From the Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
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Our previous studies have established that a
cell-surface 25-kDa elastin-binding protein of Staphylococcus
aureus (EbpS) mediates binding of this pathogen to the
extracellular matrix protein elastin. Results from binding assays
examining the activity of various EbpS fragments suggested that the
elastin recognition domain is contained within the first 59 amino
acids. In this report, we have used functional analyses with synthetic
peptides and recombinant truncated forms of EbpS to localize the
elastin binding domain to a 21-amino acid region contained within
residues 14-34 of EbpS. Further evidence for the importance of this
domain was obtained by demonstrating that the inhibitory activity of
anti-EbpS antibodies on staphylococcal elastin binding was neutralized
when these antibodies were pre-absorbed with a truncated recombinant
EbpS construct containing residues 1-34. Overlapping synthetic
peptides corresponding to EbpS residues 14-36 were then generated and
tested for elastin binding activity to define further the elastin
binding domain, and results from these studies showed that sequences
spanning amino acids Gln14-Asp23,
Asp17-Asp23, and
Thr18-Glu34 inhibit binding of
Staphylococcus aureus to elastin. Our analyses indicate
that the hexameric sequence
Thr18-Asn-Ser-His-Gln-Asp23 is the minimal
sequence common to all active synthetic peptides, proteolytic
fragments, and recombinant constructs of EbpS. Furthermore, substitution of Asp23 with Asn abrogated the blocking
activity of the synthetic peptides, demonstrating the requirement for a
charged amino acid at this location. The composite data indicate that
staphylococcal elastin binding is mediated by a discrete domain defined
by short peptide sequences in the amino-terminal extracellular region
of EbpS.
Cell-extracellular matrix
(ECM)1 interactions are
necessary events in various biological processes including embryonic
development, inflammation, tumor cell metastasis, homeostasis, and
microbial infections (1-3). Molecular interactions between ECM
components and corresponding cell-surface receptors instruct cells to
differentiate, migrate, adhere, or proliferate in directing these
biological processes. Although there are exceptions, mammalian cells
typically use the integrins, an Bacterial pathogens also interact with the host matrix through specific
cell-surface ECM-binding molecules categorized collectively as adhesins
or microbial surface components recognizing adhesive matrix molecules
(3, 6). The Gram-positive bacterial pathogen Staphylococcus
aureus has been found to interact with many ECM macromolecules
such as collagen (7, 8), fibronectin (9), laminin (10), proteoglycans
(11), fibrinogen (12), and elastin (13). Staphylococcal ECM adhesins do
not have endogenous bacterial ligands and in general are thought to be
used to assist in colonization of host tissues. For example, collagen
(14) and fibronectin (15) adhesin mutants show a reduced capacity to
cause disease in in vivo models but are otherwise
phenotypically normal.
At the molecular level, all characterized staphylococcal adhesins
function as monomers. Available evidence suggests that ligand-binding sites in staphylococcal ECM adhesins are contained within small regions
of the extracellular domain. The ligand-binding site in the
staphylococcal fibronectin adhesin, for example, has been mapped to a
repetitive 38-amino acid motif, and synthetic peptides containing this
sequence have been found to possess direct binding activity and to
inhibit bacterial binding to fibronectin (16, 17). Similarly, a
synthetic 25-amino acid peptide corresponding to the region between
Asp209 and Tyr233 of the collagen adhesin has
been shown to inhibit binding of type II collagen to S. aureus (18), suggesting that this short peptide sequence alone can
mediate staphylococcal binding to collagen.
In a previous study, we showed that specific binding between S. aureus and elastin was mediated by a 25-kDa elastin-binding protein on the surface of S. aureus (EbpS) (13). Elastin
binding activity was localized to the extracellular, amino-terminal end of EbpS within the first 59 amino acids (19). To better define the
amino acids in EbpS responsible for the elastin binding activity, we
have used overlapping synthetic peptides and truncated recombinant EbpS
constructs in elastin binding assays. Our results demonstrate that the
critical elastin recognition sequence within the amino-terminal domain
resides between Gln14 and Glu34. Sequence
comparison indicates that the minimal sequence shared by all active
EbpS constructs is the hexamer Thr-Asn-Ser-His-Gln-Asp spanning
residues 18-23.
Materials--
Restriction enzymes, calf intestinal alkaline
phosphatase, T4 DNA ligase, isopropyl- Synthetic Peptides--
Synthetic peptides containing the
deduced primary sequence of EbpS were prepared in our laboratory by
conventional solid phase synthesis on an Applied Biosystems model 431A
synthesizer using FastMoc chemistry. Peptides were purified by reverse
phase high performance liquid chromatography (Beckman C18, 0-80%
linear water/acetonitrile gradient containing 0.05% trifluoroacetic
acid). Purity of the peptides was confirmed by either amino-terminal
sequencing or electron spray mass spectrometry. All synthetic peptides
were soluble in the assay buffer at the concentrations tested.
Expression of Recombinant EbpS Proteins--
Expression of
full-length recombinant EbpS (rEbpS) as a fusion protein with a
polyhistidine tag at the amino terminus was as described previously
(19). Essentially the same protocol was followed to make truncated
recombinant EbpS-1 and -2 (trEbpS-1 and trEbpS-2), except that
different PCR primers were used. For the generation of trEbpS-1
containing EbpS residues 1-78, a 2.6-kilobase pair
HindIII/HincII cloned fragment in pBluescript KS+
which contains full-length ebpS (19) was PCR-amplified using
the oligonucleotide 5'-TGTGGATCCATAGAAAGGAAGGTGGCTGTG-3'
as the forward primer and the oligonucleotide
5'-CATTGAGCTCAGATGTTTGTGATTC-3' as the reverse primer. To
make trEbpS-2 corresponding to amino acid residues 1-34 of EbpS, the
same template and forward primer were used, whereas the oligonucleotide
5'-GTTCGAGCTCTGATTGGTCTTTTTC-3' served as the reverse
primer. The forward primer contained a BamHI site
(underlined), and A of the two ATG codons was changed to G (in bold
letters) to avoid internal initiation of translation as recommended by
Qiagen. The reverse primers contained a SacI cleavage site
(underlined). PCR amplification was performed with a Perkin-Elmer
thermocycler using standard reagents. Conditions for PCR amplification
and subsequent restriction enzyme digestion, ligation, transformation,
expression, and purification of recombinant proteins were as described
previously (19).
Direct Binding of trEbpS Proteins to Elastin--
trEbpS-1 and
trEbpS-2 were biotinylated using a commercially available kit (Pierce).
Recombinant proteins (1 mg) were incubated with sulfo-NHS-biotin
reagent (2 mg) in 1 ml of PBS for 2 h at 4 °C. Biotinylated
proteins were then separated from free biotinylating reagent by PD-10
(G-25) gel filtration chromatography.
Three micrograms of recombinant human tropoelastin and bovine serum
albumin were fractionated by 10% SDS-PAGE and transferred to
nitrocellulose membranes by Western blotting. Transferred blots were
blocked overnight at 4 °C with blocking buffer that contained 0.5%
(w/v) bovine serum albumin and 0.05% (v/v) Tween 20 in Tris-buffered saline (50 mM Tris, 150 mM NaCl, pH 7.5). The
blots were washed twice with blocking buffer and incubated for 2 h
at room temperature with either 5 µM biotinylated
trEbpS-1 or -2 in the absence or presence of 3 mg/ml elastin peptides
in blocking buffer. After washing twice with blocking buffer, the blots
were incubated with a 1:1000 dilution of avidin conjugated to
horseradish peroxidase. Membranes were developed by
4-chloro-naphthol.
EbpS Polyclonal Antibodies--
Immunization protocols and
characterization of the anti-EbpS polyclonal antibodies have been
described previously (19). To subtract the population of antibodies
recognizing EbpS residues 1-34, anti-EbpS IgGs (25 mg) purified by
caprylic acid precipitation (20) were incubated overnight at 4 °C
with trEbpS-2 (8 mg) coupled to 3 ml of Affi-Gel 10. On the following
day, the mixture was transferred to a disposable polypropylene column,
and unbound IgGs were collected by gravity flow. The efficiency of the
absorption was tested by Western immunoblotting against the full-length
and two truncated constructs of recombinant EbpS (Fig. 2C).
Fab fragments from both the original and trEbpS-2-absorbed IgG
fractions were prepared by digestion with immobilized papain as
described previously (19).
Other Procedures--
Purification and radiolabeling of
full-length recombinant human soluble tropoelastin, generation of
elastin peptides, cellular elastin binding assays, SDS-PAGE, and
Western blotting were performed as described previously (13). Automated
amino acid sequencing was performed in our laboratory using an Applied
Biosystems 473A protein sequencer. Electron spray mass spectrometry was
performed by the Protein Chemistry Laboratory at Washington University
School of Medicine (St. Louis, MO). The PROTEAN program (DNAStar,
Madison, WI) was used to predict the secondary structure of EbpS constructs.
Truncated Recombinant EbpS Constructs Bind to Elastin and Inhibit
S. aureus Binding to Elastin--
Results from our previous studies
examining elastin binding properties of various EbpS fragments (19),
and recombinant constructs are summarized in Fig.
1. A cyanogen bromide fragment containing the first 125 amino acids of EbpS showed binding activity, whereas an
EbpS degradation product lacking the first 59 amino acids, a cyanogen
bromide fragment containing the carboxyl-terminal one-third of the
protein, and a synthetic peptide corresponding to residues 1-13 did
not interact with elastin. These results suggested that the
elastin-binding site in EbpS is contained in amino acid residues 14-59
(shaded area in Fig. 1).
To identify the domain in EbpS critical for binding, two truncated
recombinant constructs of EbpS (trEbpS-1 and trEbpS-2) were generated
and tested for their ability to bind directly to elastin and to inhibit
binding of S. aureus to elastin. trEbpS-1, with a predicted
molecular mass of 12.8 kDa, contains residues 1-78 of EbpS, whereas
trEbpS-2 spans residues 1-34 and has a predicted mass of 7.5 kDa.
Characterization of the truncated constructs by mass spectrometry,
peptide microsequencing, and immunoblotting with the anti-rEbpS
antibody (Fig. 2B, lanes C and
D) confirmed that correct truncated proteins have been
expressed. Upon resuspension in a physiological buffer for analysis,
trEbpS-1 appeared to have been degraded as evident from the two bands
seen in Fig. 2. Another notable property of the two truncated EbpS
proteins is that they migrated at higher than their predicted molecular
mass when fractionated by SDS-PAGE (Fig. 2). This behavior has also
been observed with full-length recombinant EbpS (rEbpS) (19). For
reasons not fully understood, aberrant migration in SDS-PAGE appears to
be a common characteristic of Gram-positive cell-surface proteins (12,
16, 21-24).
To examine whether trEbpS-1 and -2 bind directly to elastin, the
truncated proteins were biotinylated and reacted with tropoelastin that
was transferred to nitrocellulose membranes. As shown in Fig.
3, both truncated constructs bound to the
67-kDa tropoelastin in the absence (lanes A and
C), but not in the presence (lanes B and
D), of competing soluble elastin peptides. The biotinylated proteins did not bind to bovine serum albumin under similar conditions (data not shown), supporting the specificity of the
tropoelastin-binding interaction. A 45-kDa band also reacted with the
two biotinylated trEbpS constructs. This binding, however, was observed
in all lanes of the Western ligand blot indicating that this
interaction is nonspecific.
Effects of trEbpS proteins on elastin binding at the cellular level
were tested by incubating S. aureus cells with radiolabeled elastin in the absence or presence of increasing amounts of either soluble full-length (rEbpS) or truncated forms of the adhesin. All
three proteins inhibited binding of S. aureus cells to
elastin in a concentration-dependent manner (Fig.
4). rEbpS and trEbpS-1 completely
inhibited elastin binding at the highest concentration tested. trEbpS-2
was somewhat less effective as an inhibitor, with about 20% residual
elastin binding activity at the highest inhibitor concentration.
Pre-absorption of the Anti-rEbpS Antibody with trEbpS-2 Neutralizes
Its Inhibitory Effect--
We have previously shown that Fab fragments
of a polyclonal antibody raised against rEbpS inhibit binding of
S. aureus to elastin (19), suggesting that a population of
antibodies in the immune IgG recognizes a region in EbpS critical for
elastin binding. To test this possibility, anti-rEbpS IgGs were
absorbed to the trEbpS-2 construct coupled to Affi-Gel 10, and unbound IgGs were collected. Immunoblotting revealed that the unbound immunoglobulins (trEbpS-2 negative) retained the ability to interact with full-length rEbpS and trEbpS-1 (Fig. 2C, lanes B and
C), although with reduced activity toward trEbpS-1. As
expected, the trEbpS-2-negative immunoglobulin fraction that was not
absorbed to the trEbpS-2 construct did not react with trEbpS-2 on
Western blot (Fig. 2C, lane D).
Fab fragments from both the original and trEbpS-2-negative IgGs were
generated by papain digestion and tested for their effects on
staphylococcal elastin binding. Consistent with previous findings, Fab
fragments from the original anti-rEbpS IgGs abrogated binding of
S. aureus to elastin (Fig. 5).
In contrast, Fab fragments from the trEbpS-2-negative IgGs did not
inhibit S. aureus binding to elastin at the highest
concentration tested (500 µg/ml).
Contiguous Synthetic EbpS Peptides Inhibit S. aureus Binding to
Elastin--
The findings described above suggest that the elastin
recognition domain in EbpS is contained within residues 14-34. To
define more precisely the elastin binding domain, overlapping synthetic peptides within this region were generated (Fig.
6) and tested for their ability to
inhibit staphylococcal elastin binding. We first searched for
repetitive sequences in EbpS as a candidate elastin binding domain
since several staphylococcal and streptococcal ECM adhesins have been
shown to use repetitive domains for ligand recognition (16, 22).
Although no identical repetitive sequences were identified, there are
two related sequences, 21HQDHTEDVE29 and
37HQDTIENTE45, in the amino-terminal end of the
molecule. The sequence 21HQDHTEDVE29 is within
the putative amino-terminal elastin-binding site and is contained in
all active EbpS constructs. The second sequence, 37HQDTIENTE45, is present only in full-length
EbpS and trEbpS-1, which are the most efficient elastin-binding
constructs of EbpS.
To determine whether the HQDHTEDVE sequence might participate in
elastin binding, we generated two synthetic 17 amino acid peptides, P1
and P2, corresponding to residues 18-34 (Fig. 6). The P1 peptide was
made according to the deduced sequence of EbpS. In the P2 peptide,
Asp23, Glu26, and Glu29 were
substituted with Asn, Pro, and Gln, respectively. The charged amino
acids were targeted for substitution because staphylococcal elastin
binding has been shown to involve electrostatic interactions (25). As
shown in Fig. 6, the P1 peptide inhibited S. aureus binding
to elastin in a concentration-dependent manner with an IC50 of 0.4 mM, whereas the P2 peptide failed
to inhibit elastin binding (IC50 = >1.3
mM).
To define the elastin binding domain more completely, three overlapping
10-mers spanning amino acid residues 14-36 were generated (Fig. 6) and
tested for their ability to inhibit S. aureus binding to
elastin. Peptide P4 (residues 21-30) containing the HQDHTEDVE sequence
and the P5 peptide (residues 27-36) only reduced elastin binding by
approximately 35% at the highest concentration tested, whereas the P3
peptide (residues 14-23) inhibited binding of S. aureus to
radiolabeled elastin by more than 95% in a
concentration-dependent manner, indicating that the
HQDHTEDVE sequence does not represent the elastin binding domain of
EbpS. IC50 values obtained from these experiments for P3,
P4, and P5 were 0.7, >1.6, and >2.1 mM, respectively
(Fig. 6).
Sequence comparison of P3 and other active EbpS constructs revealed
that the hexapeptide 18TNSHQD23 is the only
sequence shared by all active constructs. However, the hexapeptide
TNSHQD (P6) and its control TNSHQS (P7) did not inhibit staphylococcal
elastin binding at any concentration tested (0.1-3.0 mM).
These findings suggest that although the presence of the TNSHQD
sequence is essential for elastin binding by EbpS, additional flanking
amino acids are required for its activity. Furthermore, abrogation of
activity in the P1 peptide by substitution of Asp23 to Asn,
Glu26 to Pro, and Glu29 to Gln suggests that
the carboxyl side chain of Asp23 is critical for elastin
recognition if the 18TNSHQD23 hexapeptide
indeed plays an important role in elastin recognition.
To test these hypotheses, we generated synthetic peptides P8
corresponding to the wild type EbpS sequence spanning residues 14-25,
P9 with Asp17 in P8 substituted with Asn, P10 with
Asp23 in P8 substituted with Asn, P11 with 1 amino acid
added to TNSHQD in the amino-terminal direction, P12 as a scrambled
peptide control of P9, and P13 with 2 residues added to TNSHQD in the
carboxyl-terminal direction and tested for their ability to inhibit
staphylococcal elastin binding (Fig. 6). Both peptides P8 and P9
inhibited staphylococcal elastin binding in a
concentration-dependent manner with IC50 values
of 0.8 and 1.0 mM, respectively. However, the P10 peptide with Asn substituted for Asp23 failed to inhibit binding
(IC50 = >4.3 mM), demonstrating the importance
of a charged amino acid at this location. The P11 peptide showed
concentration-dependent inhibition of binding with an
IC50 of 0.9 mM, although both its scrambled
control (P12) and P13 were inactive (Fig. 6). These results indicate
that more than two flanking amino acids in the carboxyl-terminal
direction are required to make the TNSHQD hexapeptide active, whereas
addition of one residue in the amino-terminal direction is sufficient
to render specific elastin binding activity to the hexamer TNSHQD.
ECM adhesins are important for bacterial colonization of and
dissemination through host tissues. To understand better the mechanism
of S. aureus adhesion to elastin, we sought to identify the
elastin binding domain in EbpS, the cell-surface elastin-binding protein of S. aureus. By using overlapping EbpS fragments,
polyclonal anti-EbpS antibodies, and recombinant constructs, we mapped
the elastin binding domain in EbpS to the extracellular amino-terminal region of the molecule. Two truncated recombinant constructs spanning EbpS residues 1-34 and 1-78 inhibited staphylococcal elastin binding. When the polyclonal anti-EbpS antibody, previously shown to inhibit S. aureus binding to elastin, was absorbed to the trEbpS-2
protein spanning residues 1-34, the antibody lost its inhibitory
activity. These findings further localized the elastin binding domain
in EbpS to residues 14-34.
Eleven overlapping synthetic peptides spanning amino acids 14-36 were
then used to define better the elastin binding domain in EbpS. Among
these, only peptides corresponding to residues 14-23, 17-23, and
18-34 of EbpS specifically inhibited elastin binding by more than
95%. Our analyses revealed that the hexameric sequence
Thr18-Asn-Ser-His-Gln-Asp23 is the only
sequence common to all active synthetic peptides, proteolytic
fragments, and recombinant constructs of EbpS. Further evidence that
this sequence is important for elastin binding was the loss of activity
when Asp23 was substituted with Asn in the synthetic
peptide corresponding to residues 14-25 and 18-34. Interestingly, the
synthetic hexamer TNSHQD by itself did not inhibit staphylococcal
binding to elastin. These findings suggest that although the presence
of the TNSHQD sequence is essential for EbpS activity, flanking amino
acids in the amino- or carboxyl-terminal direction and the carboxyl side chain of Asp23 are required for elastin recognition.
A survey of the active synthetic EbpS peptides showed that four (P1,
P3, P8, and P11) are strongly acidic (pI The minimal requirements for elastin recognition by EbpS are
surprisingly similar to what has been observed for the interaction between S. aureus and fibronectin. Fibronectin binding to
S. aureus is mediated by a surface fibronectin-binding
protein, and the fibronectin-binding site in this adhesin has been
mapped to an extracellular 38-amino acid motif repeated three times and
partially a fourth time (16). A subsequent investigation by McGavin
et al. (26) has shown that essential amino acids are
contained within residues 21-33 of the 38-amino acid motif and that
flanking amino- and carboxyl-terminal amino acids are required for
activity. The carboxyl side chains of acidic amino acids are also
essential. These authors suggested that the flanking residues are
required to acquire a conformation that is favorable for fibronectin binding.
Similar to the ligand recognition mechanism of the staphylococcal
fibronectin adhesin protein, the TNSHQD hexamer by itself could be
inactive because it folds improperly, and flanking residues are
required to form a conformation that is necessary for efficient activity. This might explain why affinities of active EbpS synthetic peptides for binding to elastin are considerably lower than those of
larger elastin-binding EbpS constructs. Although the three-dimensional structure of EbpS is still unknown, the amino-terminal region of
full-length EbpS is predicted to fold into amphipathic The properties of staphylococcal elastin and fibronectin recognition
mechanisms appear to be opposite that of their corresponding mammalian
receptors. Mammalian receptors bind to their respective ligands through
the interaction of structural domains in the receptor and a short
contiguous peptide sequence in the ligand. Structural domains formed by
both the The different strategy in receptor-ligand recognition used by S. aureus also raises the interesting possibility that other elastin-binding proteins may use a similar approach for interacting with elastin. Sequence comparisons, however, failed to detect the
TNSHQD sequence in several known elastin-binding proteins, including
pancreatic and neutrophil elastases (34-36), lysostaphin (37),
microfibril-associated glycoprotein (38), or lysozyme (39).
Furthermore, none of these proteins showed significant homology to the
extended elastin-binding EbpS sequence corresponding to residues
14-34.
The exact sequence in elastin recognized by EbpS is still unknown,
although previous studies with recombinant tropoelastin fragments have
localized the recognition sequence to the amino-terminal one-third of
the protein (13). A possible binding domain in this amino-terminal
region has been suggested by peptide antibody inhibition
experiments2 to lie within a
sequence that is encoded by exons 9 and 10. Interestingly, this domain
contains a conserved Arg residue that may be critical for interaction
with the essential Asp23 in EbpS. Further studies are in
progress to characterize better this domain.
INTRODUCTION
Top
Abstract
Introduction
References
/
heterodimeric receptor complex,
to interact with the ECM (4, 5).
EXPERIMENTAL PROCEDURES
-thiogalactoside,
5-bromo-4-chloro-3-indolyl-
-D-galactoside, and
HindIII-digested
DNA markers were purchased from Promega (Madison, WI). Luria-Bertani medium and Luria-Bertani agar-medium capsules were from Bio 101 (La Jolla, CA). Tryptic soy broth (TSB) was
obtained from Remel (Lenexa, KS). Na125I was from ICN
(Costa Mesa, CA). Papain and protein A immobilized to cross-linked
agarose, Immunopure Sulfo-NHS-Biotinylation kit, and IODO-GEN were
purchased from Pierce. QIAexpress vector kit type IV and the midi-prep
plasmid purification kit were obtained from Qiagen (Chatsworth, CA).
Nitrocellulose membrane and blotting paper were from Schleicher & Schuell. Affi-Gel 10 affinity support was from Bio-Rad (Melville, NY).
S. aureus strain 12598 (Cowan) was purchased from the ATCC
and used throughout this study. All other materials were purchased from Sigma.
RESULTS
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Fig. 1.
The elastin-binding site in EbpS is contained
within residues 14-59. Elastin binding properties of various EbpS
fragments and recombinant constructs (described in the text) were
assessed by their capacity to specifically bind to tropoelastin.
Inactivity of the amino-terminal synthetic peptide was determined by
its inability to inhibit staphylococcal elastin binding. Residues
14-59 (shaded area) are common to all fragments with
elastin binding activity.
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Fig. 2.
Expression of recombinant EbpS proteins.
Recombinant EbpS proteins were purified by nickel-nitrilotriacetic acid
affinity chromatography, fractionated by 15% SDS-PAGE, and stained
with Coomassie Brilliant Blue R-250 (A) or transferred to
nitrocellulose membranes and reacted with anti-rEbpS IgG (B)
or anti-rEbpS IgG that has been pre-absorbed to trEbpS-2
(C). Lane A, ovalbumin; lane B, rEbpS;
lane C, trEbpS-1; lane D, trEbpS-2; and
lane E, lysozyme. Molecular masses of the recombinant
proteins were approximated from the migration pattern of ovalbumin,
lysozyme, and pre-stained size standards.
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Fig. 3.
Recombinant trEbpS-1 and trEbpS-2 bind
to elastin. Tropoelastin (3 µg) that was fractionated by 10%
SDS-PAGE and Western-blotted to nitrocellulose membranes was reacted
with 5 µM biotinylated trEbpS-1 (lanes A
and B) or trEbpS-2 (lanes C and D) in
the absence (lanes A and C) or presence
(lanes B and D) of 3 mg/ml elastin peptides.
Binding of truncated EbpS proteins was visualized by subsequent
incubation with avidin-horseradish peroxidase and
4-chloronaphthol.
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Fig. 4.
Truncated recombinant EbpS proteins inhibit
binding of S. aureus cells to radiolabeled
elastin. Live S. aureus cells (2 × 108) were incubated with radioiodinated elastin (10 ng) in
the absence or presence of increasing concentrations of rEbpS,
trEbpS-1, or trEbpS-2 for 1 h at room temperature in 200 µl of
TSB. The assay was terminated by centrifugation, and cell pellets were
washed twice with 1 ml of TSB. Extent of binding was quantified by
measuring radioactivity associated with the pellets. Results are
presented as mean relative percent binding ± S.D. of triplicate
determinations, with measurements obtained in the absence of
recombinant EbpS proteins defined as 100%.
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Fig. 5.
The inhibitory effect of anti-rEbpS antibody
is neutralized by pre-absorption with trEbpS-2. Fab fragments from
the original (control) and anti-rEbpS IgGs that have been pre-absorbed
to trEbpS-2 (trEbpS-2 negative) were prepared by papain digestion.
Increasing concentrations of Fab fragments were incubated with live
S. aureus cells and radiolabeled elastin as described
previously. Data are shown as mean relative percent binding ± S.D. from triplicate determinations. Fab concentration is as follows:
, 0 mg/ml; shaded box, 0.1;
, 0.3;
, 0.5.
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Fig. 6.
Elastin binding domain in EbpS defined by
peptide inhibition studies. Thirteen overlapping synthetic
peptides spanning residues 14-36 were tested for their ability to
inhibit binding of S. aureus to tropoelastin. Inhibition
activity, expressed as IC50 values, is indicated next to
the calculated pI of the peptides. Active synthetic peptides are
highlighted in bold letters. Amino acids in peptides P2, P9,
and P10 were substituted as indicated, and peptide P12 represents the
scrambled control of peptide P11. The shaded box indicates
the minimal sequence shared by all active constructs of EbpS.
DISCUSSION
5.1). This finding raised
the possibility of nonspecific inhibition in which any peptide with two
or more strongly acidic amino acid residues, such as Asp and Glu, can
interact with elastin. Our results, however, show that the highly
negatively charged P5 and P12 peptides (pI = 3.7 and 3.9, respectively) are inactive, excluding the possibility that elastin
binding is simply or solely a consequence of strong negative charge.
More importantly, our results show that the elastin binding domain is
sequence-specific in that the scrambled peptide P12, which is a control
for P11, the shortest functional elastin binding sequence, is inactive.
-helices (Eisenberg method, PROTEAN program) except for regions including residues 14-23 which is where the TNSHQD sequence resides. These predictions may imply that the flanking residues in EbpS are required to stabilize the flexible binding domain containing the TNSHQD sequence
for interaction with the ligand elastin. A similar mechanism has been
found to be utilized by integrins and their matrix ligands in that the
integrin-binding site in the ligands consist of short peptide sequences
presented on flexible loops between
-strands (27). Furthermore, the
affinities of the short integrin-binding peptides are considerably
lower than those of intact ligands as with our short elastin-binding
EbpS peptides. We are in the process of determining the crystal
structure of EbpS to study directly whether the TNSHQD epitope is
indeed stabilized by flanking
-helical domains and to define the
elastin recognition sequence.
and
integrin subunits (27), for example, interact with
short peptide sequences such as RGD (28), LDV (29), REDV (30), and
IDAPS (31). Similarly, the 67-kDa mammalian elastin-binding protein
recognizes the hydrophobic VGVAPG hexapeptide sequence in elastin (32).
In contrast, staphylococcal elastin and fibronectin adhesins appear to
interact with their ligands by fitting a small region of the adhesin
stabilized by flanking residues into a structural binding pocket formed
by the ligand. For example, the staphylococcal binding domain in
fibronectin is a structural domain that requires all five type I
modules in the amino-terminal region for binding (33). One intriguing
possibility is that these differences in ligand recognition mechanisms
may be important for pathogenesis in ensuring a lack of binding
competition between the staphylococcal and mammalian elastin and
fibronectin binding systems, thus promoting efficient binding of
staphylococci to host tissue components for colonization and dissemination.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL53325 and HL41926.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.
Current address: Neonatology, Children's Hospital, Harvard
Medical School, 300 Longwood Ave., Enders-9, Boston, MA 02115. Tel.:
617-355-7037; Fax: 617-355-7677; E-mail: park_p{at}a1.tch.harvard.edu.
§ To whom correspondence and reprint requests should be addressed: Dept. of Cell Biology and Physiology, Washington University School of Medicine, Box 8228, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-2254; Fax: 314-362-2252; E-mail: bmecham{at}cellbio.wustl.edu.
The abbreviations used are: ECM, extracellular matrix; EbpS, elastin-binding protein of S. aureus; rEbpS, full-length recombinant EbpS; trEbpS, truncated recombinant EbpS; TSB, tryptic soy broth; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.
2 P. W. Park, T. J. Broekelmann, B. R. Mecham, and R. P. Mecham, unpublished results.
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