants on the COS-7 cell transfectants were
determined using the Fc
RI
mAb 22E7, shown to detect an epitope
distant to the binding site (12). Using these results, the binding of hIgE was corrected for variation in expression between the mutant receptors, which ranged from 80 to 120% of wild-type Fc
RI levels (data not shown).
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Fig. 3.
Monomeric human IgE binding by
Fc RI
alanine point
mutants. Radiolabeled monomeric human IgE was titrated on COS-7
cells transfected with wild-type Fc
RI
or Fc
RI
containing
alanine point mutations. A, F-G loop mutants, wild-type
Fc
RI
(
), Lys154
Ala (×), Val155
Ala (
), Trp156
Ala (
), Gln157
Ala (
), Leu158
Ala (
), Asp159
Ala
(
), Tyr160
Ala (
), Glu161
Ala
(
). B, C'-E loop mutants, wild-type Fc
RI
(
),
Tyr129
Ala (
), Trp130
Ala (
),
Tyr131
Ala (
), Glu132
Ala (
),
Asn133
Ala (
), His134
Ala (
);
C, B-C loop mutants, wild-type Fc
RI
(
),
Trp113
Ala (
), Val115
Ala (
),
Tyr116
Ala (
). A comparison of the levels of IgE
binding with the Fc
RI
mutants relative to wild-type Fc
RI
is
shown. D, F-G loop mutants. E, C'-E loop mutants.
F, B-C loop mutants. The percentage of binding was
calculated from hIgE bound at a concentration of 2 µg/ml. The binding
of wild-type Fc
RI
was taken as 100% and mock-transfected cells
as 0% binding. Results are expressed as means ± S.E. To control
for variable receptor expression between the mutant Fc
RI COS-7 cell
transfectants, levels of expression were determined using a
radiolabeled monoclonal anti-Fc
RI antibody 22E7, and IgE binding was
normalized to that seen for wild-type Fc
RI (see "Experimental
Procedures").
RI (data not shown). The
substitution of Asp159 with alanine also resulted in
diminished IgE binding, this receptor exhibiting 52.7 ± 7.2%
binding of the wild-type receptor. The substitution of
Lys154, Gln157, and Leu158 with
alanine had no significant effect on the binding of IgE, these mutants
exhibiting binding comparable with wild-type Fc
RI
. In contrast,
the replacement Trp156, Tyr160, or
Glu161 with alanine produced the interesting effect of
increasing the binding of IgE (132.7 ± 14.0, 123.7 ± 11.1, and 139 ± 15.0% of wild-type Fc
RI
, respectively).
Therefore, these findings clearly identify five individual residues of
the F-G loop of Fc
RI
(Val155, Trp156,
Asp159, Tyr160, and Glu161 as
playing critical roles in the binding of hIgE. The observation that
substitution of Val155 and Asp159 decreased
binding suggests that these residues may directly interact with hIgE.
The increased binding observed upon substitution of Trp156,
Tyr160, and Glu161 also suggests an important
role for these residues, which is possibly a contribution to the
structural integrity of the binding site, although a direct role in
hIgE binding cannot be excluded.
RI
(Fig. 3, B and E). In contrast, replacement of Trp130 dramatically increased binding by
over 70% to 172.5 ± 8.8% binding of the wild-type receptor. The
substitution of Tyr129, Asn133, and
His134 had no significant effect on the binding of hIgE,
since these mutants exhibited binding comparable with that seen for
wild-type Fc
RI
(data not shown). These findings suggest that
Trp130, Tyr131, and Glu132 may play
an important role in the binding of hIgE. Again, a distinction between
a possible direct binding role or contribution to structural integrity
of the receptor cannot be made. However, as for the mutagenesis studies
of the F-G loop, these results clearly identify the C'-E loop as also
playing a role in the binding of IgE by Fc
RI
.
RI
. This was demonstrated, since the substitution of
Trp113 for alanine resulted in a dramatic loss of IgE
binding, this mutant receptor exhibiting only 18.6 ± 3.2% of IgE
binding relative to the wild-type receptor. Substitution of
Val115 or Tyr116 with alanine did not
significantly alter IgE binding (Fig. 3, C and
F).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RI
. First, to localize IgE binding regions of
Fc
RI
, a series of chimeric FcRs were engineered by exchange of
segments between the second domain of Fc
RI
and Fc
RIIa. Second, fine structure analysis of these binding regions, and an additional region likely to be in juxtaposition, was performed by generating 17 point mutants in Fc
RI
using alanine scanning mutagenesis. These
approaches have enabled the localization of IgE binding regions in
Fc
RI
to subregions of the second extracellular domain and
identified key residues putatively involved in the interaction with
IgE. Based on a molecular model of Fc
RI
-D2, these data suggest
that the IgE binding regions comprise the F-G, C'-E, and B-C loops and
adjacent strand regions of this domain. Both the F-G and C'-E loops
were directly implicated in the interaction with IgE, since insertion
of these regions into Fc
RIIa was able to impart IgE binding to this
receptor. In contrast, insertion of the B-C loop was itself
insufficient to direct the binding of IgE. However, site-directed
mutagenesis of this region identified the residue Trp113 as
playing an important binding role, which provides evidence to suggest
that the B-C loop also contributes to the interaction with IgE.
RI
-D2 suggests that the F-G, C'-E, and
B-C loops of Fc
RI
-D2 are likely to be juxtaposed at the membrane
distal end of the domain at the interface with domain 1. The
localization of the Fc
RI
-D2 IgE interactive sites to this region,
together with the finding that domain 1 also plays a key role in
maintaining high affinity binding of the receptor (9, 11), suggests
that the interdomain region between domains 1 and 2 comprises an
important region of interaction of Fc
RI
with IgE. In support of
this model, the anti-Fc
RI
mAb 15A5, which recognizes an epitope
in the B-C loop region of Fc
RI
-D2, is able to block the binding
of IgE to Fc
RI completely (12), suggesting that the multiple IgE
binding regions are likely to be situated in close proximity to one another.
RI
-D2 has also
implicated the C'-C loop (residues
Ile119-Tyr129 as playing a role in the binding
of IgE (13). A peptide encompassing this region and designed to mimic
the conformation of the C'-C loop was demonstrated to competitively
inhibit IgE binding to Fc
RI
and prevent IgE-mediated mast cell
degranulation in vitro. Thus, the inclusion of the C'-C
loop with the B-C, C'-E, and F-G loops described herein extends the
putative region of contact of Fc
RI
with IgE. These data therefore
suggest that the entire four-stranded
-sheet face of Fc
RI
-D2,
namely the C-C'-F-G strands and adjacent loops, may be important in the
interaction of Fc
RI
with IgE.
RI
-D2 has identified a number of residues that may contribute
to the binding of IgE. The substitution of amino acids Trp113, Tyr131, Glu132,
Val155, and Asp159 with alanine decreased the
binding of IgE, whereas substitution of Trp130,
Trp156, Tyr160, and Glu161
increased binding. Based on the three-dimensional model of
Fc
RI
-D2, the side chains of these residues are exposed
predominantly on the surface of the domain and contribute to a
continuous face in the C-C'-F-G region (Fig. 1B). The
majority of these residues are aromatic (Trp113,
Trp130, Tyr131, Trp156,
Tyr160) or charged (Glu132, Asp159)
and are likely candidates for direct contact with IgE.
RI
have identified a number of putative interactive sites
(32-36). The third constant domain (C
3) appears to be the principle
Fc
RI
binding domain, containing major binding sites in the
C
2/C
3 junction and the C
3 A-B loop region. Both of these regions contain a number of exposed aromatic and charged residues that
may form a complementary surface for interaction with that described
herein for Fc
RI
. Interestingly, the C
2/C
3 junction region
is located distally to the C
3 A-B loop, suggesting a discontinuous binding site in IgE-Fc. This implies that the C
2/C
3 and C
3 A-B
loop may interact with different regions of Fc
RI
. Since the
Fc
RI
binding site appears to comprise a single continuous region
in the C-C'-F-G face of domain 2, it is therefore possible that a
second binding site distant from this region (e.g. in domain 1) may also exist. The definition of the precise molecular basis of the
interaction between Fc
RI
and IgE awaits the elucidation of the
structure of Fc
RI
-IgE complexes.
RI
when compared with similar
studies of the structurally related Fc
Rs, i.e. Fc
RI (37), Fc
RIIa (25, 29), and Fc
RIII (30, 31), reveal a number of
similarities in the molecular basis of how these receptors interact
with their respective ligands. The two Ig-like domains of the
extracellular regions of Fc
RI
, Fc
RIIa, and Fc
RIII and the
first two domains of the three domains of Fc
RI clearly represent a
structurally conserved Ig binding motif of this receptor family. In
each of these receptors, it is the second extracellular domain that is
responsible for the direct binding of Ig, with the first domain making
an as yet undefined contribution to maintain optimal binding affinity.
The localization of Ig-binding regions in domain 2 of Fc
RI
,
Fc
RIIa, and Fc
RIII has identified common regions of these
receptors that are involved in the interaction with their Ig ligands
(Fig. 4). The three loop regions
identified herein as involved in the binding of IgE by Fc
RI
,
namely the F-G, C'-E, and B-C, have also been implicated as crucial in
the binding of IgG by Fc
RIIa (25, 29) or Fc
RIII (30, 31) (Fig.
4). The C'-C loop of Fc
RI
and Fc
RIII also contributes to Ig
binding in both of these receptors (13, 30, 31); however, it does not
appear to be involved in Fc
RIIa (29) (Fig. 4). Thus, the focus of
the interaction of Fc
RI
and Fc
RIII with Ig exhibits some
differences to that of Fc
RIIa. However, it is clear from all of
these studies that the above mentioned loop regions of the second
extracellular domain, which contribute to the four-strand
-sheet
(C-C'-F-G) face, constitute the major Ig interactive regions of these
receptors. Thus, despite Fc
RI
exhibiting a distinctly different
specificity and affinity for Ig to the Fc
R, structural similarities
are likely to be maintained between these receptors in their
interaction with Ig.
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Fig. 4.
Ig binding regions of leukocyte FcR. A
schematic diagram is shown of model structures proposed for domain 2 of
human Fc RI, Fc
RII, and Fc
RIII, showing the location of amino
acid residues implicated in Ig binding. The models are based on the
known structure of CD4-domain 2 (see Refs. 26 and 27) and are shown in
ribbon form with
-strands labeled and depicted as arrows.
The models are oriented with the four
-strand C'CFG face at the
front and adjoin domain 1 at the top and the
transmembrane region at the bottom. The predicted positions
of amino acids implicated in Ig binding through mutagenesis studies
(see "Fine Structure Analysis of the Fc
RI
IgE Binding Site"
for details) are indicated with red circles and labeled in single
letter code with their residue number. The C'-C loop region of Fc
RI
implicated in IgE binding using peptide inhibition studies is
highlighted in red. The data are compiled from this paper
and Refs. 13, 25, 29, 30, and 31.
Understanding the molecular basis of the interaction of FcRI
with
IgE will assist in the design of therapeutic strategies to treat
IgE-mediated allergic disease by blocking the binding of IgE by
Fc
RI
. The contribution to the definition of the IgE binding site
of Fc
RI
as described herein represents a step toward the
possibility of rational design of such therapeutic agents. The recent
demonstration that the structure-based design of a constrained peptide
of the C'-C loop of Fc
RI
-D2 can inhibit IgE binding to Fc
RI
highlights the feasibility of a rational approach (13). The IgE binding
loops of Fc
RI
-D2 identified herein, i.e. F-G and C'-E,
may represent other candidate regions for similar studies. The ability
of recombinant soluble Fc
RI
to inhibit the binding of IgE to cell
surface Fc
RI has also been demonstrated (38-40). The engineering of
higher affinity forms of soluble Fc
RI
, such as the
Trp130
Ala, Trp156
Ala,
Tyr160
Ala, and Glu161
Ala as described
in this study, may provide more effective therapeutic agents.
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ACKNOWLEDGEMENTS |
---|
We thank Jim Karkaloutsos and Ewa Witort for technical assistance.
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FOOTNOTES |
---|
* This work was supported by the National Health and Medical Research Council and Harry Triguboff.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.
§ Recipient of a National Health and Medical Research Council Australian Postdoctoral Research Award.
To whom correspondence should be addressed. Tel.:
61-3-9287-0666; Fax: 61-3-9287-0600.
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ABBREVIATIONS |
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The abbreviations used are: PCR, polymerase chain reaction; mAb, monoclonal antibody; hIgE, human IgE; EA, erythrocyte-antibody.
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REFERENCES |
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