# **Recognition of IgG by Fcγ Receptor: The Role of Fc Glycosylation and The binding of peptide inhibitors**

Sergei Radaev^1^, Wolf-Herman Fridman^2^, Catherine Sautes-Fridman^2^,
and Peter D. Sun^1\*^

^1^Structural Biology Section, Laboratory of Immunogenetics, National
Institute of Allergy and Infectious Diseases, National Institutes of
Health, 12441 Parklawn Drive, Rockville, MD 20852,USA

^2^Laboratory of Cellular and Clinical Immunology, INSERM Unit 255,
Curie Institute, Paris, France

\* Corresponding author: <psun@nih.gov>; Phone: 301-496-3230; Fax:
301-402-0284.

Running title: Binding studies of FcγRIII and Fc.

***Summary***

Recently determined crystal structures of the complex between the
immunoglobulin constant region (Fc) and their Fc receptor (FcR) revealed
the detailed molecular interactions of this receptor-ligand pair. Of
particular interest is the contribution of a glycosylation at Asn 297 of
the C~H~2 domain of IgG to the 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. The removal of
carbohydrates resulted in a non-detectable receptor binding to Fc and a
15-20 fold reduction of the receptor binding to IgG1, suggesting the
importance of the carbohydrate in the function of the FcγRIII.
Structurally, the carbohydrates attached to Asn 297 fill the cavity
between the C~H~2 domains of Fc functioning equivalent 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 the receptor-Fc recognition. To
evaluate the potential of designing small molecular ligands to inhibit
the receptor function, four lower hinge peptides were investigated in
their ability to bind to the receptor FcγRIII. These peptides bind
specifically to FcγRIII with affinities 20-100 fold lower than IgG1 and
are able to compete with Fc in receptor binding. The results of peptide
binding illustrated new ways of designing therapeutic compounds.

***Introduction***

Activation of the low affinity Fcγ receptors require the binding of
antigen activated immune complexes (1;2). The recent determination of
crystal structures of Fc receptor in complexes with Fc revealed that the
receptor binds asymmetrically at a 1:1 ratio to the lower hinge region
of its dimeric Fc ligand (3-5). This mode of binding is conserved in
both FcγRIII and FcεRI. 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, Asn 297 of the constant region of IgG~1~, 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 a stable
rather than flexible conformation existed 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 C~H~2 domains to partially fulfill the cleft. Such a unique
location of carbohydrate may contribute to the conformational stability
of Fc, specifically, the relative orientation between the two chains of
Fc. Since the receptor epitope is formed primarily by the joint hinge
segments of both chains of Fc, it makes the recognition potentially
sensitive to the relative orientation of the two C~H~2 domains. Thus, it
is conceivable that the unique position of the glycosylation at Asn 297
may contribute to the receptor binding by stabilizing the lower hinge
conformation. While earlier results on Fcγ receptor binding to IgG
demonstrated a drastic reduction on cell surface receptor affinity when
the carbohydrates at Asn 297 were removed, similar experiments on IgE
binding to Fcε receptor showed a much less effect of carbohyrate in that
receptor-ligand recognition (6-9). There are no direct contacts observed
between the receptor and the carbohydrate attached to Asn 297 in the
crystal structures of the receptor-Fc complexes to account for the
observed carbohydrate effect on the Fcγ receptor binding.

Certain autoimmune diseases, such as rheumatoid arthritis, resulted from
the activation of Fcγ 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 structure of the
complex revealed the dominance of the lower hinge residues of Fc in the
receptor binding suggesting a new way of designing small peptide ligands
that are capable to inhibit the binding of immunoglobulins to their
receptors. These receptor inhibitors may be potential candidates for the
treatment of autoimmune diseases.

In addition, what determines the immunoglobulin isotype specificities of
the low affinity Fcγ receptors remains to be clarified. This receptor
isotype specificities has been correlated to the pathogenicity of an
antierythrocyte autoantibody (14). Earlier results of chimeric IgG work
as well as mutational analysis suggest the importance of the lower hinge
region in determining the receptor preferences (15;16).

In this work, we demonstrated using solution binding studies that the
carbohydrates attached to Asn 297 of IgG~1~ are critical to Fcγ receptor
recognition. Synthetic peptides, 8 to 12 residues long that mimic the
lower hinge regions of IgG~1~, IgG~2~, IgG~4~ and IgE, were shown to
bind FcγRIII and to compete with the binding of the native Fc.

***Experimental Procedures***

#### 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 10mg/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 of 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 fragment and IgG1 were prepared by PNGase F (New
England Biolabs) digestion in water for 1.5 h at 37°C using 1 unit
enzyme/10μg protein. The extend of deglycosylation was analyzed by
SDS-PAGE, gel filtration chromatography and electrospray ionization mass
spectrometry. The gel filtration experiments were carried out using a
Superdex 200 PC 3.2/30 gel filtration column (Amersham Pharmacia
Biotech) and an Äkta HPLC 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. Electrospray ionization mass spectrometry (ESI-MS) measurements
were acquired and recorded with a Perkin-Elmer Sciex API-300 triple
quadruple system (Thornhill, Ontario, Canada) using 8 μg the native or
deglycosylated Fc fragment and 6 μg the native or deglycosylated IgG1.
The native and deglycosylated IgG1 were reduced with 100 mM DTT 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 1). All peptides were purified on a Superdex 200 HR16/60 gel filtration column (Amersham Pharmacia Biotech) with H~2~O as running buffer to remove impurities and to exchange the original buffer. A mostly poly alanine peptide (pALA) with a sequence of AAADAAAAL was used as the control. The concentration of peptides were estimated using absorbance at 220 nm and an extinction coefficient of 2.6 AU ml mg^-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 hour 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.

#### Surface plasmon resonance measurements

Surface plasmon resonance (SPR) measurements were performed using
BIAcore 2000 instrument (BIAcore AB). FcγRIII receptor was immobilized
at 15 μM and 45 μM concentrations 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 1). 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, 3mM 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 FcγRIII 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 of FcγRIII mixed with various concentrations of Fc from 10-0.02
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 5-0.15 μM were used. All
dissociation constants (*K*~D~) 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
BIAevaluation software package (BIAcore AB).

***Results***

**Deglycosylation of Fc**

Papain digest of the human IgG1 resulted in a 53 kD disulfide bonded Fc
fragment. Due to the carbohydrate attachment to Asn 297, the mass
spectrum of the native Fc fragment displays considerable heterogeneity
in mass (Figure 1A). Treatment of native Fc fragment with
peptide-N-glycosidase F under non-denaturing condition resulted in a
shift of molecular mass of the Fc from 53400 to 50345 daltons (Figure
1B). This agrees well with the predicted 50407 Dalton molecular weight
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 gelfiltration column (Amersham Pharmacia) (Figure 1C). It remains
as a disulfide bonded dimer (Figure 1D). The result of a native gel
electrophoresis shows that the deglycosylated Fc fragment appears to be
more compact than the native Fc (Figure 1E).

**Binding of FcγRIII to the native and deglycosylated ligands**

Both deglycosylated IgG1 and its Fc fragment were prepared under similar
conditions (Method) 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 - 0.02 μM and 10-0.08 μM, respectively. The
FcγRIII affinity of the native Fc fragment was essentially the same as
that of IgG1 and both were approximately 5 μM (Table 2). The
deglycosylation of Fc resulted in no detectable receptor binding (Figure
2A), and that of IgG1 resulted in a K~D~ of approximately 50 μM, a 10
fold reduction in the receptor binding affinity (Table 2, Figure 2B).

To rule out the possibility that the loss of receptor binding was due to
the global disruption of Fc structural fold by deglycosylation, binding
of both the native and deglycosylated Fc to protein G, which recognizes
the C~H~2-C~H~3 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 in the gel filtration and native gel
electrophoresis analyses, both of which show a slightly more compact
shape of the deglycosylated Fc compared to that of the native Fc (Figure
1).

**Binding of the lower hinge peptides to immobilized FcγRIII**

The crystal structure of FcγRIII 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 LLGG (residues 234-237) four
lower hinge residues. 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 1). All
four peptides were able to bind to the immobilized receptor on a CM5
sensor chip (Figure 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.

**Binding of FcγRIII to immobilized peptides**

To estimate the affinity between the peptides and FcγRIII, individual
peptides were immobilized on CM5 sensor chips at 10 mM concentration.
Serial dilutions of the receptor between 750-0.37 μM concentrations were
used as the analyte (Figure 4). Among the peptides, the disulfide-linked
cIgG1 binds the tightest, with K~D~ 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 other peptides, pIgG1, pIgG2 and pIgG4
display similar receptor binding affinity (Table 2, Figure 4).
Unexpectedly, pIgE displays significant binding to FcγRIII compared to
pALA although the affinity is 4-10 times lower than other hinge
peptides.

**Competition between the peptides and Fc to the 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 FcγRIII mixed with
various concentration 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 on 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 Biacore response will be independent of or 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 are blocked with an
increase concentration of Fc (Figure 5). Since no detectable affinities
could be observed between Fc and the peptides (Table 2), 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 FcγRIII at the same site as that of Fc. Interestingly,
the receptor binding of pIgE displays similar Fc competition curve as
other peptides indicating that pIgE also shares the same Fc binding
site. The Fc competition of pIgE is less profound compared to those of
other peptides due to a lower receptor-peptide affinity. The control
peptide, pALA, displays no measurable receptor binding nor Fc
competition.

**Discussion**

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 Fcγ receptors when compared to the native IgG2a (7;18).
Pound et al. found that aglycosylated IgG3 was capable of trigging human
phagocyte respiratory burst at 80% of the level trigged by the
glycosylated IgG3 despite a severe impairment in ADCC (8). Unlike Fcγ
receptors, the removal of carbohydrate of IgE did not cause significant
loss in FcεRI recognition(9;19). Structurally, the oligosaccharides
attached to Asn 297 of IgG are of 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 FcγRIII and IgE-Fc in
complex with FcεRI have been determined (3-5). However, no direct
interactions were observed between the carbohydrate on Fc and its
receptor in all 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 native Fc fragment and
intact IgG1 bind to the receptor FcγRIII with a similar dissociation
constant of 4 μM as measured in BIAcore experiments, agreeing with
previously published affinity constants (20;21). Upon enzymatic
deglycosylation, the IgG1-FcγRIII binding dissociation constant
increased from 4 μM to 50 μM, while the Fc- FcγRIII binding became
non-detectable. This 10-15 fold loss in the receptor binding affinity
indicates that the carbohydrate contribute significantly to the
receptor-ligand recognition. Since 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 extend 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 FcγRIII-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 Fc
undergoes from a open to a closed conformation (Figure 6). A structural
change associated with aglycosylated IgG3 was previously observed in the
vicinity of His 268 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 (Figure 1).
Alternatively, the carbohydrates may restrict the lower hinge
flexibility and their removal would result in enhanced hinge flexibility
and thus the reduced receptor binding (22).

It is interesting to note that the delycosylated Fc consistently
resulted in a 2 fold reduction in protein G binding affinity. Even
though protein G binds at the C~H~2-C~H~3 hinge region of Fc, away from
the carbohydrate moieties (23), the mild reduction in the protein G
binding affinity was observed upon deglycosylation. This suggests a
structural change in the C~H~2-C~H~3 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 mediate
autoimmune diseases generated from the response to autoantibodies such
as rheumatoid factor in rheumatoid arthritis (12;13;24). Under these
conditions, it is beneficial to block the autoantibody triggered
activation of FcR to relieve the autoinflammatory response that leads to
specific tissue damage. Since the activation of FcR requires receptor
aggregation by multi-valent antigen immune complexes, small molecular
compounds that capable to compete the binding of immune complexes to FcR
should prevent the receptor aggregation and thus inhibit FcR activation
(25). The structure of FcγRIII in complex with Fc provided 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 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
FcγRIII with an affinity approximately 20 times less than the native
immunoglobulins and 3 times better than the non disulfide-linked
peptide, pIgG1(Table 2). 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
same affinity. An unexpected result is that the IgE derived peptide,
pIgE, possess a significant binding affinity to FcγRIII. This is
particular interesting since the lower hinge sequence of IgE is quite
different from those of IgGs. It suggests that the binding of low
affinity receptors be quite promiscuous. It remains to be seen if the
observed receptor affinity to pIgE, which is approximately 3 times lower
compared to the IgG peptides, is sufficient to produce functional
binding of IgE by Fcγ receptors on cell surface. This may lead to the
FcγRIII activation by antigen-bound IgE under certain circumstances such
as in a saturated allergen condition or in the absence of Fcε receptors.

The competition results show that all lower hinge peptides compete
directly with Fc in the 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 example, pIgG analogs may
be useful in treating Fcγ receptor mediated autoimmune diseases by
blocking the activation of the receptor or pIgE-like compounds could be
used as potent inhibitors of Fcε receptors thus providing a potential
treatment for allergy.

The study on the binding affinity of the lower hinge peptides also
allows us to examine the issue of the receptor isotype specificity. The
low affinity human FcγRIII binds to IgG1 and IgG3 much better than it
does to IgG2 and IgG4 (16). Previous mutations on IgG2 and their binding
to human high affinity receptor FcγRI concluded that the entire lower
hinge sequence was required to restore the IgG1 binding affinity in IgG2
whereas the 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 of either the direct binding of the receptor to the immobilized
peptide chips or from the Fc competition of the receptor binding show
that pIgG2 and pIgG4 have near equal affinity to FcγRIII as does pIgG1.
Replacing a Phe to Leu in pIgG4 or a Pro-Val-Ala to Glu-Leu-Leu-Gly in
pIgG2 that contains a single residue deletion makes little difference in
their affinity toward the receptor. This suggests that factors other
than the lower hinge amino acid composition play important role in
determining the weaker binding affinity of IgG2 and IgG4 to FcγRIII. It
has been proposed that the overall length of the lower hinge may be
important to the receptor IgG subtype specificity (16), since the hinges
of IgG1 and 3 are about three residues longer than those of IgG2 and 4.
It is possible, however, that each IgG subtype vary in its glycosylation
at Asn 297 and the differences in carbohydrate may contribute to the
observed receptor specificity. Some preference in glycosylation of IgG
was 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, Pro 329 of Fc is sandwiched between Trp
90 and Trp 113 of the receptor, providing important van der waals
contacts between the receptor and Fc. Although Pro 329 is conserved
among the IgG subtypes, residue 327 displays a dimorphism with an Ala in
IgG1, 3 and a Gly in IgG2 and 4.

In conclusion, the present study suggests that glycosylation at Asn 297
of Fc play an important role in the binding of low affinity receptor
FcγRIII. The role of carbohydrate is mostly to stabilize the receptor
epitope conformation. We also demonstrated that small peptide ligands
can be designed to inhibit the binding of FcγRIII to its natural ligand
Fc.

***Acknowledgements***

We thank Drs. 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.

***Figure Legends***

Figure 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-PAGE (20% polyacrylamide PhastGel,
Amersham Pharmacia Biotech AB) analysis of the native and deglycosylated
Fc fragments of IgG1 after digestion with PNGase F (New England
Biolabs). Lane 1, molecular weight standard; 2 and 3, the native and
deglycosylated Fc; 4 and 5, the native and deglycosylated Fc under
reduced conditions. (E) Native gel. Lane 1 and 2, the native and
deglycosylated Fc fragments.

Figure 2. Effect of deglycosylation on FcγRIII 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.

Figure 3. Binding of the lower hinge peptides to immobilized FcγRIII.
Peptides are labeled as 1,2,4,E for pIgG1,pIgG2,pIgG4 and pIgE,
respectively. The three sets of experiments correspond to 5, 2.5 and
1.25mM peptide concentrations (from left to right). No detectable
binding were observed for pALA at all three concentrations.

Figure 4. FcγRIII binding to peptides immobilized on CM5 sensor chips.
Measurements were done by using serial dilutions of FcγRIII from 750 to
0.37 μM as analytes. All RUs were normalized against their level of
peptide immobilizations using cIgG1 as the standard.

Figure 5. Competition between Fc and the immobilized peptides on FcγRIII
binding. Mixtures of 10 μM FcγRIII with various concentrations of Fc
from 10 to 0.02 μM were used as analytes.

Figure 6. Proposed role of glycosylation in stabilizing the Fc
conformation.

Table 1. Immobilization of the lower hinge pepetides and FcγRIII

Immobilization

Concentration Response (RU)

pIgG1 12 mer CPAPE**LLGG**PSV 10 mM 295

cIgG1 586

pIgG2 8mer PPVAGPSV 10mM 362

pIgG4 9mer PEFLGGPSV 10mM 313

pIgE 9mer DSNPRGVSA 10mM 473

pALA 9mer AAADAAAAL 10mM 449

FcγRIII Flow cell 2 15 μM 10182

> Flow cell 3 45 μM 11731

Table 2. Dissociation constants for the binding of FcγRIII

**Immobilization Analyte K~D~ (μM)\***

Binding between the receptor and Fc

Flow Cell 2 Flow Cell 3

FcγRIII Native Fc 4.6 6.7

#  Deglycosylated Fc \> 200 \> 150

> Native IgG~1~ 4.8 3.1

Deglycosylated IgG~1~ 69.2 43.3

Binding between the receptor and peptides

K~D~ (μM)

cIgG1 FcγRIII 116

pIgG1 489

pIgG2 352

pIgG4 384

pIgE 1324

pALA \>4000

cIgG1 Fc ND

pIgG1 ND

pIgG2 ND

pIgG4 ND

pIgE ND

pALA ND

Competition between Fc and peptides to FcγRIII binding

\*FcγRIII were immobilized to a CM5 sensor chip in flow cell 2 and 3 via
primary amine attachment at approximate concentrations of 15 and 45 μM,
respectively. Each binding experiment was carried out with a serial 2x
dilution of 8 to 10 concentration points of the analyte starting at 10
μM.

The dissociation constant for the binding of the native Fc and IgG1
(K~D~^n^ ) to the CM5 chip was estimated from the steady state curve
fitting. The dissociation of deglycosylated Fc and IgG1 (K~D~^d^ ) was
estimated from the equation :

Req^d^ / Req^n^ = (K~D~^n^ + C) / (K~D~^d^ + C)

where Req and C are equilibrium response unit and the concentration of
analyte.

ND: not detectable.

Reference List

1\. Daeron, M. (1997) *Annu.Rev.Immunol.* **15,** 203-234

2\. Kato, K., Fridman, W. H., Arata, Y., and Sautes-Fridman, C. (2000)
*Immunol.Today* **21,** 310-312

3\. Garman, S. C., Wurzburg, B. A., Tarchevskaya, S. S., Kinet, J. P.,
and Jardetzky, T. S. (2000) *Nature* **406,** 259-266

4\. Sondermann, P., Huber, R., Oosthuizen, V., and Jacob, U. (2000)
*Nature* **406,** 267-273

5\. Radaev, S., Fridman, W.-H., Sautes-Fridman, C., and Sun, P. D. The
Structure of Fc Receptor and Fc complex. 2000.\
Ref Type: Generic

6\. Jefferis, R., Lund, J., and Pound, J. D. (1998) *Immunol.Rev.*
**163,** 59-76

7\. Lund, J., Tanaka, T., Takahashi, N., Sarmay, G., Arata, Y., and
Jefferis, R. (1990) *Mol.Immunol.* **27,** 1145-1153

8\. Pound, J. D., Lund, J., and Jefferis, R. (1993) *Mol.Immunol.*
**30,** 233-241

9\. Kulczycki, A., Jr. and Vallina, V. L. (1981) *Mol.Immunol.* **18,**
723-731

10\. Deisenhofer, J. (1981) *Biochemistry* **20,** 2361-2370

11\. Harris, L. J., Larson, S. B., Hasel, K. W., Day, J., Greenwood, A.,
and McPherson, A. (1992) *Nature* **360,** 369-372

12\. Vaughan, J. H. (1993) *Arthritis Rheum.* **36,** 1-6

13\. Tighe, H. and Carson, D. A. (1997) Rheumatoid Factor. In Kelley, W.
N., Harris, E. D., Ruddy, S., and Sledge, C. B., editors. *Textbook of
Rheumatology*, W.B. Saunders,

14\. Fossati-Jimack, L., Ioan-Facsinay, A., Reininger, L.,
Chicheportiche, Y., Watanabe, N., Saito, T., Hofhuis, F. M., Gessner, J.
E., Schiller, C., Schmidt, R. E., Honjo, T., Verbeek, J. S., and Izui,
S. (2000) *J.Exp.Med.* **191,** 1293-1302

15\. Chappel, M. S., Isenman, D. E., Everett, M., Xu, Y. Y., Dorrington,
K. J., and Klein, M. H. (1991) *Proc.Natl.Acad.Sci.U.S.A* **88,**
9036-9040

16\. Tamm, A. and Schmidt, R. E. (1997) *Int.Rev.Immunol.* **16,** 57-85

17\. Rousseaux, J., Rousseaux-Prevost, R., and Bazin, H. (1983)
*J.Immunol.Methods* **64,** 141-146

18\. Leatherbarrow, R. J., Rademacher, T. W., Dwek, R. A., Woof, J. M.,
Clark, A., Burton, D. R., Richardson, N., and Feinstein, A. (1985)
*Mol.Immunol.* **22,** 407-415

19\. Basu, M., Hakimi, J., Dharm, E., Kondas, J. A., Tsien, W. H.,
Pilson, R. S., Lin, P., Gilfillan, A., Haring, P., and Braswell, E. H.
(1993) *J.Biol.Chem.* **268,** 13118-13127

20\. Galon, J., Robertson, M. W., Galinha, A., Mazieres, N., Spagnoli,
R., Fridman, W. H., and Sautes, C. (1997) *Eur.J.Immunol.* **27,**
1928-1932

21\. Huizinga, T. W., Kerst, M., Nuyens, J. H., Vlug, A., dem Borne, A.
E., Roos, D., and Tetteroo, P. A. (1989) *J.Immunol.* **142,** 2359-2364

22\. Burton, D. R. (1985) *Mol.Immunol.* **22,** 161-206

23\. Sauer-Eriksson, A. E., Kleywegt, G. J., Uhlen, M., and Jones, T. A.
(1995) *Structure* **3,** 265-275

24\. Ravetch, J. V. and Clynes, R. A. (1998) *Annu.Rev.Immunol.* **16,**
421-432

25\. McDonnell, J. M., Beavil, A. J., Mackay, G. A., Jameson, B. A.,
Korngold, R., Gould, H. J., and Sutton, B. J. (1996) *Nat.Struct.Biol.*
**3,** 419-426