From the Department of Laboratory Medicine, Lund
University, Sölvegatan 23, S-223 62 Lund, Sweden and the
§ Department of Microbiology, University of Alabama at
Birmingham, Birmingham, Alabama, 35294
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ABSTRACT |
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Surface proteins that bind to the Fc part of
human IgA are expressed by many strains of Streptococcus
pyogenes, a major human pathogen. Studies of these proteins have
been complicated by their size and by their ability to bind human
plasma proteins other than IgA. Here, we describe a synthetic
50-residue peptide, derived from streptococcal protein Sir22, that
binds human IgA but not any of the other plasma proteins known to bind
to Sir22. The peptide binds serum IgA and secretory IgA and binds IgA
of both subclasses. Evidence is presented that the peptide folds
correctly both in solution and when it is immobilized and that it
readily renatures after denaturation. Together, these data indicate
that the peptide corresponds to a protein domain that binds IgA with
high specificity. This is the first report of an IgA-binding domain
that retains its properties in isolated form.
An important property of human IgA is the ability of the Fc part
to interact with different receptors. Binding to one of these receptors, CD89, is believed to be essential for the effector functions
of serum IgA (1, 2), whereas binding to the poly-Ig receptor on mucosal
epithelial cells is required for secretion of dimeric IgA (3).
The Fc part of human IgA also binds to surface proteins expressed by
some pathogenic streptococci (4, 5). These proteins are of interest for
analysis of pathogenetic mechanisms in bacterial infections and are
also of potential value as model systems and analytical tools for
studies of IgA. The most extensively studied of these bacterial
IgA-binding proteins are those expressed by Streptococcus
pyogenes (group A streptococcus), a major human pathogen.
IgA-binding proteins expressed by this bacterium are members of the M
protein family (6-8), a family of coiled-coil proteins that are
important for virulence. However, characterization of several of the
IgA-binding proteins of S. pyogenes has shown that they not
only bind IgA but also bind other human plasma proteins (9-11). This
property is of interest with regard to pathogenetic mechanisms but has
complicated studies of the IgA binding property of the proteins. For
structural analysis, it would also be desirable to have access to an
IgA-binding molecule of smaller size.
In this study, we explored the possibility of studying the IgA-binding
region of an S. pyogenes protein in isolated form. We show
that a 50-residue synthetic peptide, derived from streptococcal protein
Sir22,1 binds IgA but not any
of the other plasma proteins that bind to Sir22. The properties of this
peptide indicate that it corresponds to a protein domain that binds IgA
with high specificity.
Synthetic Peptide and Purified Proteins--
The 50-residue
synthetic peptide (see Fig. 1A) was purchased from the
Department of Clinical Chemistry (Malmö General Hospital, Sweden). It corresponds to amino acid residues 35-83 of protein Sir22
(10) and includes a C-terminal cysteine residue not present in Sir22.
The peptide was Immunoglobulins and Human Serum--
All immunoglobulins were of
human origin. Polyclonal serum IgA was from Cappel-Organon Teknika
(Turnhout, Belgium). S-IgA was purified from colostrum (12). Monoclonal
IgA proteins were isolated from serum of patients with IgA multiple
myeloma (12). Three of the monoclonal IgA proteins and all monoclonal
IgG proteins were kindly provided by Dr. F. Skvaril (Bern,
Switzerland). Monoclonal IgM and IgD were from The Binding Site
(Birmingham, United Kingdom), and IgE was the gift of Dr. I. Olsson
(Lund University). Polyclonal IgG was from Kabi (Stockholm, Sweden).
Fab and Fc fragments of an IgA1 protein were purified after cleavage
with IgA protease from Haemophilus influenzae (12). Serum
from an IgA-deficient individual was kindly provided by Dr. A. Sjöholm (Lund University).
Binding Assays and Competitive Inhibition--
Binding of
radiolabeled human proteins was analyzed after immobilization of
peptide or Sir22 in microtiter wells. The wells were coated overnight
at 4 °C with 50 µl of a solution of peptide or Sir22 (100 µg/ml
in PBS). After washing and blocking with PBSAT (PBS with 0.02% sodium
azide and 0.05% Tween 20), radiolabeled ligand (~25,000 cpm) was
added to each well, and the plates were incubated for 2 h at room
temperature. After washing with PBSAT, the radioactivity associated
with each well was determined. Nonspecific binding (<0.7%) was
determined for wells coated only with PBSAT. The Igs used were polyclonal.
For inhibition tests, wells of microtiter plates were coated with the
peptide (5 µg/ml for tests with serum IgA and 50 µg/ml for tests
with S-IgA). After blocking, radiolabeled serum IgA or S-IgA (~25,000
cpm) was added together with inhibitor as indicated. The final
concentration of radiolabeled IgA was ~0.3 nM. Binding was analyzed as described above. Nonspecific binding was <0.25%.
Other Methods--
Western blot and dot-blot analysis on
polyvinylidene difluoride membranes were performed as described (10).
Protein concentrations were determined with the MicroBCA kit from
Pierce using BSA as a reference. Radiolabeling of peptides and proteins
with 125I was performed with the chloramine-T method.
Design of a Synthetic Peptide Including the IgA-binding Region of
Protein Sir22--
Previous studies of the S. pyogenes
protein Arp4 demonstrated that the IgA-binding region of this protein
is localized in a 29-residue region in the N-terminal part of the
molecule (8, 13). An attempt was made to demonstrate IgA binding for a
33-residue synthetic peptide including this region from Arp4, but no
binding was observed (data not shown). A longer peptide was therefore tested as described below. This new peptide was derived from the Sir22
protein, which is closely related to Arp4 (10) and is expressed by a
bacterial strain that is suitable for analysis of pathogenetic
mechanisms (14).
Protein Sir22 is a dimeric coiled-coil molecule that binds at least
three different human plasma proteins, IgA, IgG, and C4BP (10, 11, 15)
(Fig. 1A). The regions in
Sir22 that bind these ligands have been partly identified. The
29-residue IgA-binding region (defined by homology with Arp4) overlaps
with an upstream region that includes a C4BP-binding site (16). Because
IgA and C4BP do not compete for binding to Sir22 (11), these ligands may bind to separate domains. The IgA-binding region also overlaps with
a downstream region including an IgG-binding site (10, 17). The
bindings of IgA and IgG to Sir22 are mutually exclusive, indicating
shared or contiguous binding sites (10). In addition to these ligands,
serum albumin may bind to the central C repeat region of Sir22
(18).
The design of an IgA-binding peptide was based on the hypothesis that
C4BP, IgA, and IgG bind to separate domains in Sir22 and that the
inhibition of IgA binding by IgG (10) was due to steric hindrance not
shared binding sites. The synthetic 50-residue peptide covers the
predicted IgA-binding region of Sir22 and also includes 10 residues on
either side, added to enhance the probability for correct folding.
Because the Sir22 protein must form a coiled-coil dimer to bind IgA
(15, 19), a C-terminal cysteine residue was included to ensure that
dimerization could occur. However, it also seemed possible that the
peptide would be able to form a coiled-coil by itself (20).
The Peptide Specifically Binds IgA--
Wells of microtiter plates
were coated with the peptide or with Sir22 and analyzed for the ability
to bind radiolabeled ligands (Fig. 1B). As expected,
immobilized Sir22 bound all four ligands. For unknown reasons, the
binding of IgG was lower than for serum IgA in this test, although
Sir22 has similar affinity for these two ligands (10). The immobilized
peptide bound serum IgA and S-IgA, but not C4BP or IgG, indicating that
it specifically binds IgA. The binding of serum IgA and S-IgA to the
immobilized peptide could be completely inhibited by unlabeled IgA but
not by BSA, confirming that the binding of IgA was specific and showing
that the binding was not due to the radiolabeling (Fig. 1C).
Moreover, binding of serum IgA or S-IgA to immobilized peptide could be inhibited by free peptide, indicating that the ability to bind IgA was
not due to a conformational change in the peptide upon immobilization
(Fig. 1C). Finally, the binding of serum IgA and S-IgA to
immobilized peptide could be inhibited by a purified monoclonal IgA1
protein, showing that the binding of IgA was independent of the
antigen-binding site (data not shown).
The binding properties of the peptide were further studied by Western
blot analysis under nonreducing conditions (Fig.
2A). Radiolabeled peptide,
used as the probe, bound to serum IgA but not to IgG. Thus, the peptide
in solution retains its binding ability after radiolabeling. Striking
evidence for the specificity of the peptide came from analysis of whole
normal serum and serum from an IgA-deficient individual. The peptide
lacked reactivity with any of the many proteins present in
IgA-deficient serum, but bound to IgA present in normal serum. Similar
results were obtained in a dot-blot analysis (Fig. 2B). In
saliva, the peptide recognized a high molecular weight protein with the
mobility of S-IgA and also detected some monomeric IgA (Fig.
2A), giving further support for the specificity of the
binding. The peptide even recognized separated The Peptide Binds IgA-Fc and Binds IgA of Both Subclasses--
As
expected, the peptide binds to the Fc part of IgA (Fig.
3A). To analyze whether the
peptide binds IgA of both subclasses, a dot-blot analysis was performed
with purified monoclonal proteins (Fig. 3B). The analysis
also included analysis of monoclonal IgG, IgM, IgD, and IgE proteins,
and the binding properties of the peptide were compared with those of
the parental Sir22 molecule. In agreement with previous results (10),
Sir22 was found to bind most of the IgA and IgG proteins. The peptide
only bound IgA proteins, and binding was observed for 8 of 9 IgA1
proteins and for 10 of 11 IgA2 proteins. However, this
semi-quantitative analysis indicated that the affinity varied
considerably for different IgA proteins. Possible reasons for this
variation are differences in glycosylation and loss of binding ability
during purification of monoclonal proteins. The ability of the peptide
to bind different IgA proteins was similar to that of protein Sir22,
supporting the hypothesis that the peptide corresponds to an
IgA-binding domain in Sir22.
Western Blot Analysis and Effect of Heating--
Western blot
analysis of the peptide under nonreducing conditions showed that it
bound radiolabeled IgA (Fig.
4A). This analysis included an
initial step, in which the peptide was boiled in SDS-containing buffer,
a step that most likely caused denaturation. Thus, these results
suggest that at least some peptide molecules renatured during the
analysis. The peptide, probably a dimer, moved more slowly than
expected, a common property in streptococcal surface proteins (10).
Under reducing conditions, when the peptide migrated as a monomer, it
also bound IgA after blotting. In this regard, the peptide behaved like
protein Sir22, which also binds IgA in Western blot analysis, after
having migrated as a monomer (10). Because Sir22 must form a
coiled-coil dimer to bind IgA (15, 19), it seems likely that
dimerization of Sir22 can occur on the blotting membrane, allowing
binding of IgA. Similarly, the ability of the peptide to bind IgA in
Western blot analysis during reducing conditions might have been due to
formation of coiled-coil dimers on the blotting membrane. Thus, the
results of this analysis cannot be taken as evidence that a monomeric
form of the peptide binds IgA. Structural analysis of the peptide will
now be of considerable interest and will show whether it has a
coiled-coil structure, as suggested by the available data (15, 16, 19).
Interestingly, the C-terminal part of the peptide includes a sequence
(LEEKEKNLEKK) that is similar to a consensus "trigger" sequence
implicated in the formation of coiled-coils (21).
The ability of the peptide to bind IgA after heating was further
analyzed in an inhibition test (Fig. 4B). In this test, the ability of radiolabeled IgA to bind to immobilized peptide was inhibited by free peptide. Boiling of the peptide for 5 min, which most
likely caused denaturation, had no effect on the ability of the peptide
to subsequently inhibit binding, implying that most peptide molecules
refolded into the native form after denaturation. Taken together, the
data in Fig. 4 indicate that the peptide is a very stable molecule. The
radiolabeled form of the peptide was also stable, because it could be
kept frozen for 4 months without loosing the ability to specifically
bind IgA (data not shown).
Concluding Remarks--
The properties of the peptide studied here
indicate that it corresponds to an IgA-binding domain that binds human
IgA-Fc with high specificity. To our knowledge, this represents the
first example of an IgA-binding domain that retains its binding
properties in isolated form. Importantly, the peptide binds human IgA
of both subclasses and binds both serum IgA and S-IgA, suggesting that
it may become a useful tool for studies of human IgA. With regard to
applications, it is of interest that the peptide showed specificity for
IgA both when it was immobilized and when it was present in soluble
form. Previous studies of Sir22 and other IgA-binding streptococcal
proteins suggest that the peptide may have lower affinity for S-IgA
than for serum IgA, due to the presence of secretory component in S-IgA
(10, 22), but the peptide could readily detect S-IgA in Western blot
analysis, implying that it may become valuable also for studies of this
form of IgA.
A bacterial IgA-Fc-binding protein unrelated to Sir22 has been
described in group B streptococcus (5, 23, 24), and a fusion protein
including a 73-residue sequence from this protein was found to bind IgA
(25). Direct comparison of this 73-residue sequence with the peptide
described here did not disclose any similarity. The group B
streptococcus protein has little or no ability to bind S-IgA, which may
limit its use as an immunological tool (26), but structural comparisons
of the different IgA-binding regions would be of interest.
The size and stability of the peptide described here suggest that it
may be amenable to structural analysis and could become a useful model
for studies of human IgA receptors. An interesting parallel has already
been identified, because human CD89 was found to bind to a site on
IgA-Fc analogous to the site on IgG-Fc that is recognized by
staphylococcal protein A (2), stressing the value of bacterial proteins
as model systems. Further characterization of the peptide will also be
of interest for analysis of pathogenetic mechanisms in S. pyogenes infections.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
95% pure. Recombinant Sir22 was purified as
described (10). Purified human
C4BP2 was the kind gift of
Dr. B. Dahlbäck (Malmö General Hospital).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Sequence and binding properties of the
IgA-binding peptide. A, schematic representation of
protein Sir22 and sequence of the derived peptide. The regions in Sir22
known to include binding sites for C4BP, IgA, and IgG are indicated.
The ligand-binding sites may actually be nonoverlapping (see text). The
peptide corresponds to residues 35-83 of the processed form of Sir22
and includes the predicted 29-residue IgA-binding region (8), 10 additional residues on either side, and a C-terminal cysteine not
present in Sir22. B, specificity of binding. Microtiter
wells coated with Sir22 (left) or peptide (right)
were analyzed for ability to bind radiolabeled human proteins.
C, competitive inhibition. Microtiter wells were coated with
the peptide. Binding of radiolabeled serum IgA (left) or
S-IgA (right) was inhibited with unlabeled protein or
peptide, as indicated. Inhibition by BSA was tested at one
concentration. In the experiments in B and C,
each value is the average of duplicate samples. All experiments were
performed twice with similar results.
chains, as shown by
Western blot analysis of IgA under reducing conditions (Fig.
2C).
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Fig. 2.
Binding specificity analyzed with radioactive
peptide. A, the preparations indicated were analyzed by
Western blot under nonreducing conditions. B, dot-blot
analysis of normal human serum and serum from an IgA-deficient
individual. C, Western blot analysis of serum IgA and IgG
under reducing conditions. The probe used in all panels was
radiolabeled peptide. These experiments were performed twice with
similar results.
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Fig. 3.
The peptide binds IgA-Fc and binds IgA of
both subclasses. A, dot-blot analysis of an IgA1
protein and its Fab and Fc fragments. The membrane was probed with
radiolabeled peptide. B, dot-blot analysis of purified human
monoclonal Igs. The indicated amounts of Ig were applied to two
identical membranes, which were probed with radiolabeled Sir22 or
peptide. Each vertical row corresponds to one monoclonal
protein. These experiments were performed twice with similar
results.
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Fig. 4.
Western blot analysis of the peptide and
effect of heating. A, samples of the peptide were
subjected to Western blot analysis using a radiolabeled IgA1 protein as
the probe. B, binding of radiolabeled serum IgA to peptide,
immobilized in microtiter wells, was inhibited with untreated peptide
( ), or with peptide that had been boiled in PBS for 5 min and kept
at room temperature for 30 min (
). These experiments were performed
twice with similar results.
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FOOTNOTES |
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* This work was supported by grants from Swedish Medical Research Council Project 9490, the Royal Physiographic Society in Lund, the Swedish Society for Medical Research, the Alfred Österlund Trust, the Crafoord Trust, and the Johan and Greta Kock Trust.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: Dept. of Laboratory Medicine, Lund University, Sölvegatan 23, S-223 62 Lund, Sweden. Tel.: 46-46-173244; Fax: 46-46-189117; E-mail: Gunnar.Lindahl{at}mig.lu.se.
1 In an alternative nomenclature (14), Sir22 is designated Emm22.
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ABBREVIATIONS |
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The abbreviations used are: C4BP, C4b-binding protein; S-IgA, secretory IgA; PBS, phosphate-buffered saline; BSA, bovine serum albumin.
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