(Received for publication, October 12, 1995; and in revised form, December 11, 1995)
From the
We have investigated the capacity of N- and C-terminally
truncated and chimeric human (h) IgE-derived peptides to inhibit the
binding of I-labeled hIgE, and to engage cell lines
expressing high and low affinity receptors (Fc
RI/II). The peptide
sequence Pro
-Ser
of the hC
3
domain is common to all h
-chain peptides that recognize
hFc
RI. This region in IgE is homologous to the A loop in C
2
that engages the rat neonatal IgG receptor. Optimum Fc
RI occupancy
by hIgE occurs at pH 6.4, with a second peak at 7.4. N- or C-terminal
truncation has little effect on the association rate of the ligands
with this receptor. Dissociation markedly increases following
C-terminal deletion, and hFc
RI occupancy at pH 6.4 is diminished.
His residue(s) in the C-terminal region of the
-chain may thus
contribute to the high affinity of interaction. Grafting the homologous
rat
-chain sequence into hIgE maintains hFc
RI interaction
without conferring binding to rat Fc
RI. hFc
RII interaction is
lost, suggesting that these residues also contribute to hFc
RII
binding. h
-chain peptides comprising only this sequence do not
block hIgE/hFc
RI interaction or engage the receptor. Therefore,
sequences N- or C-terminal to this core peptide provide structures
necessary for receptor recognition.
Antibodies of the immunoglobulin (Ig)E isotype sensitize target
cells expressing the class-specific Fc receptors for antigen-induced
mediator release, by binding through residues located in the Fc portion
of the molecule(1, 2) . The potent pharmacologically
active substances that are released in response to this stimulus cause
the clinical symptoms of allergy. Strategies that block the initial
sensitization of target cells with antigen-specific IgE have been
explored following the demonstration that human (h) ()myeloma IgE-derived Fc
fragments generated by
proteolytic cleavage with papain(1, 2) , which
produces peptides comprising h
-chain residues 1-226 and
227-547, can competitively inhibit the binding of IgE to cells
expressing high affinity receptor (Fc
RI)(1, 2) ,
whereas cleavage products of pepsin digestion, which generates
fragments spanning residues 1-338, 339-349, and
350-547 (3) do not inhibit binding. This observation
initiated the quest for progressively smaller peptides as potential IgE
antagonists (reviewed in (4) ). In early studies, the
inhibition of passive cutaneous anaphylaxis in human skin was used to
assess the Fc
RI-blocking activity of proteolytic fragments or
recombinant IgE-derived peptides expressed in Escherichia
coli(1, 5, 6) . This led to the proposal
that sequences N- and C-terminal to Val
contribute
structures necessary for Fc
RI interaction(6) . More recent
studies aimed at the identification of the receptor binding site(s)
employed chimeric human/mouse IgE antibodies,
/
chimeras,
site-specific mutagenesis, anti-IgE antibodies, or IgE-derived
peptides(7, 8, 9, 10, 11, 12, 13, 14, 15) .
They indicate that the site(s) in IgE that interact(s) with the Fc
receptors depend(s) on structures associated with residues located in
the C
3 domain, although C
4 involvement has also been
invoked(11, 12) . Furthermore, it has been suggested
that IgE/Fc
RI interaction is mediated primarily by electrostatic
interactions (14) and dependent on the entire C
3 in its
native conformation(10) , while the C
4 domains are
essential for the maintenance of the active conformation of the C
3
domain(7, 16) . Our earlier investigations showed that
while IgE/Fc
RII interaction is critically dependent on C
4 or
its homologue C
3(16) , it is possible to delete the entire
C
4 domain and more than 60% of residues in C
3 and still
maintain the Fc
RI-blocking capacity of the recombinant
-chain
fragment(6, 17) .
Based on our demonstration of the
parallel nature of the inter--chain disulfide bonds in
hIgE(18) , we developed a structural model that predicts that
an exposed and probably flexible segment connects the globular portions
of the C
2 and C
3 domains(18, 19) .
Subsequently, Gould et al.(20, 21) claimed
that the N-terminal 11 residues in C
3, which are included in this
segment, are essential for Fc
RI binding. This proposal relied on
studies where the biological activity of recombinant
-chain
fragments was tested by blocking the binding of ragweed-specific IgE to
mast cells in the skin of the senior investigator conducting the
study(6) . When the fallibility of the passive cutaneous
anaphylaxis reaction in assessing the biological activity of
recombinant IgE-derived fragments emerged (17, 22) ,
we re-assessed the biological activity of these and additional
truncated fragments using our recently developed receptor binding
assay, which allowed us to study direct binding of IgE-derived ligands
to rat (r) basophilic leukemia cells (RBL-2/2/C) transfected with the
-chain of hFc
RI(23) .
In the present investigation
we describe the capacity of a series of overlapping N- and C-terminally
truncated and chimeric /
-chain derived fragments, expressed
as glutathione S-transferase (GST) fusion proteins in E.
coli to bind directly to and block the binding of hIgE to
RBL-2/2/C cells. We show that the peptide sequence spanning amino acid
residues Pro
-Ser
is common to all
recombinant
-chain fragments capable of binding to Fc
RI.
Deletion of this sequence is associated with a complete loss of
receptor recognition, confirming earlier observation by others that
grafting the homologous sequence from IgG1 into hIgE reduces Fc
RI
binding by 97%(14) . Replacing this sequence in hIgE by the
homologous rat sequence maintains binding to hFc
RI, but there is a
loss of hFc
RII interaction, confirming earlier observations by
others that rodent IgE recognizes only hFc
RI but not
hFc
RII(10) . Since recombinant GST
-chain fusion
proteins containing this sequence do not block IgE/Fc
RI
interaction, we conclude that sequences N- or C-terminal to this core
peptide are essential for the provision of additional structural
scaffolding in order to generate a receptor binding conformation.
Viewed in the context of our model structure for
IgE(18, 19) , this core peptide has been computed to
form a loop proximal to the interface between the C
3/4 domains
that is homologous to the site in rodent IgG involved in the binding to
the groove formed by the
1 and
2 domains of the neonatal
Fc
Rn(24) . Interestingly, as for IgG/Fc
Rn
interaction, we also observe two pH optima at pH
6.4 and 7.4 for
hIgE/Fc
RI
interaction. While N- or C-terminal truncation has
little effect on the association rate, deletion of C-terminal sequences
increases the rate of dissociation several hundred-fold and reduces
receptor occupancy at pH 6.4. The slow dissociation of IgE from
Fc
RI
therefore may be due, at least in part, to the
stabilization of the interaction by His residues in the C-terminal
region of the ligand.
For the construction of the chimeric h/r IgE molecule we employed
the -chain expression plasmids
pSV-V
h
/r
(18) . A
construct where the sequences known to be essential for hFc
R1
interaction had been replaced by the homologous rat sequence encoding
residues 341-356 was also generated by overlap extension
PCR(25) . The template for PCR was a 3.4-kilobase pair IgE
C
1-4 genomic DNA cassette cloned into the BamHI
site in pUC19 (pH
). A 719-base pair fragment coding essentially
for C
2-3 was generated by PCR.
This involved two rounds of PCR and four primers, two external (5` BglII, CGTGAAGATCTTACAGTCGTC; 3` NcoI, CCTGCCCATGGCTCACCG) and two internal primers (5` h-r16, CCTCGACCTGTATGAAAATGGGACTCCCAAACTTACCTGTCTGGTGGTGGACCTG; 3` h-r16, CCATTTTCATACAGGTCGAGGGGACTGGGTGGGATTAGGTAGGCGCTCACCCCTCT).
For each
PCR, reaction mixtures contained 200 ng of template, 2 µg of each
primer, 1 mM dNTPs, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl in 100 µl, and following
a hot start 1 unit of Taq polymerase was added. An initial
denaturation cycle at 96 °C for 6 min, 64 °C for 2 min, 72
°C for 1 min 30 s was followed by 30 cycles at 94 °C for 1 min
30 s, 64 °C for 1 min 30 s, 72 °C for 1 min 30 s. The resultant
719-base pair fragment was cloned into pH
using BglII and NcoI sites to give a chimeric C
1-4 cassette, which
was subcloned, using the BamHI sites, into the mammalian
expression vector pSV-V
(18) . The orientation of
this cassette was checked by PCR.
The identity of all gene constructs was confirmed by sequencing the DNA of both strands.
The chimeric pSV-V construct was linearized
using PvuI and electroporated into the J558L plasmacytoma cell
line(18) . J558L cells were cultured in Dulbecco's
modified Eagle's medium (10% fetal calf serum,
penicillin/streptomycin, gentamicin) and selection medium
(Dulbecco's modified Eagle's medium, 10% fetal calf serum,
penicillin/streptomycin, gentamicin, mycophenolic acid, xanthine, and
hypoxanthine) was added 48 h after electroporation(18) . High
secreting clones were selected by enzyme-linked immunosorbent assay.
The conditions for maintenance of
RBL-2H3 cell lines transfected with the -chain of hFc
RI
(RBL-2/2/C) have been described(23) . RBL-2/2/C clones were
plated into 48-well plates at an initial plating density of 10
cells/well and incubated with 10
M dexamethazone for 24 h at 37 °C. In preliminary experiments,
RBL-2/2/C cells were incubated with increasing concentrations of
I-labeled ligands (0.1-7.5 µg/ml) to determine
the minimum saturation concentrations for Fc
RI binding. The
proportion of molecules capable of binding to Fc
RI was
78-91% for
I-labeled h and h/r IgE V
,
while the bindable portion of the
I-labeled
GST
-(226-547), GST
-(326-547),
GST
-(340-547), and GST
-(226-354) was
41, 56, 53, and 27%, respectively. The number of rFc
molecules
bound per cell was calculated on the basis that
GST
-(326-547) and GST
-(340-547) are
dimers, while GST
-(226-354) is a monomer.
Nonspecific binding was determined using a 50-100-fold molar
excess of nonlabeled hIgE over I-hIgE, and the same
amount of GST was used as a negative control. The binding of
recombinant proteins to Fc
RI
was determined indirectly, after
correcting for nonspecific binding (7-17%), by calculating the
percentage of inhibition of
I-IgE binding to cells. To
measure the inhibition (IC
) of
I-IgE binding
to RBL-2/2/C or the 8866 lymphoblastoid cell line (18) by
native and chimeric h/r IgE V
and recombinant
-chain
fragments, cells were preincubated with increasing concentrations
(10
-10
M) of each
of the unlabeled peptides in 125 µl of binding buffer or, as
control, binding buffer alone at 22 °C for 1 h, before the addition
of 50 µl of binding buffer containing
I-hIgE (1
nM). After 45 min, the cells were washed twice with 0.5 ml of
ice-cold binding buffer and lysed with 0.5 ml of lysis buffer (0.5 M NaOH, 1% Triton X-100). Samples (0.25 ml) were removed and
counted for 5 min on a LKB1277
-counter.
The kinetics of
association between RBL-2/2/C cells and I-labeled h and
h/r chimera, GST
-(226-547),
GST
-(326-547), GST
-(340-547) (2
µg/ml, in binding buffer), and GST
-(226-354) (0.7
µg/ml, in binding buffer) were measured at 22 °C during the
first 300 s of incubation at pH 7.4. Rate constants were calculated on
the basis that GST
-(226-547),
GST
-(326-547), and GST
-(340-547) are
dimers, while GST
-(226-354) is a monomer. The forward
rate constant (k
) was calculated as V
/C
R
, where V
represents the
initial rate of binding, and C
and R
represent the concentration of ligand and
receptor number (
130,000 h
-chains/cell(23) ). To
determine the dissociation rate constant (k
), cells were preincubated for 1 h at 22
°C with 125 µl of the following
I-labeled
ligands: h and rat IgE, the h/r chimera,
GST
-(226-547), GST
-(326-547),
GST
-(340-547), and GST
-(226-354/7)
(ligand concentration as for the determination of k
). Cells were washed twice with 0.5 ml of
binding buffer before 125 µl binding buffer containing a 50-fold
molar excess of unlabeled hIgE or binding buffer was added. At t
and after 15-, 30-, 60-, 120-, and 180-min
intervals, the cells were washed twice with 0.5 ml of ice-cold binding
buffer, solubilized in 0.5 ml of lysis buffer (0.5 M NaOH, 1%
Triton X-100), and 0.25-ml samples were assayed for cell-bound
I.
The pH optimum for the binding of I-labeled hIgE and the GST
-chain fragments to
Fc
RI was determined by incubating RBL-2/2/C cells (23) in
48-well plates with 100 µl of 50 mM phosphate-buffered
saline containing 0.2% BSA (pH range 5.9-8.1) for 10 min at 37
°C before adding 50 µl of 2 µg/ml
I-hIgE or
0.7 µg/ml of the
I-GST
-chain fragments.
Cells were incubated for 30 min, and the excess protein was removed by
washing with saline containing 0.2% BSA before measurement of
cell-bound label. Results were corrected for nonspecific binding.
In the present study, we focused on the identification of the
site(s) that determine the interaction of hIgE with its cellular
receptors. The strategies employed for the expression of an overlapping
family of chimeric GSTh
-chain fusion proteins are outlined
in Fig. 1. Panel A summarizes the receptor-binding
capacities of the GST
h
-chain fusion proteins, that of a
chimeric
/
peptide, and that of a chimeric h/r IgE molecule.
The assignment of biological activities is based on (i) competition and
(ii) direct binding studies detailed in Fig. 2and Table 1. In Fig. 1, panels C and D show
the electrophoretic mobilities of C- and N-terminally truncated
recombinant GST
h
-chain fusion proteins immunoprecipitated
with a rabbit anti-GST antiserum, followed by PAGE analysis under
nonreducing conditions and immunoblotting with a horseradish
peroxidase-labeled rabbit anti-IgE serum. As shown in Fig. 1,
C-terminal truncation yields a number of
-chain peptides for each
construct. As judged by PAGE (Fig. 1C) and column
chromatography (data not shown), approximately one-third of the
peptides in each set corresponds to the full-length fusion peptide as a
monomeric fragment. None of these fragments show any propensity to
dimerize, although biologically inactive polymeric aggregates form at
concentrations >1.3 mg/ml. A set of identical fragments is observed
following analysis under reducing conditions (data not shown). Most of
the smaller
-chain fragments represent proteolytic cleavage
fragments that are recognized by monoclonal antibodies specific for the
C
2 domain. (
)In contrast, deletion of N-terminal
sequences gives rise to two
-chain fragments and their apparent
molecular weight under nonreducing (Fig. 1D) and
reducing conditions (data not shown) indicates that they correspond, in
almost equal quantities, to monomeric and dimeric GST
h
-chain
fusion proteins.
Figure 1:
Gene constructs, expression products,
and mapping of receptor binding regions in human IgE. Recombinant
-chain gene fragments were subcloned into the multiple cloning
site (MCS) of the bacterial expression plasmids pGEX-3X and
pGEX-KG (25) and expressed in E.
coli(6, 18) . The
-chain expression plasmids
pSV-V
h
/r
were employed for the construction of
mutant and chimeric IgE molecules and expressed in the J558L myeloma
cell line(18) . Panels A and B summarize the
ability of the truncated, chimeric, and mutant
-chain variants to
bind to Fc
RI
expressed on RBL-2H3.1 and RBL-2/2/C cells (23, 27) and to Fc
RII expressed on the 8866
lymphoblastoid cell line(18) . Initial screening for biological
activity was determined by assessing the capacity of
GST
-chain fusion proteins to inhibit the binding of
I-labeled hIgE (1 nM) to the receptors. The
degree of inhibition effected by nonbinders was identical, within
limits of experimental error, to that observed with GST, which was
included as a negative control (see Fig. 2). Purification of
truncated recombinant GST
-chain fusion proteins and mutant
and chimeric IgE molecules was carried out as described (see
``Experimental Procedures''). Ligands were labeled with
I for direct binding studies (see Table 1).
Nonbinders showed no binding above background even at concentrations
above 10
M. Panels C and D show GST
-chain fusion proteins that were
immunoprecipitated with a rabbit anti-GST serum, followed by SDS-PAGE
(12%) separation under nonreducing conditions and immunoblotting with a
horseradish peroxidase-labeled rabbit anti-human IgE serum. Panel
C, lanes 1-6, GST
-(226-547),
GST
-(226-361), GST
-(226-357),
GST
-(226-354), GST
-(226-352),
GST
-(226-340). Panel D, lanes
1-6, GST
-(326-547),
GST
-(340-547), GST
-(343-547),
GST
-(345-547), GST
-(350-547),
GST
-(226-440)-C
3. +, receptor binding;
-, loss of receptor binding; n.d., not
determined.
Figure 2:
Percentage of inhibition of I-hIgE binding to RBL-2/2/C cells by native and
recombinant hIgE-derived
-chain fragments. To measure the
inhibition (IC
) of 1
I-hIgE to RBL-2/2/C cells
by native IgE and recombinant
-chain fragments, cells were
preincubated at 22 °C for 1 h with increasing concentrations
(10
-10
M) of each
of the unlabeled GST fusion peptides in 125 µl of binding buffer
or, as a negative control, GST or binding buffer.
I-hIgE
was then added (1 nM). After 45 min, the cells were washed
twice with 0.5 ml of binding buffer and lysed in the same volume of
lysis buffer, and aliquots were removed for
-counting. The
IC
values for GST
-(226-547) and
GST
-(226-357) (data not shown) were identical to those
observed for hIgE (
) and GST
-(226-354)
(
). GST
-(226-340) (
) and
GST
-(355-547) (
), where the sequence common to
all fragments that can engage Fc
RI has been deleted by either the
N- or C-terminal truncation, show inhibition levels similar to that
obtained with GST (
) and all other fragments classified as
nonbinders in Fig. 1.
, GST
-(340-547);
, GST
-(440-547). Data shown represent the means
of at least three separate experiments carried out in
duplicate.
The present investigation confirms our previous
observations, which show that only those peptides that contain C4
or the homologous C
3 domain can engage both Fc
RI and
Fc
RII(6, 16) , while C-terminal truncation of the
-chain results in elimination of binding to Fc
RII. As
summarized in Fig. 1A, sequences common to all
fragments capable of binding to Fc
R1
comprise residues
Pro
-Ser
in the C
3 domain.
Further deletion from either the C- or N-terminal end beyond these
residues is associated with a loss of Fc
RI binding. As shown in Fig. 2and Table 1, GST
-(340-547) and
GST
-(226-354), which comprise the core peptide,
inhibit the binding of hIgE with an IC
in the nanomolar
range. In contrast, blocking of IgE/Fc
RI interaction by the GST
control, GST
-(226-340),
GST
-(355-547), and GST
-(440-547) is
identical and cannot be detected even above micromolar concentrations.
These results confirm observation by others who find that recombinant
IgE-derived fragments comprising residues 355-547 do not block
hIgE binding to hFc
RI (11) and that substitution of
residues 346-353 by the homologous sequence from IgG1 reduces
binding of the chimera to background levels(14) .
As shown
in our model structure of hIgE-Fc (Fig. 3) (18) , this
sequence forms a loop that is homologous to the loop in rIgG shown to
bind to the neonatal FcRn(24) . A further similarity
emerged when we investigated the pH dependence of the binding of hIgE
to Fc
RI. As shown in Fig. 4two pH optima are observed for
the binding of hIgE to Fc
R1, and occupancy of the receptor is
almost twice as high at pH 6.4 as at pH 7.4. Although the significance
of this is not known, it is tempting to speculate that hIgE has evolved
the lower pH optimum as a result of its physiological importance in the
fight against parasitic infestations in the lumen of the intestine at
acid pH.
Figure 3:
Drawing of the -carbon trace of a
model structure (18, 19) for hIgE with various
fragments and domains indicated. The light chains are drawn with thin lines and the heavy chains with thick lines, one
thicker than the other. The interchain Cys at position 328 is labeled.
The 11-amino acid segment 343-353, which is common to all
IgE-derived peptides that bind to Fc
RI, is drawn with bigger
circles and wide, empty bonds in both heavy
chains.
Figure 4:
pH
profile for the binding of native IgE and recombinant IgE-derived
peptides to RBL-2/2/C cells. h-chain-transfected RBL-2/2/C clones
were distributed into 48-well plates at 10
cells/well and
incubated with 10
M dexamethazone for 24 h
at 37 °C. Prior to the assay cells were washed twice with 0.5 ml of
saline containing 0.4% BSA and preincubated with 125 µl of 50
mM phosphate-buffered saline containing 0.4% BSA (pH range
5.9-8.1) for 10 min at 37 °C before adding 50 µl of 2
µg of
I-labeled IgE (
), 0.7 µg of
GST
-(340-547) (
), and
GST
-(226-354) (&cjs3409;). Cells were incubated for 30
min, after which unbound ligand was removed by washing with binding
buffer before measurement of cell-bound label. Results were corrected
for nonspecific binding. (Data shown represent the means of two
determinations carried out in duplicate)
Data summarized in Table 1show that N- or C-terminal
truncation has a negligible effect on the rate of association of
biologically active -chain fragments with Fc
RI. In contrast,
the rate of dissociation increases several hundred-fold following the
deletion of residues from the C-terminal end, and, as shown in Fig. 3, this is associated with a concomitant decrease in
receptor occupancy at pH 6.4. Taken together, these data suggest that
His residues in the C-terminal region of the IgE molecule make a
contribution toward the maintenance of the high affinity interaction
between IgE and Fc
RI
since this is largely determined by the
slow rate of dissociation of the ligand from the receptor.
Results
obtained in the current study differ in one significant respect from
those in our previous investigation(6) , where the
FcRI-blocking capacity of IgE-derived fragments was evaluated by
the senior investigator, who performed passive cutaneous anaphylaxis
tests in his own skin. Employing a well defined cellular assay
system(23) , we demonstrate here that N-terminal IgE sequences
can be deleted beyond residue 340 without any significant effect on the
kinetics of ligand/receptor interaction. Our data show that the
essential determinant for hIgE/Fc
RI recognition depends on a
consecutive sequence comprising 11 amino acids computed to form a loop
at the interface between the C
3 and C
4 domain(18) .
In accord with others(7, 8, 9, 10) ,
our observations exclude any direct contribution of C
4-specific
residues as proposed by Stanworth et al.(12) . Our
results confirm and extend those made by Nissim et
al.(8, 9, 10) , who demonstrated that
the receptor binding site in IgE is located in the C
3 domain. They
differ from the claims of Hamburger (29) and Gould et
al.(20, 21) , who propose, respectively, that
residues 330-334 and 329-340 in the switch region between
C
2 and C
3 are essential for IgE/Fc
RI binding. As the
results of our study clearly demonstrate, these sequences can be
deleted without any major influence on the kinetics of hIgE/Fc
RI
interaction.
It is interesting to note that the active core sequence
identified by us corresponds closely to the hIgE-derived peptide
generated by Nio et al.(15) , who report its
capability to block the binding of antigen-specific IgE to cells
expressing FcRI at concentrations in the mM range(15) .
Although the results of our study indicate
that fragments containing the C2 domain show an increased
susceptibility to proteolysis (Fig. 1C), the inclusion
of the protease inhibitor phenylmethylsulfonyl fluoride during the
isolation procedure facilitates the purification of peptides that
engage Fc
RI/II. Using our published method, others have been
unable to generate h
-chain fragments in E. coli that
retain Fc
RI-binding capacity and have attributed this failure to
folding problems(11) . At least one other laboratory has
expressed
-chain fragments in E. coli which are
biologically active(30) .
Based on the outcome of FcRI
binding studies with chimeric and mutant hIgE molecules, Presta and
co-workers (14) proposed that six amino acid residues located
in three loops, C-D, E-F, and F-G, computed to form the outer ridge on
the most exposed side of the C
3 domain, are involved in receptor
binding primarily by electrostatic interactions(14) . These
conclusions were based on the observation that replacement of these
residues reduced the binding of variant molecules to Fc
RI
relative to native hIgE. We disagree with their conclusion in view of
the fact that most of the mutations at Arg
(408),
Ser
(411), Lys
(415), Glu
(452), Arg
(465), and Met
(469)
(Presta et al.(14) numbering scheme in parentheses),
which affect IgE/Fc
RI interaction to a greater or lesser extent,
are invariably due to replacements by residues of opposite charge or by
a Pro, changes which could cause structural rearrangements. In
contrast, more conservative substitutions of these residues either have
little effect or cause an apparent enhancement of binding ((14) , Table 1). Our own study shows that e.g. a single point mutation involving Cys
, which by
itself is not required for either Fc
RI or Fc
RII
binding(18) , can have a dramatic effect on the conformation of
the IgE molecule. Its substitution by Met, but not Ser, destroys
binding to both receptors(18) . When we compared the intrinsic
fluorescence of Trp residues in the native and IgE Met
molecule, we found that on average native IgE has 41% of its Trp
residues exposed to solvent, while IgE Met
was found to
have only 22% of Trp residues exposed, although both molecules were
recognized by a conformation-dependent monoclonal antibody directed
against the C
2 domain. This observation shows that the
substitution of a single amino acid that is not involved in receptor
recognition can induce a significant deformation in structure and
profoundly affect ligand/receptor association.
Presta et al.(14) have also claimed that the grafting of loops C-D,
E-F, and F-G and the inter-C2/3 switch region into hIgG (which
they refer to as IgGEL), conferred Fc
RI binding to
hIgG
. Their own data, however, on the binding of variant
IgE do not support this interpretation. It is important to point out
that their chimeric IgGEL construct still retains the endogenous
IgG
loop A-B sequence, which when grafted into hIgE ((14) , Table 1, variant 1) effected a 97% decrease in
binding. This represents a greater reduction in activity than any other
loop replacements described in their study. Data in their Fig. 3, which is interpreted by them to support their claim that
the IgGEL chimera can recognize Fc
R1, demonstrate the opposite
since they show that when CHO 3D10 cells are incubated with IgGEL at a
concentration of 1 µg/ml, less than 2% of cells become labeled.
Since these are the criteria that they applied for the assignment of
the receptor binding activity of all other chimeric and mutant IgE
molecules described in their investigation ((14) , Table 1), we conclude that IgGEL has the same affinity for
Fc
RI as variant 1, which is less active than any other chimera or
mutant reported in their study. Although Fig. 3of their paper (14) appears to suggest
40% binding at 20 µg/ml IgGEL,
there is no evidence for saturation, and the slope of the line is
indicative of nonspecific binding.
In our opinion, the data of
Presta et al. provide compelling evidence that the A-B loop in
hIgE comprises the essential structural determinant for
IgE/FcRI
interaction because the only difference between
their variant 1 and native hIgE is that in variant 1 the core sequence,
which we have shown to be common to all hIgE fragments that can engage
hFc
R1
, is replaced by the IgG1 homologue.
As our study
shows, sequences N- or C-terminal to this core peptide are necessary to
provide structural scaffolding for the maintenance of a receptor
binding conformation since the core peptide alone cannot engage the
receptor. Deletion of C-terminal, but not N-terminal, sequences
diminishes receptor occupancy at pH 6.4 and increases the dissociation
of the ligand from the receptor. We conclude that residues, including
His, in the C-terminal domain make an important contribution toward the
maintenance of the high affinity of interaction between IgE and
FcRI
.
Although human and rodent IgE are highly homologous,
rodent IgE can engage only hFcRI but not hFc
RII(10) .
In contrast, hIgE cannot bind to either of the rodent receptors. The
structural basis for this phenomenon is unknown. In the current study
we have replaced the human A loop by the rat homologue and find that
the chimera still recognizes hFc
RI. This graft, however, does not
confer binding to rFc
RI, indicating that conformational
determinants outside the A loop in hIgE inhibit recognition of
rFc
RI. The fact that this replacement destroys the binding of hIgE
to hFc
RII indicates exquisite species specificity and suggests
that residues in this motif contribute to the binding to both
receptors. Nissim et al.(10) have demonstrated that
the species specificity for recognition of Fc
RII by murine and
hIgE is contained in the C
3 domain and proposed that part of the
binding energy for hFc
RII interaction is contributed by amino acid
residues between 346 and 356.
At present, limited structural
information is available regarding the interaction between IgE and its
receptors. Unlike IgG/FcRn interaction, where the ligand can
engage two receptors, IgE molecules bind to Fc
RI
in a 1:1
stoichiometry, although bilateral symmetrical protection to proteolysis
has been observed when rodent IgE is complexed to the
-chain(24, 31, 32) . This has been
explained in terms of a bent conformation of IgE(33) , where
the second
-chain becomes inaccessible to an additional copy of
the receptor, or to antibodies directed against epitopes in IgE that
become masked following receptor engagement. There is little evidence
for a beneficial role for IgE antibodies except in parasitic diseases,
and such epitopes may therefore have an application as immunogens for
the therapy of IgE-mediated allergies, since naturally occurring and
monoclonal antibodies (33, 34, 35, 36) have been described
that block the binding of IgE to cells expressing Fc
R1 but do not
trigger mediator release. An improved understanding of the docking of
hIgE to its receptors will provide the structural information needed
for the rational design of such immunogens. The identification of the
binding site constitutes a major step in this direction.