From the Department of Molecular and
Cellular Pathology, University of Dundee Medical School, Ninewells
Hospital, Dundee DD1 9SY, United Kingdom and the
Department of
Laboratory Medicine, Lund University, Sölvegatan 23, S-223 62 Lund, Sweden
Received for publication, October 16, 2000, and in revised form, November 22, 2000
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ABSTRACT |
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Certain pathogenic bacteria express surface
proteins that bind to the Fc part of human IgA or IgG. These bacterial
proteins are important as immunochemical tools and model systems, but
their biological function is still unclear. Here, we describe studies of three streptococcal proteins that bind IgA: the Sir22 and Arp4 proteins of Streptococcus pyogenes and the unrelated Human IgA is abundant in the seromucous secretions that bathe
mucosal surfaces, such as those lining the lungs, gut, and
genitourinary tracts. These surfaces represent major potential sites of
invasion, and the immune protection offered by secretory IgA, as the
predominant antibody at these sites, serves as a critical "first line
of defense" against many bacteria and viruses (1). Moreover, evidence
is accumulating that IgA present in serum plays an important role in a
"second line of defense" against microorganisms that have penetrated the mucosal barrier (2, 3).
IgA performs the dual role of all antibodies, of both recognizing
foreign invaders and triggering their elimination. For IgA, this latter
effector function involves the interaction of its Fc region with an
Fc Surface proteins that bind human IgA-Fc have also been identified in
many strains of Streptococcus pyogenes (group A
streptococcus) and group B streptococcus (GBS), two important
human pathogens (10-12). Despite the importance of streptococci as
pathogens, it is unclear what advantage the ability to bind IgA-Fc
offers a bacterium during the establishment of an infection. However,
even in the absence of information concerning their exact biological role, the IgA-binding proteins (IgA-BPs) are of considerable interest as immunochemical tools and model systems. A similar situation prevails
for the well known bacterial IgG-binding proteins, staphylococcal protein A and streptococcal protein G, which have been extensively characterized (13), but whose biological function is unknown.
In S. pyogenes, the two IgA-BPs that have been studied in
most detail are the Arp4 and Sir22 proteins (14, 15), which are members
of the heterogeneous M protein family (16, 17). These streptococcal
proteins have 29-residue IgA-binding regions that are related, but not
identical, making comparisons of interest (18, 19). Importantly, these
IgA-BPs bind human IgA of both subclasses and bind both serum IgA and
secretory IgA (15, 20).
In GBS, binding of IgA is due to the Here, we report experiments aimed at analyzing the interaction between
IgA-Fc and streptococcal IgA-BPs. Experiments with domain swap
antibodies and mutant IgAs indicate that binding of the different
IgA-BPs, and a 50-residue synthetic IgA-binding peptide derived from
the Sir22 protein (25), depend on almost identical sites in the Fc
interdomain region of IgA, the binding region also used by CD89. In
agreement with these results, we demonstrate that the streptococcal
IgA-BPs inhibit interaction of IgA with CD89, a property that may allow
IgA-BPs to inhibit IgA effector function.
Bacterial Strains--
The S. pyogenes strain AL168
expresses the Sir22 protein (15). Strain
AL168mrp22-sir22 Purified Bacterial Proteins and Peptides--
The S. pyogenes proteins Arp4 and Sir22 were purified after expression in
Escherichia coli (15). Two deletion derivatives of Arp4 with
nonoverlapping deletions in the IgA-binding region, the Arp4
Analysis of purified bacterial proteins by Western blot was performed
as described (29), using rabbit anti-Sir22 serum and mouse anti- Human IgG/IgA Domain Swap and Mutant IgA1 Antibodies--
All of
the recombinant anti-NIP antibodies used were chimeric (heavy chains
comprised human constant domains and murine variable domain; light
chains ( Analysis of Ig Binding by ELISA--
Nunc Maxisorp microtiter
plates, coated overnight with 0.25 µg of appropriate streptococcal
protein in fresh coating buffer (0.05 M sodium carbonate
buffer, pH 9.6), were blocked with PBS containing 0.25% (w/v) gelatin
and 0.1% (v/v) Tween 20. After three washes with water, 100 µl of
appropriately diluted wild-type or mutant IgA1 or IgG1 in Glasgow
minimum essential medium with 10% (v/v) fetal calf serum were added
per well and incubated for 2 h at room temperature. Following
washing, 100 µl of rat monoclonal anti-mouse
The binding of the detecting antibody in the absence of IgA or IgG was
used as the value for nonspecific binding and was subtracted from total
binding to give specific binding values. Results from different
experiments were normalized such that the fractional binding of
wild-type IgA at 1 × 10 Binding Assays with Whole Bacteria--
Washed suspensions of
bacteria (~5 × 109/ml) were prepared as described
(26), and identical samples (200 µl) were added to each of a series
of tubes. Different amounts of a purified Ig (in a volume of 50 µl)
were added, giving the final concentrations indicated. After incubation
for 2 h, the bacteria were washed twice with PBSAT (PBS with
0.02% NaN3 and 0.05% Tween 20), and the presence of bound
Ig was analyzed by the addition of ~15,000 cpm of
125I-labeled rat anti-mouse
Binding to Sir22 on the bacterial cell surface was analyzed with
S. pyogenes strain AL168, using the non-IgA-binding mutant AL168mrp22-sir22 Inhibition of Rosetting--
Human erythrocytes were derivatized
with NIP and sensitized with wild-type IgA1 at 200 µg/ml as described
(7). Neutrophils were isolated as described (7) and resuspended in PBS
containing 0.1% (w/v) BSA (PBS/BSA). In the inhibition assay, which
was essentially a modification of a previously described method (33),
diluted coated erythrocytes and inhibitor protein (peptide, bacterial proteins, or their control peptide or proteins), both in PBS/BSA, were
incubated at room temperature for 1 h (except for
The results were normalized so that the mean rosetting level seen in
the absence of inhibitor gave 0% normalized rosette inhibition. For
each inhibitor, experiments were performed at least twice, each time
using neutrophils from a different donor.
Inhibition of Neutrophil Respiratory Burst--
The inhibition
assay was essentially a modification of a previously described
chemiluminescence assay of respiratory bursts (7). Wells of a
chemiluminescence microtiter plate (Dynex Technologies, Ashford, UK)
were coated with NIP-BSA with subsequent incubation with wild-type IgA1
in PBS (100 µl/well at 50 µg/ml) for 1-2 h at room temperature.
After washing, appropriately diluted inhibitor in Hanks' buffered
saline solution containing 20 mM HEPES and 0.1% (w/v)
globulin-free BSA (Hanks' buffered saline solution/BSA) was added.
After incubation for 1 h at room temperature to allow prebinding
of the inhibitor to the IgA, neutrophils in Hanks' buffered saline
solution/BSA containing 260 µg/ml luminol were added (giving a final
suspension of 0.25 × 106/ml), the plate was
transferred to a Microlumat LB96P luminometer, and the
chemiluminescence was measured at regular intervals.
Immunochemical Comparison of Streptococcal IgA-binding
Proteins--
The ~40-kDa Arp4 and Sir22 proteins from S. pyogenes are both members of the M protein family and share
structural similarities, including a high degree of residue identity in
the 29-residue IgA-binding region (Fig.
1). As expected, antibodies to Sir22 were
found to cross-react with Arp4 (Fig. 1). However, the ~125-kDa Contribution of IgA-Fc Domains to Interaction with IgA-binding
Proteins--
To analyze the contribution of the C
Because intact Sir22 binds both IgA and IgG (15), the IgA/IgG domain
swap antibodies could not be used to provide information on the IgA
binding requirements of Sir22. However, the Sir22-derived peptide,
Arp4, and
To analyze whether the findings using purified bacterial IgA-binding
proteins give a true reflection of the reactivity of the proteins when
expressed on the bacterial cell surface, we performed binding studies
with whole bacteria. Due to the IgG binding ability of the S. pyogenes strains expressing Sir22 and Arp4 (34), we were unable to
analyze whole S. pyogenes bacteria for ability to bind the
IgA/IgG domain swap antibodies but could perform studies with whole GBS
bacteria expressing the
Although the data described above indicate that the C Use of Point Mutations in the C Effect of Mutations in the Predicted LLG Loop of the C
When the two C Effect of Mutations in the Predicted PLAF Loop of the C
Mutations in the PLAF loop of C Streptococcal IgA-BPs Inhibit Binding of IgA to Human
CD89--
The data reported above revealed that the PLAF and LLG loops
at the IgA-Fc interdomain region, which are critical for CD89 binding
(6, 7), also appear to be important for binding to the bacterial
IgA-BPs. Therefore, we investigated the capacity of the bacterial
proteins to inhibit the ability of IgA to bind to and activate CD89.
Using a rosetting assay, we found that Sir22 and the Sir22-derived
peptide inhibited binding of IgA1 to human CD89, the former producing
half-maximal inhibition at concentrations of ~1 × 10
As an additional, physiologically relevant test for function, we
assessed the ability of the bacterial IgA-BPs to inhibit the
IgA-mediated triggering of a respiratory burst in neutrophils, characterized by NADPH oxidase activation and degranulation, which can
be assessed in a chemiluminescence assay. The oxidative burst is a
biological response of major importance in host defense, and an ability
to inhibit it would clearly be advantageous to a pathogen. Although we
were unable to perform such assays with Sir22, the Sir22-derived
peptide, or Surface proteins that bind to the Fc part of human IgA or IgG are
expressed by many pathogenic bacteria, in particular by Gram-positive
pathogens (38). While IgA-binding proteins have been less extensively
studied than those binding IgG, more is known about their biological
properties. For example, the IgA-binding Sir22 and Arp4 proteins of
S. pyogenes are known to be important virulence factors
(26), and the IgA-binding To analyze regions in IgA-Fc critical for interaction with
streptococcal IgA-BPs, we employed domain swaps and point mutants. As
discussed before (7), these mutant proteins are unlikely to have
undergone any gross structural aberrations, allowing conclusions to be
drawn on the relative contributions of different domains and mutated
residues to the binding of streptococcal proteins. Our results indicate
that the C It may be argued that mutations in the PLAF loop of C The PLAF loop in C protein of group B streptococcus. Analysis of IgA domain swap and point mutants indicated that two loops at the C
2/C
3 domain interface are critical for binding of the streptococcal proteins. This region is
also used in binding the human IgA receptor CD89, an important mediator
of IgA effector function. In agreement with this finding, the three
IgA-binding proteins and a 50-residue IgA-binding peptide derived from
Sir22 blocked the ability of IgA to bind CD89. Further, the Arp4
protein inhibited the ability of IgA to trigger a neutrophil respiratory burst via CD89. Thus, we have identified residues on IgA-Fc
that play a key role in binding of different streptococcal IgA-binding
proteins, and we have identified a mechanism by which a bacterial
IgA-binding protein may interfere with IgA effector function.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptor (Fc
RI,1
CD89) expressed on neutrophils, eosinophils, or macrophages (4, 5).
After binding to antigen, IgA can interact with CD89 and elicit an
array of potent eradication mechanisms, including phagocytosis, superoxide generation, and release of enzymes and inflammatory mediators (5). The molecular basis of this important interaction between IgA and CD89 is now emerging, with the demonstration of the
critical role played by two loops lying at the interface of the two
domains of IgA-Fc (6, 7) and with the identification of the binding
region in CD89 (8, 9).
protein, which is unrelated to
the IgA-BPs of S. pyogenes (21, 22) and has a 73-residue IgA-binding region that does not vary in sequence between strains (23).
The
protein binds human serum IgA of both subclasses and has the
remarkable property that it binds poorly to secretory IgA, the
molecular form of IgA that predominates on mucous membranes (24).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
is a
derivative of AL168 lacking expression of the two M proteins Mrp22 and
Sir22 (26). The M-negative S. pyogenes strain
JRS145 and its derivative JRS145/pJRS264, which expresses the Arp4
protein, have been described (27). The GBS strain A909, which expresses the
protein (11), was obtained from Dr. J. Michel (Channing Laboratory, Boston, MA). A
-negative derivative of A909 was
constructed by replacement of the
gene with a kanamycin resistance
cassette.2 Streptococcal
strains were grown in Todd-Hewitt broth (Oxoid, Basingstoke, Hampshire,
UK) at 37 °C without shaking.
450 and
Arp4
451 proteins, were constructed and purified as described (18,
28). The group B streptococcal
and Rib proteins were purified from
streptococcal extracts (29). Protein G was purchased from
Calbiochem-Novagen Sciences (Nottingham, UK). A synthetic 50-residue
IgA-binding peptide, derived from the Sir22 protein, has been described
(25). This peptide contains a C-terminal C residue, not present in
Sir22, that promotes dimerization and thereby enhances IgA-binding
(30). A 53-residue synthetic peptide (M5-N), derived from the
N-terminal region of the non-IgA-binding streptococcal M5 protein, was
purchased from the Department of Clinical Chemistry, Malmö
General Hospital, Sweden. This peptide includes 50 residues derived
from the M5 protein and also includes the sequence YYC added at the
C-terminal end, allowing radiolabeling at the tyrosine residues and
dimerization via the cysteine residue.
serum for analysis of cross-reactivity. The blotting membranes were
incubated with antiserum diluted 1000-fold, followed by incubation with
radiolabeled protein G (for rabbit IgG) or protein A (for mouse IgG)
and autoradiography. Control membranes were incubated with preimmune serum.
) were murine). They included previously described wild-type
human IgA1 and IgG1 (7, 31) and the IgG1/IgA1 domain swap antibodies
1
2
3 and
1
2
3 (7). IgA1 mutants with single or double
residue substitutions in the two interdomain loops (L257R, G259R,
P440R, P440A, A442R, F443R, and LA441-442MN) (numbering
according to the commonly adopted scheme used for IgA1 Bur (32)) have
also been described (7). Recombinant antibodies were purified from
supernatants of CHO-K1 transfectants by affinity chromatography on
NIP-Sepharose (7).
(
1
and
2) light chain-biotin conjugate (Pharmingen, San
Diego, CA) diluted 1:2000 in PBS with 0.1% Tween 20 (PBST) were added
to each well and incubated for 1 h at room temperature. This
monoclonal antibody was used, since the light chains of the Igs studied
here were of mouse origin. After washing, wells were incubated for
1 h with 100 µl of streptavidin-alkaline phosphatase (Dako)
diluted 1:2000 in PBST. After further washing, reactions were developed
by incubating with substrate (Sigma Fast p-nitrophenyl phosphate tablets made up according to the manufacturer's instructions).
6 M = 1.0.
light chain (Pharmingen).
After incubation for 1 h and two washes with PBSAT, the
radioactivity associated with the pelleted bacteria was determined in a
-counter. All incubations were performed at room temperature.
Binding is expressed as a percentage of the radioactivity added to each
tube. Binding to control bacteria (
1%) has been subtracted. All
determinations were made in duplicate.
as a
negative control. Binding to Arp4 on the surface of S. pyogenes was performed with strain JRS145/pJRS264, with JRS145 as
the negative control, and binding to
on the surface of GBS was
analyzed with strain A909, using a
-negative mutant of this strain
as the negative control.
protein and
protein G, where overnight incubation was used) in wells of a
V-bottomed microtiter plate. Neutrophils (~50,000) in PBS/BSA were
added and mixed carefully; the plates were incubated for 10 min, centrifuged at 50 × g for 5 min, and further incubated for 50 min. Following the addition of acridine orange solution (6 µg/ml final concentration) to stain nucleated cells, the suspensions were examined by fluorescence microscopy, defining a rosette as a
fluorescent neutrophil with three or more erythrocytes attached.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
protein of GBS lacks residue identity with Arp4 and Sir22 and did not
react with anti-Sir22 antibody; nor did anti-
antibodies recognize
Arp4 or Sir22. These data confirm that the IgA-BPs of S. pyogenes and GBS are unrelated, underlining the interest in comparing their functional properties.
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Fig. 1.
Comparison of streptococcal IgA-binding
proteins. A, Western blot analysis of purified
preparations of the Sir22 and Arp4 proteins of S. pyogenes
and the protein of group B streptococcus. Blotting membranes were
incubated with anti-Sir22 serum or anti-
serum, as indicated, and
bound antibodies were detected with radiolabeled protein A or protein
G, as described under "Experimental Procedures." No bands were seen
in control blots incubated with preimmune serum. An equivalent
Coomassie-stained SDS gel is shown on the left.
B, alignment of the IgA-binding regions of Sir22 and
Arp4.
2 and C
3
domains of IgA-Fc to the binding of streptococcal IgA-BPs, we used two
domain swap antibodies in which homologous domains are exchanged
between IgA1 and IgG1. These domain swaps are designated
1
2
3
(constant domain structure C
1, C
2, C
3), and
1
2
3
(constant domain structure C
1, C
2, C
3) (Table
I). The ability of the constructs to bind the three streptococcal IgA-BPs and the IgA-binding peptide derived from Sir22 was analyzed by ELISA (Fig.
2).
Mutant antibodies used
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Fig. 2.
Binding of wild-type IgA1, wild-type IgG1 and
domain swaps to streptococcal IgA-binding molecules. The
antibodies were diluted as indicated and analyzed by ELISA for ability
to bind to the Sir22-derived IgA-binding peptide and to the Arp4 and
proteins, immobilized in microtiter wells. Results were normalized
by expressing ELISA absorbance as a fraction of mean absorbance seen
with wild-type IgA1 at 1 × 10
6
M. The controls for the IgA-binding peptide was the M5-N
peptide, the control for Arp4 was the Arp4
451 mutant, and the
control for
was the Rib protein from GBS. All of these control
molecules lack ability to bind IgA. The control binding shown is in
each case the mean of the fractional binding seen for the panel of
antibodies, all at 1 × 10
6
M. This value varied very little from antibody to antibody
(S.D. values of ±0.02 for M5-N, ±0.06 for Arp4
451, and ±0.19 for
Rib). The experiment was performed twice with very similar
results.
protein did not bind wild-type IgG1 in the ELISA, so this
approach was useful to illustrate the relative contributions of the two
IgA-Fc domains to the interaction site. For all of these three
IgA-binding molecules, we observed that the
1
2
3 antibody bound
with an apparent affinity generally comparable with wild-type IgA1
(Fig. 2). In contrast, no binding was observed for the
1
2
3
antibody. Several streptococcal proteins/peptides that do not bind IgA
were used as controls, and all were unable to bind to either wild-type
IgA1 or the swap antibodies. Together, these results suggest that the
C
3 domain makes a major contribution to the binding site for these
IgA-binding molecules.
protein (Fig.
3). Neither IgG1 nor the domain swap
1
2
3 bound to the
-expressing GBS, while both wild-type IgA1
and the
1
2
3 antibody bound to these bacteria. Indeed, the
1
2
3 construct bound even better than IgA1 in this test. None
of the constructs showed significant binding to an isogenic GBS mutant
lacking expression of the
protein (data not shown). These results
are consistent with a major role for the C
3 domain of IgA in binding
to the
protein.
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Fig. 3.
Use of Ig domain swap mutants to analyze
binding of IgA to protein expressed on the
surface of GBS. The analysis was performed with whole A909
bacteria and purified Ig proteins, as described under "Experimental
Procedures." Each value represents the average of duplicate
determinations. This experiment was performed twice, with very similar
results.
3 domain is of
major importance for the binding of streptococcal IgA-BPs to IgA, they
do not rule out the possibility that the C
2 domain also makes a
contribution, since regions in the C
2 domain in the
1
2
3
antibody may be able to adequately substitute for the corresponding
parts of the C
2 domain in the binding process. Data reported below
suggest that this is indeed the case.
2/C
3 Interdomain Region of
IgA-Fc for Characterization of Sites That Bind Streptococcal
Proteins--
Since the binding regions for the IgG-binding bacterial
proteins staphylococcal protein A and streptococcal protein G have been
localized to the Fc interdomain region of IgG (35, 36), we analyzed
whether the interdomain region of IgA-Fc might be directly involved in
interaction with the streptococcal IgA-binding proteins. Studies of
this region in IgA were also of interest because recent work has
implicated two loops at the C
2/C
3 interface in the binding of IgA
to human CD89 (6, 7). We used a panel of IgA1 antibodies (7), each with
a single or double amino acid substitution located in either of two
predicted loops in the interdomain region, corresponding to
Leu257-Gly259 in the C
2 domain and
Pro440-Phe443 in the C
3 domain (Table I).
These two predicted loops will be referred to as the LLG and PLAF
loops. Molecular modeling (37) suggests that these two loops occupy
positions in IgA analogous to interdomain loops in IgG that are
essential for binding of staphylococcal protein A (35). The IgA
proteins mutated in the two loops have Arg substitutions, since
conversion to this bulky side chain in a critical residue was thought
likely to be sufficient to ablate binding. However, several lines of
evidence indicate that no gross conformational changes have been
introduced into the IgA mutants (7).
2
Domain--
Two mutants with single point mutations in the LLG loop,
the L257R and G259R mutants, were assessed by ELISA for binding to the
four streptococcal IgA-binding molecules (Fig.
4A). The G259R mutant had
apparent affinities similar to those of wild-type IgA1. In contrast,
the L257R mutant displayed slightly decreased binding to the
Sir22-derived peptide, and more markedly decreased binding to intact
Sir22 and to
protein, but was not affected in its ability to bind
the Arp4 protein. Together, these data suggest that residues in the
C
2 region may play a role in the binding of streptococcal IgA-BPs
and that the Arp4 and Sir22 proteins, which are closely related, may
not have completely identical IgA-binding properties when tested in
purified form.
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Fig. 4.
Binding of IgA1s with point mutations in the
predicted LLG loop in C 2. A,
binding to the streptococcal Sir22, Arp4, and
proteins and the
Sir22-derived IgA-binding peptide. The results were normalized by
expressing ELISA absorbance as a fraction of mean absorbance seen with
wild-type IgA1 at 1 × 10
6
M. The following non-IgA-binding streptococcal
peptide/proteins served as negative controls: M5-N peptide for the
IgA-binding peptide, Arp4
451 for Arp4, and Rib for
protein. The
control binding shown is in each case the mean of the fractional
binding seen for the panel of antibodies, all at 1 × 10
6 M. This value varied very
little from antibody to antibody (S.D. values of ±0.01 for M5-N,
±0.04 for Arp4
451, and ±0.08 for Rib). The experiment was
performed twice with very similar results. B, binding to
whole streptococci expressing one of the IgA-binding proteins Sir22,
Arp4, or
protein. The protein expressed and the bacterial species
are indicated above each graph. Each
graph shows results obtained with the IgA1 wild type protein
(IgA1) and different mutant proteins, as indicated. Each value
represents the average of duplicate determinations, and each experiment
was performed at least twice, with very similar results.
2 mutants were analyzed for binding to IgA-BPs
expressed on the bacterial cell surface, a similar pattern emerged (Fig. 4B). Thus, G259R bound to bacteria expressing Sir22,
Arp4, or
with apparent affinities similar to those of wild-type
IgA1. In contrast, L257R displayed strongly decreased ability to bind to each strain. The binding observed in this analysis with whole bacteria was due to the IgA-BPs, since bacterial mutants lacking expression of the different IgA-BPs were completely unable to bind any
of the IgA proteins (data not shown). Together, these results suggest
that the Gly259 residue does not play a role in interaction
with any of the streptococcal IgA-BPs when they are expressed on the
bacterial cell surface but that residue Leu257 may be
involved. It is noteworthy that the L257R mutant showed strongly
reduced binding to Arp4 expressed on bacteria, but not to purified Arp4
(Fig. 4A), stressing the importance of comparing binding tests
performed with purified proteins and tests performed with whole
bacteria. However, it should be noted that L257R is the only protein
for which we have noted a clear difference between results obtained
with purified proteins and with whole bacteria.
3
Domain--
The IgA antibodies with mutations in the C
3
interface-proximal PLAF loop all had single amino acid substitutions
with the exception of LA441-442MN (Table I). The effects of the
substitutions on binding to purified proteins were similar for the
three molecules originating from S. pyogenes,
i.e. Sir22, the Sir22-derived peptide, and Arp4 (Fig.
5A). The A442R mutant bound
all three molecules with affinity generally similar to wild-type IgA1,
while the LA441-442MN mutant displayed binding consistent with a
decrease in affinity of around 10-fold for Sir22 and the peptide and
around 2-5-fold for Arp4. Mutant P440R showed only weak binding to the
three IgA-binding molecules originating from S. pyogenes,
with apparent reductions in affinity of greater than 100-fold, while
the P440A and F443R mutants showed essentially no binding. Analysis of
binding of these mutants to Sir22 or Arp4 expressed on the surface of
S. pyogenes produced a similar picture (Fig. 5B).
Thus, the binding of A442R to whole S. pyogenes bacteria was
similar to that observed with wild-type IgA1. In contrast, P440R and
F443R displayed markedly reduced binding, while no binding was observed
for P440A, i.e. the effect on binding was even more dramatic
for P440A than for P440R. Together, these results suggest that the PLAF
loop in C
3, and residues Pro440 and Phe443
in particular, are critical for binding of Sir22, its peptide derivative, and Arp4. As described above, some contribution to binding
is also apparently made by the close-lying Leu257 residue
in the LLG loop of C
2.
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Fig. 5.
Binding of IgA1s with point mutations in the
predicted PLAF loop in C 3. A,
binding to the streptococcal Sir22, Arp4, and
proteins and the
Sir22-derived IgA-binding peptide. The results were normalized by
expressing ELISA absorbance as a fraction of mean absorbance seen with
wild-type IgA1 at 1 × 10
6
M. Controls were as for Fig. 4. The control binding shown
is in each case the mean of the fractional binding seen for the panel
of antibodies, all at 1 × 10
6
M. This value varied very little from antibody to antibody
(S.D. values of ±0.007 for M5-N, ±0.02 for Arp4
451, and ±0.08 for
Rib). The experiment was performed twice with very similar results.
B, binding to whole streptococci expressing one of the
IgA-binding proteins Sir22, Arp4, or
protein. The protein expressed
and the bacterial species are indicated above each
graph. Each value represents the average of duplicate
determinations, and each experiment was performed at least twice, with
very similar results.
3 also had dramatic effects on
binding to
protein. The results were reminiscent of those obtained
with the S. pyogenes proteins/peptide but with important distinctions. As observed for the S. pyogenes proteins,
mutant A442R had an apparent affinity similar to that of wild-type
IgA1, and LA441-442MN showed decreased binding consistent with a drop in affinity of around 10-fold (Fig. 5A). However, unlike the
S. pyogenes proteins, mutant P440R was almost completely
negative, while mutant F443R retained some binding, with an apparent
reduction in affinity of around 10-fold. Thus, both of the P440R and
P440A mutations appeared to virtually ablate binding to
protein,
suggesting that the Pro440 residue plays a highly critical
role in binding of
to IgA. Binding tests with
expressed on the
bacterial cell surface confirmed the ELISA data (Fig.
5B).
8 M and the latter at ~1 × 10
7 M (Fig.
6A). This effect appears to be
specific, since a control peptide (M5-N) did not display inhibitory
ability at concentrations up to 5 × 10
6
M. Similarly, we found that Arp4 was capable of inhibiting
the IgA1-CD89 interaction, with half-maximal inhibition at ~1.6 × 10
8 M (Fig. 6B).
This inhibition required the presence of an intact IgA-binding region
in Arp4, since two non-IgA-binding Arp4 derivatives with short
deletions in the IgA-binding region, Arp4
450 and Arp4
451, did not
cause any inhibition. The
protein also appeared able to inhibit IgA
binding to CD89, but much greater concentrations were required, with
half-maximal inhibition only being reached at ~2 × 10
6 M (Fig. 6C).
However, the protein G control did not influence rosette formation at
equivalent concentrations. Together, these data indicate that all four
of the IgA-binding bacterial molecules studied here are able to block
the binding of IgA-Fc to CD89.
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Fig. 6.
Inhibition of binding of IgA1 to CD89 on
neutrophils, assessed by rosette formation. A,
inhibition test with Sir22 and the Sir22-derived IgA-binding peptide
and with the non-IgA-binding M5-N peptide. B, inhibition
test with Arp4 and with the non-IgA-binding deletion mutants Arp4 450
and Arp4
451. C, inhibition test with
protein and with
the non-IgA-binding protein G. The results were normalized and
expressed such that rosette formation in the absence of IgA1 coating
was used to provide the value for 0% rosettes (equivalent to 100%
inhibition), while that in the absence of inhibitor was used as the
value for 100% rosettes (equal to 0% inhibition).
protein due to unexpected generalized effects on
neutrophil bursts, we did observe that Arp4 was capable of inhibiting
the IgA-triggered oxidative burst at concentrations greater than 5 × 10
8 M (Fig.
7). This inhibition appears to be due to
the ability of Arp4 to bind IgA, since the two non-IgA-binding Arp4
deletion mutants Arp4
450 and Arp4
451 caused little or no
inhibition. Moreover, Arp4 did not inhibit a phorbol 12-myristate
13-acetate-stimulated respiratory burst (data not shown). Together,
these data indicate that Arp4 can inhibit a respiratory burst triggered
by the binding of IgA-Fc to CD89.
View larger version (27K):
[in a new window]
Fig. 7.
Analysis of inhibition of an IgA1-mediated
neutrophil respiratory burst by Arp4 and its non-IgA-binding deletion
mutants Arp4 450 and
Arp4
451. Chemiluminescence
(CL, arbitrary units) was induced by IgA1 alone (
) or in
the presence of bacterial inhibitor, as indicated. The negative control
(
) shows chemiluminescence observed in the absence of IgA1 and
inhibitor. Inhibitor at 5 × 10
10
M (
), 5 × 10
9
M (
), 5 × 10
8
M (
), 5 × 10
7
M (
). The results of representative experiments are
shown. The experiments were performed at least twice with very similar
results.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
protein of GBS has been shown to be a
target for protective antibodies (39). However, the exact role of
IgA-BPs in streptococcal pathogenesis remains unknown. This situation,
and the potential usefulness of IgA-BPs as immunochemical tools and
model systems, prompted us to characterize the binding site in
IgA-Fc for different IgA-BPs.
3 domain of IgA-Fc makes a major contribution to binding
for all the streptococcal proteins, with the C
2 domain possibly
playing a less important role. This result is in agreement with a
previous study, which implicated the C
3 domain in the binding of the
Arp4 protein (40). The present study indicates that the PLAF loop
(residues 440-443), predicted to lie on the surface of the C
3
domain, is of particular importance for the binding of the bacterial
IgA-BPs, but the LLG loop in C
2 also appears to contribute. The data
on proteins/peptide from S. pyogenes suggest that they bind
to identical or very similar sites in IgA-Fc, and binding of the
protein from GBS appears to depend on essentially the same residues.
However, Phe443 is less critical for interaction with
than with the IgA-binding molecules from S. pyogenes. Thus,
the IgA-BPs of S. pyogenes bind to a site that appears to be
very similar to the site used by the unrelated
protein of GBS, but
the sites are probably not identical. This conclusion is in good
agreement with previously reported inhibition experiments, which
indicated that the Arp4 and
proteins bind to the same region in IgA
(24).
3 and the LLG
loop of C
2 produced their effects either by perturbing direct
binding interactions or by triggered alterations in the conformation of
close-lying residues that provide the binding contacts. The finding
that mutation of Gly259 in the LLG loop and of
Ala442 in the PLAF loop did not reduce binding, while that
of adjacent residues produced marked effects, may indicate that the
latter possibility is less likely. In either case, these interdomain loops may be considered as important markers of the binding sites for
the streptococcal IgA-BPs.
3 and the LLG loop in C
2 are predicted to lie
close in three-dimensional space, as highlighted on a molecular model
of IgA based on solution structural studies (37) (Fig. 8). These loops are proposed to play a
key role for binding of IgA-Fc to CD89 (6, 7) and to streptococcal
proteins (this study), but there are clear differences between the
sites interacting with the human and bacterial proteins. In particular,
domain swap experiments indicated that binding of CD89 to IgA-Fc
requires both of the C
2 and C
3 domains, while the C
2 domain
can be replaced with the C
2 domain without affecting binding of the
streptococcal proteins. Further, mutation in the PLAF loop of
Ala442 to Arg was associated with loss of detectable
binding to CD89 (7), while it had no impact on binding to the bacterial
proteins. Together, these data indicate that CD89 and the bacterial
IgA-BPs have overlapping, but not identical, binding sites in
IgA-Fc.
View larger version (54K):
[in a new window]
Fig. 8.
Molecular model of human IgA1 Fc,
highlighting residues at the
C 2/C
3 interface
critical for binding to streptococcal IgA-binding proteins. This
model of IgA1 Fc is adapted from Ref. 37. (The atomic coordinates for
the molecular model of human IgA1 are available in the Protein Data
Bank, Research Collaboratory for Structural Bioinformatics, Rutgers
University, New Brunswick, NJ, accession number 1iga). The two heavy
chain backbones are shown as cyan ribbons, with the LLG loop
(Leu257-Gly259) of the C
2 domain and the
PLAF loop (Pro440-Phe443) of the C
3 domain
represented in yellow. Residues Leu257,
Pro440, and Phe443, which are predicted to play
particularly important roles for binding of the streptococcal proteins,
are highlighted in green. The C-terminal tailpieces are
omitted.
The proposed overlapping nature of the binding sites in IgA-Fc for CD89 and bacterial IgA-BPs is strongly supported by the observed ability of the IgA-BPs to inhibit the binding of IgA to CD89, as measured by rosetting, and by the ability of the Arp4 protein to inhibit an IgA-triggered respiratory burst in CD89-expressing neutrophils. It could be argued that the observed blockade is rather a gross effect, since intact IgA-BPs might be expected to mask an appreciable area of the IgA-Fc surface due to their molecular size. However, we found that the much smaller 50-residue IgA-binding peptide, representing an isolated IgA-binding domain (25), was still capable of specifically blocking interaction with CD89. This peptide would be anticipated to adopt a three-dimensional structure of relatively small size, so its ability to inhibit CD89 binding is most likely explained by close proximity of their respective binding sites on IgA-Fc.
The ability of streptococcal IgA-BPs to inhibit binding of IgA to CD89
suggests that such disruption may also occur during a bacterial
infection. We can therefore propose a mechanism by which possession of
an IgA-BP may confer on a bacterium the ability to evade clearance
mediated by specific IgA antibodies. According to this mechanism,
specific binding of an IgA molecule to a bacterial surface antigen is
followed by binding of the Fc part of the IgA molecule to a bacterial
IgA-BP also present on the bacterial surface, thereby allowing the
bacterium to evade the elimination processes that would normally be
triggered via binding of IgA-Fc to CD89 (3). Such bridging of a bound
Ig molecule has also been proposed to explain the mechanism of action
of the IgG-Fc receptor of herpes simplex virus type 1, but it is not
known if this viral Fc receptor blocks binding of IgG to human Fc
receptors or if it exerts its function by some other mechanism (41,
42).
The finding that unrelated IgA-BPs, expressed by S. pyogenes
or GBS, bind to similar sites in IgA-Fc is reminiscent of the situation
described for the two unrelated bacterial IgG-binding molecules,
staphylococcal protein A and streptococcal protein G, both of which
bind to the Fc domain interface in IgG (13, 35, 36, 43). Taken
together, these data imply that convergent evolution has favored the
appearance of bacterial proteins that bind to the CH2/CH3 interdomain
region in IgA or IgG. Interestingly, accessibility and sequence
comparison analyses and a recent study exploiting random peptides
indicate that the interdomain region of IgG-Fc has intrinsic properties
that favor binding to other proteins (44, 45). This conclusion is
supported by evidence that the Fc receptor of herpes simplex virus
type 1 binds at the C
2/C
3 domain interface (46, 47) and by
localization of the binding site for CD89 to the interdomain region of
IgA-Fc (6, 7). These data raise the question whether one biological function of protein A, protein G, and the Fc
receptor of herpes simplex virus type 1 might be to inhibit IgG effector function in a
manner analogous to that proposed here for bacterial IgA-BPs, i.e. by interfering with the binding of IgG to Fc
receptors on phagocytic cells. However, the majority of available
evidence appears to argue against such a mechanism, since protein A has been shown not to inhibit the binding of IgG to human Fc
RI and Fc
RII (48) and all Fc
R receptors bind at the N-terminal end of
the Fc, well away from the Fc interdomain region (49-54). Further work
will be required to clarify the various mechanisms that may afford
evolutionary advantage to microbes that possess proteins that bind IgA
or IgG.
In summary, we have demonstrated that unrelated bacterial IgA-BPs bind
in the interdomain region of IgA-Fc, at sites overlapping with that
used by the human IgA-receptor CD89. These findings have allowed us to
propose a possible mechanism by which bacterial IgA-BPs may interfere
with IgA effector function, thereby contributing to bacterial
virulence. This study also highlights the potential of the bacterial
IgA-BPs, and the IgA-binding peptide in particular, as tools for
studies of the structure and function of IgA.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Eskil Johnsson and Margaretha Stålhammar-Carlemalm for providing purified bacterial proteins and to Stephen Perkins for generating the molecular modeling figure.
![]() |
FOOTNOTES |
---|
* This work was supported by Wellcome Trust Grant 049785 (to J. M. W.) and by grants (to G. L.) from The Swedish Medical Research Council (Grant 09490), the Medical Faculty of Lund University, the Royal Physiographic Society in Lund, the Swedish Society for Medical Research, and the Trusts of Crafoord, Kock, and Österlund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
¶ A Wellcome Trust Advanced Training Fellow.
** To whom correspondence may be addressed. gunnar.lindahl@ mmb.lu.se.
To whom correspondence may be addressed. Tel.: 44 1382 660111 (ext. 33540); Fax: 44 1382 633952; E-mail: j.m.woof@dundee.ac.uk.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M009396200
2 T. Areschoug, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
FcR, Fc
receptor;
GBS, group B streptococcus;
IgA-BP, IgA-binding protein;
NIP, 3-hydroxy-4-nitro-5iodophenylacetate;
PBS, phosphate-buffered
saline;
BSA, bovine serum albumin;
ELISA, enzyme-linked
immunosorbent assay.
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