From the Structural Biology Section, Laboratory of Immunogenetics, NIAID, National Institutes of Health, Rockville, Maryland 20852
Received for publication, January 16, 2001
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ABSTRACT |
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Recently determined crystal structures of the
complex between immunoglobulin constant regions (Fc) and their
Fc-respective receptors (FcR) have revealed the detailed molecular
interactions of this receptor-ligand pair. Of particular interest is
the contribution of a glycosylation at Asn297 of the
CH2 domain of IgG to receptor recognition. The carbohydrate moieties are found outside the receptor·Fc interface in all
receptor·Fc complex structures. To understand the role of
glycosylation in FcR recognition, the receptor affinities of a
deglycosylated IgG1 and its Fc fragment were determined by solution
binding studies using surface plasmon resonance. The removal of
carbohydrates resulted in a non-detectable receptor binding to the Fc
alone and a 15- to 20-fold reduction of the receptor binding to IgG1, suggesting that the carbohydrates are important in the function of the
Fc Activation of low affinity Fc Certain autoimmune diseases, such as rheumatoid arthritis, result from
the activation of Fc In addition, the properties that determine the immunoglobulin isotype
specificities of the low affinity Fc In this work, we demonstrate, using solution binding studies that the
carbohydrates attached to Asn297 of IgG1 are critical for
Fc Preparation of Fc by Papain Digestion--
The Fc fragment of a
human monoclonal IgG1 was isolated by papain digestion (17). In brief,
IgG1 at a concentration of 10 mg/ml was incubated for 2 h at
37 °C with 6.6% (w/w) papain at pH 7.1 in the presence of 1 mM cysteamine. This lead to complete cleavage of IgG1. The
Fc fragment was separated from the Fab fragments on a Protein A
affinity column (Amersham Pharmacia Biotech) with MAPS II (Bio-Rad)
binding and elution buffers at a flow rate of 0.5 ml/min. The Fc
fragment was further purified on a Superdex 200 HR 10/30 gel filtration
column (Amersham Pharmacia Biotech) with 50 mM NaCl, 50 mM Tris at pH 8.0 as a running buffer at a 0.5 ml/min flow rate.
Deglycosylation of Fc--
Deglycosylated Fc fragments and IgG1
were prepared by peptide-N-glycosidase F (New England
BioLabs) digestion in water for 1.5 h at 37 °C using 1 unit of
enzyme/10 µg of protein. The extent of deglycosylation was analyzed
by SDS-polyacrylamide gel electrophoresis, gel filtration
chromatography, and electrospray ionization mass spectrometry (ESI-MS).
The gel filtration experiments were carried out using a Superdex 200 PC
3.2/30 gel filtration column (Amersham Pharmacia Biotech) and an
Äkta high pressure liquid chromatography purifier (Amersham
Pharmacia Biotech) with 50 mM NaCl, 50 mM Tris at pH 8.0 as running buffer at a 0.1 ml/min flow rate. ESI-MS measurements were acquired and recorded with a PerkinElmer Life Sciences Sciex API-300 triple quadruple system (Thornhill, Ontario, Canada) using 8 µg of native or deglycosylated Fc fragment and 6 µg
of native or deglycosylated IgG1. The native and deglycosylated IgG1
were reduced with 100 mM dithiothreitol prior mass
spectrometry measurements.
Preparation of the Lower Hinge Peptides--
Peptides of 8 to 12 amino acids in length with the sequences of the lower hinge receptor
binding region of IgG1, IgG2, IgG4, and IgE were synthesized (Table
I). All peptides were purified on a
Superdex 200 HR16/60 gel filtration column (Amersham Pharmacia Biotech)
with H2O as the running buffer to remove impurities and to
exchange the original buffer. A mostly polyalanine peptide (pALA) with
a sequence of AAADAAAAL was used as the control. The concentrations of
peptides were estimated using absorbance at 220 nm and an extinction
coefficient of 2.6 absorbance units per ml mg Surface Plasmon Resonance Measurements--
Surface
plasmon resonance (SPR) measurements were performed using BIAcore
2000 instrument (BIAcore AB). Fc Deglycosylation of Fc--
Papain digestion of the human IgG1
resulted in a 53-kDa disulfide-bonded Fc fragment. Due to the
carbohydrate attachment to Asn297, the mass spectrum of the
native Fc fragment displays considerable heterogeneity in mass (Fig.
1A). Treatment of the native
Fc fragment with peptide-N-glycosidase F under
non-denaturing condition resulted in a shift of molecular mass of the
Fc from 53,400 to 50,345 Da (Fig. 1B). This agrees well with
the predicted 50,407-Da molecular mass of the polypeptide
backbone of Fc, indicating a complete enzymatic removal of
carbohydrates. The deglycosylated Fc has an apparent molecular weight
similar to that of the native Fc in a Superdex 200 SMART gel filtration
column (Amersham Pharmacia Biotech) (Fig. 1C). It remains as
a disulfide bonded dimer (Fig. 1D). The result of a native
gel electrophoresis shows that the deglycosylated Fc fragment appears
to be more compact than the native Fc (Fig. 1E).
Binding of Fc
To rule out the possibility that the loss of receptor binding was due
to a global disruption of the Fc structural fold induced by
deglycosylation, binding of both the native and deglycosylated Fc to
protein G, which recognizes the CH2-CH3
junction region of Fc, were also measured on a immobilized protein G
sensor chip. The protein G binding dissociation constant is 47 nM for the native Fc and 130 nM for the
deglycosylated Fc. The ability of deglycosylated Fc to bind protein G
suggests no global disruption in the structure upon removal of the
carbohydrate. This is also evident from the gel filtration and native
gel electrophoresis analyses, both of which show a slightly more
compact shape of the deglycosylated Fc compared with that of the native
Fc (Fig. 1, C and E).
Binding of the Lower Hinge Peptides to Immobilized
Fc Binding of Fc Competition between the Peptides and Fc for Receptor
Binding--
To further investigate whether the peptides share the
same receptor binding site as Fc, direct binding competition
experiments were performed for the peptides using the native Fc
fragment as a competitor. In each experiment, an analyte consisting of
10 µM of Fc The contribution of glycosylation of Fc to the function of
immunoglobulins has been debated over the years. Early studies have
demonstrated that aglycosylated IgG2a caused a mild reduction in
the activation of complement component C1 but a drastic reduction in
the activation of FcRIII. Structurally, the carbohydrates attached to
Asn297 fill the cavity between the CH2 domains
of Fc functioning equivalently as a hydrophobic core. This may
stabilize a favorable lower hinge conformation for the receptor
binding. The structure of the complex also revealed the dominance of
the lower hinge region in receptor·Fc recognition. To evaluate the
potential of designing small molecular ligands to inhibit the receptor
function, four lower hinge peptides were investigated for their ability
to bind to the receptor Fc
RIII. These peptides bind specifically to
Fc
RIII with affinities 20- to 100-fold lower than IgG1 and are able
to compete with Fc in receptor binding. The results of peptide binding
illustrate new ways of designing therapeutic compounds to block Fc
receptor activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptors requires the binding of
antigen-activated immune complexes (1, 2). The recent determination of
crystal structures of Fc
and Fc
receptors in complexes with Fc
fragments have revealed that these receptors bind asymmetrically
at a 1:1 ratio to the lower hinge region of their dimeric Fc ligands
(3-5). This mode of binding is conserved in both Fc
RIII and
Fc
RI.1 However, a number
of questions still remain to be addressed. The first is the role of
carbohydrate in the receptor·Fc recognition. In particular, the
contribution of a conserved glycosylation site, Asn297 of
the constant region of IgG1, remains controversial (6-9). The
carbohydrate attached to this glycosylation site is partially ordered
in all known structures of Fc (10), intact antibody (11), and in the
receptor·Fc complexes, suggesting that a stable rather than a
flexible conformation exists for the carbohydrate. Furthermore, unlike
most glycosylations that attach to surfaces of the molecules, the
carbohydrate attached to Fc is located in a cleft between the two
CH2 domains to partially fulfill the cleft. This unique
location of carbohydrate may contribute to the conformational stability
of Fc, specifically, the relative orientation between the two chains of
Fc. Because the receptor epitope is formed primarily by the joint hinge
segments of both chains of Fc, receptor recognition is potentially
sensitive to the relative orientation of the two CH2
domains. Thus, it is conceivable that the unique position of the
glycosylation at Asn297 may contribute to the receptor
binding by stabilizing the lower hinge conformation. Although earlier
investigations of Fc
receptor binding to IgG demonstrated a drastic
reduction on cell surface receptor affinity when the carbohydrates at
Asn297 were removed, similar studies on IgE binding to
Fc
receptor revealed a much weaker influence of carbohydrate on
receptor-ligand recognition (6-9). There are no direct contacts
observed between the receptor and the carbohydrate attached to
Asn297 in the crystal structures of the receptor·Fc
complexes to account for the observed effect of carbohydrate on the
Fc
receptor binding.
receptors by auto-antibodies (12, 13). The
ability to inhibit the receptor activation in this case should help to
control the antibody-mediated auto-inflammatory response. The
structures of the receptor·Fc complexes revealed the dominance of the
lower hinge residues of Fc in the receptor binding, suggesting a new
way of designing small peptide ligands that can inhibit the binding of
immunoglobulins to their receptors. These receptor inhibitors may be
potential candidates for the treatment of autoimmune diseases.
receptors remains to be
identified. The isotype specificities of this receptor have been
correlated to the pathogenicity of an anti-erythrocyte auto-antibody (14). Earlier results of chimeric IgG work as well as mutational analysis suggest that the lower hinge region is important in
determining the receptor preferences (15, 16).
receptor recognition. Synthetic peptides, 8 to 12 residues long
and that mimic the lower hinge regions of IgG1, IgG2, IgG4, and IgE,
were also shown to bind Fc
RIII and to compete with the binding of
the native Fc.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
All peptides were confirmed by mass using ESI-MS. Peptide pIgG1 includes a lower hinge cysteine residue that forms the conserved disulfide bond between the immunoglobulin heavy chains. The
disulfide-bonded dimeric form of the peptide was generated by
incubating the peptide under a mild base condition of pH 8.0 for 1 h at room temperature. This disulfide-bonded form, resistant to
alkylation by iodoacetamide (data not shown), was designated as cIgG1.
The free cysteine in the monomeric pIgG1 was blocked by alkylation
with iodoacetamide to prevent the dimerization.
Immobilization of the lower hinge peptides and FcRIII
RIII receptor was immobilized at
concentrations of 15 and 45 µM in 10 mM
sodium acetate, pH 6.0, on a CM5 sensor chip using
N-hydrosuccinimide/1-ethyl-3(-3-dimethylaminopropyl)carbodiimide hydrochloride (NHS/EDC) at a flow rate of 5 µl/min (Table I). Flow
cell 4 of every sensor chip was mocked with NHS/EDC as a negative
control of binding. All peptides were immobilized at 10 mM
concentration on CM5 sensor chips in 100 mM EDTA at pH 8.0 with NHS/EDC at a flow rate of 2 µl/min. The binding buffer consisted of 20 mM NaCl, 3 mM EDTA, 0.005% surfactant
P20, 10 mM HEPES at pH 7.4 mixed with various
concentrations of analyte. Binding of the native and deglycosylated Fc
fragments to the immobilized Fc
RIII was measured using serial
dilutions of the analyte from 10 to 0.078 µM at a flow
rate of 5 µl/min. The binding of IgG1 and its deglycosylated form to
the immobilized receptor was measured with the analyte concentrations
varying from 10 to 0.02 µM. For the binding of the
receptor to immobilized peptides, the analyte consisted of a serial
dilution of the receptor between 750 and 0.37 µM. The
same immobilized peptide chips were used for competition experiments in
which the analyte consisted of 10 µM Fc
RIII mixed with
various concentrations of Fc from 10 to 0.02 µM. To
measure the binding between protein G and Fc, protein G was immobilized on a CM5 sensor chip at 50 µM concentration. The native
or the deglycosylated Fc at concentrations between 5 and 0.15 µM were used. All dissociation constants
(KD) were obtained either from a linear regression
of steady state 1/Response versus 1/C plots using ORIGIN 3.0 (MicroCal Software, Inc.) or from kinetic rate constants fitted with
the BIAevaluation software package (BIAcore AB).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Characterization of deglycosylated
Fc. Mass spectrometry measurements of the native Fc (A)
and the deglycosylated Fc fragment (B) of IgG1. The
molecular weights (horizontal axis) of the respective Fc
fragments are labeled. C, size exclusion chromatography of
the native (solid line) and deglycosylated (dashed
line) Fc. The molecular weight standards are indicated.
D, SDS-polyacrylamide gel electrophoresis (20%
polyacrylamide PhastGel, Amersham Pharmacia Biotech AB) analysis of the
native and deglycosylated Fc fragments of IgG1 after digestion with
peptide-N-glycosidase F (New England BioLabs).
First lane, molecular weight standard; second and
third lanes, the native and deglycosylated Fc;
fourth and fifth lanes, the native and
deglycosylated Fc under reduced conditions. E, native gel.
First and second lanes, the native and
deglycosylated Fc fragments. The three bands on the native gel of
deglycosylated Fc may reflect either conformational heterogeneity or
partial degradation.
RIII to the Native and Deglycosylated
Ligands--
Both deglycosylated IgG1 and its Fc fragment were
prepared under similar conditions (under "Experimental Procedures")
and purified by Superdex 200 HR 10/30 size exclusion chromatography. Solution binding experiments were performed using a BIAcore 2000 instrument with the receptor immobilized on a CM5 sensor chip. The
analyte consisted of serial dilutions of IgG1 or Fc with concentrations from 10 to 0.02 µM and 10 to 0.08 µM,
respectively. The affinity of Fc
RIII for the native Fc fragment was
essentially the same as that for IgG1, ~5 µM (Table
II). Deglycosylation of the Fc fragment
resulted in no detectable receptor binding (Fig.
2A), and that of IgG1 resulted
in a KD of ~50 µM, a 10-fold reduction in the receptor binding affinity (Table II, Fig.
2B).
Dissociation constants for the binding of FcRIII
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Fig. 2.
Effect of Fc deglycosylation on
Fc RIII recognition. A, binding
of the native Fc (solid square) and deglycosylated Fc
(open square) to the immobilized receptor. B, the
binding of IgG1 (solid diamond) and its deglycosylated form
(open diamond) to the receptor. Each data point represents a
steady-state SPR response measured in resonance units (RU).
RIII--
The crystal structure of Fc
RIII in complex with Fc
illustrates that the receptor primarily recognizes the lower hinge
region of the Fc with 60% of the interface area contributed by the
four lower hinge residues, Leu-Leu-Gly-Gly (234). To test whether
the lower hinge alone can be recognized by the receptor, peptides with
the corresponding lower hinge sequences of IgG1/3, IgG2, IgG4, and IgE
were synthesized (Table I). All four peptides were able to bind to the
immobilized receptor on a CM5 sensor chip (Fig.
3), although pIgG1 binds consistently
better than other peptides at all concentrations tested. Due to the low
receptor immobilization and the weak binding affinity, the dissociation constant of the receptor-peptide binding could not be derived from
these experiments.
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Fig. 3.
Binding of the lower hinge peptides to
immobilized Fc RIII. The three sets of
experiments correspond to 5, 2.5, and 1.25 mM peptide
concentrations (from left to right). No
detectable binding were observed for pALA at all three
concentrations.
RIII to Immobilized Peptides--
To estimate the
affinity between the peptides and Fc
RIII, individual peptides were
immobilized on CM5 sensor chips at 10 mM concentration, and
SPR measurements were recorded (Table I). Serial dilutions of the
receptor between 750 and 0.37 µM concentrations were used
as the analyte (Fig. 4). Among the
peptides, the disulfide-linked cIgG1 binds the tightest, with a
KD of 113 µM. This is about 20 times
less than the affinity of the native Fc and two time less than that of
the deglycosylated IgG1. Among the other peptides, pIgG1, pIgG2, and
pIgG4 display similar receptor binding affinity (Table II and Fig. 4).
Unexpectedly, pIgE displays significant binding to Fc
RIII compared
with pALA, although the affinity is 4-10 times lower than other hinge
peptides.
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Fig. 4.
Fc RIII binding to
peptides immobilized on CM5 sensor chips. Measurements were
performed by using serial dilutions of Fc
RIII from 750 to 0.37 µM as analytes. All RUs were normalized against their
level of peptide immobilizations using cIgG1 as the standard.
RIII mixed with various concentrations of
Fc was used to bind to the individual peptides immobilized on CM5
sensor chips. If peptides recognize the same receptor region as that of
Fc, the receptor binding to the immobilized peptides will decrease as
the concentration of Fc in analyte increases. On the other hand, if the
peptides bind to a separate site on the receptor as that of Fc, the SPR
response will be independent of or will increase with the Fc
concentration due to the higher molecular weight of receptor·Fc
complex. The results of competition experiments show that the binding
of the receptor to cIgG1, pIgG1, pIgG2, and pIgG4 peptides is blocked
by increasing concentrations of Fc (Fig.
5). Because no detectable affinities
could be observed between the Fc and the peptides (Table II), the
effect of competition resulted directly from the titration of the
receptor rather than from the masking of the peptides by Fc. The amount
of Fc required to completely block the receptor·peptide interaction
is about 10 µM, in agreement with the Fc·receptor
binding affinity. This suggests the peptides bind to Fc
RIII at the
same site as that of Fc. Interestingly, the receptor binding of pIgE
displays a similar Fc competition curve as other peptides, indicating
that pIgE also shares the same Fc binding site. However, the Fc
competition of pIgE is less profound compared with those of other
peptides due to a lower receptor·peptide affinity. The control
peptide, pALA, displays no measurable receptor binding nor Fc
competition.
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Fig. 5.
Effect of Fc competition on
Fc RIII binding to immobilized peptides.
Mixtures of 10 µM Fc
RIII with various concentrations
of Fc from 10 to 0.02 µM were used as analytes. RU
indicates binding of Fc
RIII to peptide.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptors when compared with the native IgG2a
(7, 18). Pound et al. (8) found that aglycosylated IgG3 was
capable of triggering human phagocyte respiratory burst at 80% of the
level triggered by the glycosylated IgG3 despite a severe
impairment in antibody-dependent cellular cytotoxicity. Unlike Fc
receptors, the removal of carbohydrate of IgE did not cause significant loss in Fc
RI recognition (9, 19). Structurally, the oligosaccharides attached to Asn297 of IgG are a
biantennary type with a core heptasaccharide consisting of three
N-acetylglucosamine (GlcNac) and three manose (Man) and variable fucose additions to the core (6). Unlike most surface-attached glycosylations, these carbohydrates occupy a unique space between the
two chains of Fc and appear to have well organized conformations in all
crystal structures of Fc with electron densities visible to most of the
core sugar moieties. Recently, the crystal structures of IgG1-Fc in
complex with Fc
RIII and IgE-Fc in complex with Fc
RI have been
determined (3-5). However, no significant interactions were observed
between the carbohydrate on Fc and its receptor in the complex
structures. To understand the apparent discrepancy between the known
functional importance of this glycosylation and the lack of a
structural engagement at the receptor·Fc interface, we have examined
the biophysical and binding properties of a deglycosylated IgG1 and its
Fc fragment. The Fc fragment binds to Fc
RIII at essentially the same
affinity as that of an intact IgG1 as measured by BIAcore experiments.
Both have a dissociation constant of 4 µM. This agrees
well with the previously published ~1 µM for
KD (20, 21). Upon enzymatic deglycosylation, the
IgG1-Fc
RIII binding dissociation constant increased from 4 to 50 µM, whereas the Fc·Fc
RIII binding became
non-detectable. This 10- to 15-fold loss in the receptor binding
affinity indicates that the carbohydrate contributes significantly to
the receptor-ligand recognition. Because the sugar moieties make no
direct contact with the receptor at the receptor·Fc interface, the
most likely role for the carbohydrate is to stabilize the IgG lower
hinge in an active receptor binding conformation. The extent of epitope
stability provided by the glycosylation is also evident when the
affinity of the deglycosylated IgG1 is compared with that of a
disulfide-linked peptide, cIgG1. The affinity of the deglycosylated
IgG1 is only two times higher than that of cIgG1, which presumably has
no defined epitope conformation in solution. It is conceivable that
this conformational stability may result from the interaction between
the carbohydrate moieties which clearly visible in the electron density
of the Fc
RIII·Fc complex structure, and may serve as a substitute
hydrophobic core. The removal of the carbohydrates by deglycosylation
may cause a conformational change in the relative orientation of the
two CH2 domains such that the Fc transitions from an open
to a closed conformation (Fig. 6). A
structural change associated with aglycosylated IgG3 was previously
observed in the vicinity of His268 within the
CH2 domain as detected by NMR experiments (7). Analysis of
native gel electrophoresis is also consistent with the deglycosylated
Fc being more compact than the native Fc (Fig. 1E).
Alternatively, the carbohydrates may restrict the lower hinge flexibility, and their removal would result in enhanced hinge flexibility and thus reduced receptor binding (22).
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Fig. 6.
Proposed role of glycosylation in stabilizing
the Fc conformation.
It is interesting to note that the deglycosylated Fc consistently resulted in a 2-fold reduction in protein G binding affinity. Even though protein G binds at the CH2-CH3 hinge region of Fc, away from the carbohydrate moieties (23), a mild reduction in the protein G binding affinity was observed upon deglycosylation. This suggests a structural change in the CH2-CH3 hinge angle as the result of deglycosylation. It is also consistent with the hypothesis that the carbohydrate-free Fc adopts a different hinge conformation.
Besides the normal function of FcR in triggering cellular inflammatory
response to clear antigen-bound immune complexes, FcR also mediates
autoimmune diseases generated from the response to autoantibodies such
as rheumatoid factor in rheumatoid arthritis (12, 13, 24). Under these
conditions, it would be beneficial to block the autoantibody-triggered
activation of FcR to relieve the auto-inflammatory response that leads
to specific tissue damage. Because the activation of FcR requires
receptor aggregation by multivalent antigen immune complexes, small
molecule compounds that are capable of competing with the binding of
immune complexes to FcR should prevent the receptor aggregation and
thus inhibit FcR activation (25). The structure of FcRIII in complex
with Fc provides a detailed map of the molecular interface between the
receptor and Fc (5). In particular, the dominance of the lower hinge of
Fc, which occupies 60% of the interface area, suggests that peptides
resembling the lower hinge conformation could be good candidates to
inhibit the receptor function. In an attempt to evaluate such peptide
inhibitors and their ability to compete with the receptor binding to
the native ligand, we designed four peptides with sequences
encompassing the receptor binding region of the lower hinges of IgG1,
-2, -4, and IgE. Among these peptides, the disulfide-linked IgG1 hinge
peptide, cIgG1, binds the tightest to Fc
RIII with an affinity ~20
times less than the native immunoglobulins and three times better than
the non-disulfide-linked peptide, pIgG1 (Table II). The observed
difference in receptor binding between cIgG1 and pIgG1 suggests that
the disulfide bond located at the lower hinge region of Fc contributes
to its conformational stability. The three IgG-derived peptides bind to
the receptor with approximately the same affinity. An unexpected result
is that the IgE-derived peptide, pIgE, possesses a significant binding affinity to Fc
RIII. This is particularly interesting, because the
lower hinge sequence of IgE is quite different from those of IgGs. It
suggests that the binding of low affinity receptors may be quite
promiscuous. It leads to the possibility of Fc
RIII activation by
antigen-bound IgE under certain circumstances such as in a saturated
allergen condition or in the absence of Fc
receptors. In fact, the
binding of IgE-immune complexes to the low affinity Fc
receptors on
mast cells has been observed to trigger the release of serotonin
(26).
The competition results show that all lower hinge peptides compete
directly with Fc in receptor binding. The ability of these lower hinge
peptides to inhibit Fc binding to the receptor opens potential new ways
of designing therapeutic compounds. For examples, pIgG analogs may be
useful in treating Fc receptor-mediated autoimmune diseases by
blocking the activation of the receptor, or pIgE-like compounds could
be used as potent inhibitors of Fc
receptors thus providing a
potential treatment for allergy.
This study on the binding affinity of the lower hinge peptides has also
allowed us to examine the issue of the receptor isotype specificity.
The low affinity human FcRIII binds to IgG1 and IgG3 much better
than it does to IgG2 and IgG4 (16). Previous mutation studies of IgG2
and its binding to human high affinity receptor Fc
RI allow us to
conclude that the entire lower hinge sequence was required to restore
the IgG1 binding affinity in IgG2, whereas point mutations in IgG1
hinge residues resulted in a loss of the receptor binding (15). In this
work, the lower hinge peptides instead of the antibodies were used in
the study of the receptor binding. This enables us to separate the
individual amino acid contribution to the receptor affinity from the
effect of their environment, namely the length of lower hinge in an
intact antibody. The results from studies of solution binding between the receptor and immobilized peptides and from Fc competition assays
show that pIgG2 and pIgG4 have nearly the same affinity to Fc
RIII as
does pIgG1. Replacing Leu with Phe in pIgG4 or changing Glu-Leu-Leu-Gly
to Pro-Val-Ala in pIgG2 in addition to a single residue deletion makes
little difference in the affinity toward the receptor. This suggests
that factors other than the lower hinge amino acid composition play an
important role in determining the weaker binding affinity of IgG2 and
IgG4 to Fc
RIII (relative to IgG1). It has been proposed that the
overall length of the lower hinge may be important to the receptor IgG
subtype specificity (16), because the hinges of IgG1 and -3 are about
three residues longer than those of IgG2 and -4. It is also possible,
however, that each IgG subtype varies in its glycosylation at
Asn297 and that the differences in carbohydrate may
contribute to the observed receptor specificity. Some preference in
glycosylation of IgGs is known to exist (6). Residues outside the lower
hinge region but in the vicinity of receptor interface could also
influence the receptor binding preference. For example,
Pro329 of Fc is sandwiched between Trp90 and
Trp113 of the receptor, providing important van der Waals
contacts between the receptor and Fc. Although Pro329 is
conserved among the IgG subtypes, residue 327 displays a dimorphism with an Ala in IgG1 and -3 and a Gly in IgG2 and -4.
In conclusion, the present study suggests that glycosylation at
Asn297 of Fc fragments plays an important role in
the binding of Fc fragments to the low affinity receptor
FcRIII. The role of carbohydrate appears to be primarily to
stabilize the receptor epitope conformation. We also demonstrated that
small peptide ligands can be designed to inhibit the binding of
Fc
RIII to its natural ligand Fc.
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ACKNOWLEDGEMENTS |
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We thank Drs. C. Sautes-Fridman and W. H. Fridman for providing the antibody sample and for constructive discussions, L. Boyd and D. Margulies for providing the BIAcore 2000 instrument, C. Hammer for ESI-MS measurements, J. Lukszo for peptide synthesis, and M. Garfield for N-terminal amino acid sequencing, J. Boyington and S. Garman for their comments to the manuscript.
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FOOTNOTES |
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* This work was supported by intramural funding of NIAID, National Institutes of Health.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.
To whom correspondence should be addressed: the Structural Biology
Section, Laboratory of Immunogenetics, NIAID, National Institutes of
Health, 12441 Parklawn Dr., Rockville, MD 20852. Tel.: 301-496-3230;
Fax: 301-402-0284; E-mail: psun@nih.gov.
Published, JBC Papers in Press, January 31, 2001, DOI 10.1074/jbc.M100351200
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ABBREVIATIONS |
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The abbreviations used are: Fc, immunoglobulin constant regions; RcR, Fc receptor; ESI-MS, electrospray ionization-mass spectrometry; SPR, Surface plasmon resonance; NHS, N-hydrosuccinimide; EDC, 1-ethyl-3(-3-dimethylaminopropyl)carbodiimide hydrochloride.
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REFERENCES |
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