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J. Biol. Chem., Vol. 277, Issue 35, 32109-32115, August 30, 2002
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From the
Received for publication, April 19, 2002, and in revised form, June 5, 2002
Salivary agglutinin is encoded by
DMBT1 and identical to gp-340, a member of the scavenger
receptor cysteine-rich (SRCR) superfamily. Salivary agglutinin/DMBT1 is
known for its Streptococcus mutans agglutinating
properties. This 300-400 kDa glycoprotein is composed of conserved
peptide motifs: 14 SRCR domains that are separated by
SRCR-interspersed domains (SIDs), 2 CUB (C1r/C1s Uegf Bmp1) domains,
and a zona pellucida domain. We have searched for the peptide domains
of agglutinin/DMBT1 responsible for bacteria binding. Digestion with
endoproteinase Lys-C resulted in a protein fragment containing
exclusively SRCR and SID domains that binds to S. mutans. To define more closely the S. mutans-binding domain,
consensus-based peptides of the SRCR domains and SIDs were designed and
synthesized. Only one of the SRCR peptides, designated SRCRP2, and none
of the SID peptides bound to S. mutans. Strikingly, this
peptide was also able to induce agglutination of S. mutans
and a number of other bacteria. The repeated presence of this peptide
in the native molecule endows agglutinin/DMBT1 with a general bacterial binding feature with a multivalent character. Moreover, our studies demonstrate for the first time that the polymorphic SRCR domains of
salivary agglutinin/DMBT1 mediate ligand interactions.
Salivary agglutinin is a 300-400 kDa blood group reactive
glycoprotein (1), which has been implicated in the oral clearance of
microorganisms because of its bacteria-agglutinating properties (2, 3).
Due to its ability to bind and agglutinate the cariogenic bacterium
Streptococcus mutans, salivary agglutinin has been
considered to play an important role in the innate protection against
dental caries (4, 5). Salivary agglutinin, which is encoded by the same
gene as DMBT11 and the lung
protein gp-340 (6-11), binds also to surfactant protein-D (8).
Genetic analysis has demonstrated that salivary agglutinin/DMBT1 is a
member of the scavenger receptor cysteine-rich (SRCR) superfamily (6,
9), which is highly conserved, crossing species boundaries. This group
of glycoproteins comprises cell surface molecules as well as secreted
proteins that are characterized by the presence of multiple SRCR
domains showing broad ligand binding spectra (12). SRCR proteins,
e.g. the macrophage scavenger receptor, Mac-2-binding
protein, CD5, CD6, WC1, Ebnerin, CRP (cyclic AMP receptor
protein)-ductin, and lung gp-340, are associated with host defense
systems (7, 8, 11-17).
Until now, studies dealing with agglutinin-bacteria interactions have
been focused mainly on the characterization of bacterial receptors (18,
19) and identification of their cognate carbohydrate ligands on
agglutinin (1, 20-22). These studies revealed that carbohydrate
residues play only a partial role in binding and aggregation of
bacteria by agglutinin (1, 19, 20). For example, chemical modification
of the carbohydrate residues of agglutinin only slightly impaired its
agglutinating properties (20). On the other hand, treatments affecting
the polypeptide moiety abolished binding to S. mutans
completely, suggesting a dominant role for peptide domains (1, 20, 22).
The present study was directed on identification of the peptide domains
on agglutinin that are responsible for its bacteria-agglutinating properties.
From the DNA sequence of the gene encoding salivary agglutinin,
DMBT1 (9, 10), the architecture of the polypeptide chain of
agglutinin was deduced. Salivary agglutinin (see Fig. 1A) is composed of 13 highly homologous SRCR domains (13, 23) separated by
SIDs (SRCR-interspersed domains), 2 CUB (C1r/C1s Uegf Bmp1) domains
(24, 25) separated by a 14th SRCR domain, and a ZP (zona pellucida)
domain (26). The biological function of the highly conserved SRCR
domains has not yet been established, but their presence in proteins
with broad spectrum binding properties suggests a role in ligand
binding or adhesion. We recently noted genetic polymorphism within
DMBT1. These polymorphisms lead to DMBT1 alleles, giving rise to
polypeptides with interindividually different numbers of SRCR domains
and SIDs. Based on analogies to mucins, we have proposed that these
polymorphisms may lead to a differential efficacy in mucosal protection
(10, 27). However, the major drawback of this hypothesis is that it
remains to be shown that the SRCR domains and/or SIDs are involved in ligand binding.
The search for the peptide domains on salivary agglutinin/DMBT1
involved in bacteria binding was therefore initially directed to this
part of the molecule. On basis of the deduced amino acid sequence of
agglutinin, we designed a digestion strategy to obtain a fragment
consisting exclusively of SRCR and SID domains. This fragment preserved
the S. mutans binding properties. To characterize the
binding domain in greater detail, peptides were synthesized covering
the complete SRCR and SID consensus sequence, and their binding to
S. mutans was analyzed. Only one 16-mer peptide
(QGRVEVLYRGSWGTVC) of the SRCR domain was found to bind to
S. mutans and to mediate agglutination.
Purification of Agglutinin--
Human parotid saliva was
collected with a Lashley cup. Twenty-five ml of parotid saliva was kept
on ice water for 30 min to promote the formation of a precipitate. This
precipitate was collected by centrifugation at 5,000 × g for 20 min at 4 °C. The resulting pellet was dissolved
in 2.5 ml of PBS. The pellet was ~10-fold enriched in agglutinin.
This crude agglutinin fraction was used as starting material for
digestion studies and in the various binding studies. For further
purification, the pellet was dissolved in buffer A (10 mM
Tris, 10 mM EDTA, pH 6.9, 0.05% CHAPS) and applied
on an UNO Q-6 column (Bio-Rad) equilibrated in buffer A and linked to
fast protein liquid chromatography equipment (Amersham Biosciences).
Proteins were eluted with a linear gradient from 0 to 0.5 M
NaCl in buffer A. The eluate was monitored at 280 nm and analyzed by
SDS-PAGE and Western blotting. The isolated agglutinin preparation
contained no detectable protein impurities (less than 5%). Protein
concentrations were determined with the BCA Protein Assay Kit (Pierce)
according to the manufacturer's instructions.
SDS-PAGE and Western Blotting--
SDS-PAGE, conducted on
an Amersham Biosciences Phast System (Amersham Biosciences) using
4-15% polyacrylamide gels, and Western blotting were performed as
described (28). Blots were probed with monoclonal antibody 213-6 directed against gp-340 (6, 29), kindly provided by Dr. Uffe Holmskov
(University of Southern Denmark, Odense, Denmark) using immunoenzymatic detection.
Bacteria S. mutans (Ingbritt), Streptococcus
gordonii (HG 222), Streptococcus sanguis (NY 584),
Streptococcus oralis (NY 582), Streptococcus
sobrinus (HG 456), Streptococcus mitis I (SK 271), Streptococcus mitis II (HG 168), Actinobacillus
actinomycetemcomitans (NY 673), Prevotella intermedia
(OB 51), Escherichia coli (F7), Bacteroides
fragilis (clinical isolate), Moraxella catarrhalis (clinical isolate), Peptostreptococcus micros (clinical
isolate), and Staphylococcus aureus (clinical isolate) were
cultured on blood agar plates under anaerobic conditions with 5%
CO2 for 48 h at 37 °C. Lactobacillus
casei (clinical isolate) was cultured on blood agar plates under
microaerophilic conditions for 48 h at 37 °C.
Helicobacter pylori (ATCC 43504) was cultured on selective Dent plates (Oxoid, Hampshire, UK) for 72 h at 37 °C. Other
bacteria strains were cultured in brain heart infusion (Difco
Laboratories, Detroit, MI) or Todd Hewitt medium (Oxoid) overnight
in air/CO2 (19:1) at 37 °C. Cells were harvested and
washed twice in PBS or Tris-buffered saline (50 mM Tris, pH
7.5, containing 150 mM sodium chloride). Bacteria were
diluted in buffer to a final OD700 of 0.5 or 1.0 corresponding with ~5 × 108 and 109
cells/ml, respectively.
Protein Digestion--
0.5 µg of endoproteinase Lys-C,
sequencing grade from Lysobacter enzymogenes (Roche
Diagnostics GmbH), was added to 50 µl of crude agglutinin (50 µg/ml), dissolved in 25 mM Tris, 1 mM EDTA
(pH 8.5). After 18 h of incubation at 37 °C, digestion was stopped by adding a mixture of proteinase inhibitors (Complete Mini
tablets, Roche Diagnostics). For reduction, agglutinin was incubated
with 10 mM dithiothreitol for 4 h at 4 °C, and
subsequently carboxymethylated with 20 mM 2-iodoacetamide
for 4 h at 4 °C.
Exclusion of Genetic Polymorphism for Lys-1812--
The triplet
coding for Lys-1812 is located at the splice fusion site of
exons 45 and 46. To exclude that partial digestion at residue Lys-1812
originates from genetic polymorphism, the respective exons and their
immediately flanking intronic sequences were amplified by PCR using the
primers dmbt1ex45dsf2 (5'-GTGCAGAAGATGAAACTGGATG-3') and
dmbt1ex45dsr2 (5'-GCCCAGGACACAGTCTAAAC-3') (exon 45) and
dmbt1ex46dsf2 (5'-ACCTGTATTCAATGGCATCCC-3') and
dmbt1ex46dsr2 (5'-TGCCCCCAAAGAGGCAGC-3') (exon 46) under the
conditions described elsewhere (30). Forward and reverse strands were
sequenced with the primers as depicted herein.
Liquid-phase Binding Assay--
Two hundred µl of an S. mutans suspension (109 bacteria/ml in PBS) was
centrifuged at 3,000 × g for 10 min. The pellet was mixed with 100 µl of digested agglutinin (50 µg/ml) supplemented with 5 mM calcium chloride and incubated for 1 h at
37 °C. Next, bacteria were collected by centrifugation at 3,000 × g for 2 min, washed twice in PBS, and transferred to a
new vial. Bacteria-bound components were extracted by incubation with
10 mM EDTA in PBS for 1 h at 37 °C. The extracts
were examined by SDS-PAGE and Western analysis. Control extracts were
obtained from bacteria that had not been incubated with (digested) agglutinin.
Overlay Adherence Assay--
Adhesion of S. mutans to
the Lys-C proteinase-digested crude agglutinin immobilized on
nitrocellulose was studied using an overlay adhesion assay.
Briefly, 40-µl samples of digested agglutinin (50 µg/ml) were
separated by SDS-PAGE and blotted onto nitrocellulose membranes. The
membranes were blocked with PBS supplemented with 0.1% Tween 20 and
2% bovine serum albumin (PBS-T-BSA) and incubated with S. mutans (109 bacteria in PBS-T-BSA) for 16 h at
4 °C. The nitrocellulose membranes were washed twice with PBS-T-BSA.
Bound bacteria were visualized with monoclonal antibody OMVU37,
directed against S. mutans (31) with alkaline
phosphatase-conjugated rabbit anti-mouse immunoglobulins (DAKO,
Glostrup, Denmark) as the chromogenic substrate.
Peptide Design and Synthesis--
Based on the published amino
acid sequence of gp-340 (7), consensus sequences of the 13 SRCR domains
and 11 SIDs were determined using alignment software (Vector NTI,
InforMax Inc., Oxford, UK). Nine peptides, together spanning the
complete sequence, were synthesized by solid-phase peptide synthesis
using the T-bag method adapted for Fmoc
(N-(9-fluorenyl)methoxycarbonyl) chemistry (32).
Peptide Purification--
Peptides were purified by
reversed-phase HPLC on a JASCO HPLC System (Tokyo, Japan). Peptides
were dissolved in 0.1% trifluoroacetic acid and applied on a
VYDAC C18-column (218TP, 1.0 × 25 cm, 10-µm particles, Vydac, Hesperia, CA) equilibrated in 0.1% trifluoroacetic acid. Elution was performed with a linear gradient from 30 to 45%
acetonitrile containing 0.1% trifluoroacetic acid in 20 min at a flow
rate of 4 ml/min. The absorbance of the column effluent was monitored
at 214 nm, and peak fractions were pooled, lyophilized, and reanalyzed
by reversed-phase-HPLC and by capillary electrophoresis on a
Biofocus 2000 apparatus (Bio-Rad). The authenticity of the (monomeric)
peptides was confirmed by quadrupole-time of flight mass spectrometry
(Q-TOF MS) on a tandem mass spectrometer (Micromass Inc., Manchester,
UK) as described previously (33). Despite the presence of cysteine
residues, exclusively monomeric peptides were detected, indicating that
peptide multimerization by disulfide bond formation had not occurred.
The purity of the peptides was at least 90%.
Adhesion Assays--
Bacterial adhesion was examined using a
microtiter plate method based on labeling of microorganisms with
cell-permeable DNA binding
probes.2 Microtiterplates
Fluotrac 600 (Greiner, Recklinghausen, Germany) were coated with
various amounts of synthetic peptides, crude agglutinin, or purified
agglutinin. For reduction, the peptides and agglutinin were incubated
with 10 mM dithiothreitol for 4 h at 4 °C and
subsequently carboxymethylated with 20 mM 2-iodoacetamide for 4 h at 4 °C. The peptides were dissolved in coating buffer (100 mM sodium carbonate, pH 9.6) to a starting
concentration of 40 µg/ml and diluted serially. After incubation at
4 °C for 16 h, plates were washed twice with Tris-buffered
saline containing 0.1% Tween 20 and 1 mM Ca2+
(TTC). Subsequently, 100 µl of an S. mutans suspension
(5 × 108 bacteria/ml TTC) were added to each well and
incubated for 2 h at 37 °C. Plates were washed three times with
0.1% Tween 20 using a plate washer (Mikrotek EL 403, Winooski, VT).
Bound bacteria were detected using 100 µl/well of 1 mM
SYTO-9 solution (Molecular Probes, Leiden, The Netherlands), a
cell-permeable fluorescent DNA binding probe. Plates were incubated in
the dark for 15 min at ambient temperature and washed three times with
0.1% Tween 20. Fluorescence was measured in a fluorescence microtiter
plate reader (Fluostar Galaxy, BMG Laboratories, Offenburg, Germany) at
488-nm excitation and 509-nm emission wavelength.
Agglutination Assays--
150 µl of an S. mutans
suspension (5 × 108 bacteria/ml TTC) were mixed with
150 µl of peptide solution at final peptide concentrations of 0-200
µg/ml in a 96-well microtiter plate (low affinity, PS Microlon-F,
Greiner) and incubated for 2 h at 37 °C. After agglutination and subsequent sedimentation of the bacteria, 10 µl of the sediment was transferred on a microscopic slide. After heat fixation, bacteria were stained with a 20% crystal violet solution (Merck) and examined by light microscopy. Turbidometric analysis of the agglutination process was carried out using a spectrophotometer (UVICON 930, Kontron
Instruments, Watford, UK). Bacterial suspensions (5 × 108 bacteria/ml TTC) supplemented with either calcium
chloride or EDTA were preincubated for 2 h at 37 °C before
peptides were added. The optical density of the bacterial suspensions
was monitored at 700 nm for 100 min at 37 °C. These experiments were
repeated at least three times.
Lys-C Cleavage of Agglutinin/DMBT1--
To investigate the role of
the repeating SRCR and SID domains of agglutinin/DMBT1 with respect to
S. mutans binding, a selective proteolytic cleavage
procedure was conducted using endoproteinase Lys-C (34-36).
Agglutinin/DMBT1 contains 10 lysine residues, which are all located in
the C-terminal region (Fig.
1A). One lysine residue is
located in the 13th SRCR domain, 3 are located in the first CUB domain,
1 is located in the second CUB domain, 4 are located in the ZP domain,
and 1 is located in the unique sequence, located C-terminally of the ZP
domain. Hydrolysis after the 1st and 2nd lysine residues (Lys-1722,
Lys-1791) will not yield distinct cleavage products because the
resulting fragments are held together by disulfide linkages between
Cys-1665 and Cys-1729 and between Cys-1766 and Cys-1792, respectively
(23, 37). Hydrolysis at Lys-1812 will generate a fragment that contains
13 of the 14 SRCR domains as well as the interspersing SID domains
(Fig. 1A). The remaining C-proximal ZP and CUB domains
contain additional Lys-C cleavage sites. Thus, Lys-C digestion would
liberate a large SRCR/SID stretch in addition to a number of smaller
fragments. SDS-PAGE analysis of the Lys-C-treated agglutinin revealed a
new protein band with slightly lower molecular mass than the native
agglutinin/DMBT1 (Fig. 2A).
This band, the putative SRCR/SID stretch of agglutinin/DMBT1, was
recognized by monoclonal antibody 213-6 directed against a peptide
epitope of salivary agglutinin/DMBT1 (29) (Fig. 2A, lane 2). Immunoreactive protein bands of lower molecular
mass were not detected. Reduction and subsequent
carboxymethylation virtually did not show different protein patterns on
Western blot (not shown). Next to the putative SRCR/SID stretch, the
native agglutinin/DMBT1 was still present, even after prolonged
incubation with excess Lys-C or addition of 0.01% SDS. The triplet
encoding for Lys-1812 spans the fusion site of exons 45 and 46 after
splicing. Sequencing did not reveal heterozygosity of the saliva donor
in the exon or intron sequences. Thus, partial digestion of salivary agglutinin/DMBT1 is not based on genetic polymorphism. Sequencing of 25 cDNA clones further ruled out that alternative splicing leads to
omission of Lys-1812 (not shown).
Effect of Lys-C Digestion on Binding of Agglutinin to S. mutans--
Whether binding sites for S. mutans were
still present after digestion was examined using an overlay adherence
assay and a soluble-phase binding assay (Fig. 2). Nitrocellulose
membranes containing native agglutinin and Lys-C-digested agglutinin
overlaid with bacteria showed that S. mutans adhered to both
agglutinin bands (Fig. 2B). This demonstrates that Lys-C
digestion had not destroyed the binding domains for this bacterium.
Controls, in which either bacteria incubation or antibody incubation
was omitted, were negative. This was confirmed by the results of
liquid-phase binding assays in which bacteria were incubated with a
solution containing the digested agglutinin (Fig. 2C).
Western analysis of the bacterial extracts and the corresponding
supernatants demonstrated that both native agglutinin and the digested
fragment were found in the bacterial pellets (Fig. 2C, lane
2). In contrast, the supernatants were completely devoid of
agglutinin (Fig. 2C, lane 3). In control experiments in which the bacteria were omitted, agglutinin and the
digested fragment were only present in the supernatants.
Determination of Consensus Sequence of SIDs and SRCR
Domains--
To more specifically characterize the bacteria-binding
region, we synthesized a series of peptides spanning the SIDs and SRCR domains of agglutinin. Using alignment software, the consensus sequences of the SIDs and SRCR domains were determined. This resulted in two consensus sequences for the SIDs, one 20-residue long sequence and one 22-residue long sequence (Fig. 1, B and
C). Peptides representing these sequences were synthesized
(designated SID20 and SID22, Table I).
The consensus sequence of the SRCR domain had a length of 109 amino
acids (Fig. 1D). To cover this sequence, seven synthetic peptides were synthesized, representing loops in the native protein that run in between disulfide bridges (23, 37) (SRCR peptide (SRCRP)
1-7, Table I).
Bacteria Binding Properties of Synthetic SID and SRCR
Peptides--
Binding of S. mutans to these peptides was
determined in a solid-phase adherence assay in which adhered bacteria
were quantified using a fluorescent DNA stain. Crude agglutinin and
purified agglutinin were included as controls. Besides binding to
agglutinin, S. mutans adhered to only one of the nine
peptides tested, SRCRP2 (Fig. 3). None of
the other peptides tested, viz. SRCRP1, SRCRP3-7, SID20,
and SID22, mediated adhesion of S. mutans. To examine the specificity of the adherence, inhibition studies were conducted by
preincubating S. mutans suspensions with all peptides at
various concentrations. Also in these experiments, only SRCRP2
inhibited adhesion of S. mutans to agglutinin (Fig.
4), suggesting that the
binding sites on S. mutans for SRCRP2 and agglutinin are
identical or are located in close vicinity.
Since the SRCRP2 fragment contains a cysteine residue, this peptide
potentially forms disulfide-bonded species. Several results, however,
indicate that monomeric SRCRP2 was the active species in our
experiments. Mass analysis revealed that the SRCRP2 preparation used in
the binding studies was composed of a single species with a molecular
mass of the monomer. Furthermore, neither reduction/carboxymethylation of SRCRP2 nor substitution of its cysteine to alanine to prevent formation of disulfide-bonded species had any effect on its bacteria binding properties (not shown).
Other bacteria, including S. gordonii, S. sanguis, S. oralis,
S. sobrinus, S. mitis I, S. mitis II, A. actinomycetemcomitans, P. intermedia, E. coli, B. fragilis, M. catarrhalis, P. micros, S. aureus,
L. casei, and H. pylori were also bound by
salivary agglutinin as well as by SRCRP2 but not by the other peptides tested (results not shown). These findings suggest that the SRCRP2 domain endows agglutinin with broad spectrum binding properties.
Agglutination Assays--
Varying peptide concentrations were
mixed with S. mutans suspensions and incubated for 2 h
to allow precipitation of aggregates formed. Precipitates were examined
by light microscopy, revealing that only SRCRP2 induced agglutination
of S. mutans (Fig. 5). At
increased calcium concentrations, lower peptide concentrations were
required to induce agglutination, and the aggregates formed were
clearly larger in size (Fig. 5C). In contrast, incubation in
EDTA-containing buffer required higher concentrations of SRCRP2 and
resulted in smaller aggregates (Fig. 5A). A similar
dependence of calcium was found for the agglutinin-mediated bacteria
agglutination. These findings were confirmed by the turbidometric assay
(Fig. 6).
In the present study, we have identified a peptide domain on
salivary agglutinin/DMBT1 that is involved in bacteria binding and
agglutination. To confine the binding domain, we proteolytically cleaved the protein into smaller parts. The low prevalence of lysine
residues in the repeating SID/SRCR domains, a single lysine at
the C terminus of the 13th SRCR domain, allowed the use of Lys-C to
separate the SID/SRCR part from the rest of the molecule. A
complicating factor is the presence of disulfide bridges spanning potential Lys-C cleavage sites, which prevents liberation of the hydrolysis products. Residue Lys-1812 is the first Lys-C cleavage site
resulting in distinct cleavage fragments. The SID/SRCR stretch of
agglutinin/DMBT1 obtained after Lys-C digestion most probably contains
a part of the first CUB domain. In theory, by reduction of the
disulfide bridges, this small CUB fragment could be removed. However, the bacteria binding properties of agglutinin/DMBT1 are also lost (22), hampering further identification of the binding domain
using this approach. Next to the band for the putative SID/SRCR
stretch, the native agglutinin/DMBT1 was still present (Fig.
2A), even after prolonged incubation with excess Lys-C with the addition of 0.01% SDS or after reduction and carboxymethylation of
agglutinin/DMBT1 prior to digestion. This was not due to genetic heterogeneity of agglutinin/DMBT1 since it was obtained from a donor
who was homozygous within the respective exons. Alternative splicing
using donor/acceptor sites varying by one or few nucleotides cannot
explicitly be ruled out but is unlikely to occur because none of 25 cDNA clones sequenced over the site showed a corresponding phenomenon. We speculate that the propensity of agglutinin/DMBT1 to
form complexes may render a part of the lysine residues inaccessible to
Lys-C. This is reasonable because if the SRCR domains are
involved in ligand binding, it is likely that the constant C terminus, i.e. the CUB and ZP domains, is responsible for
oligomerization. Alternatively, resistance to Lys-C digestion may be
caused by differential N- and O-glycosylation of
potential glycosylation sites close to the presumed cleavage site. In
this context, it should be noted that CUB1 contains 21 potential
O-glycosylation sites and 3 potential
N-glycosylation sites (7).
Only one of the SRCR consensus peptides, SRCRP2, bound to a wide
variety of Gram-positive and Gram-negative bacteria. Absence of binding
by the other peptides does not necessarily mean that the corresponding
domains are not involved in bacteria adherence to agglutinin/DMBT1
per se. It is possible that these peptides are derived from
conformational domains of agglutinin/DMBT1 and that their lack of
conformational restraints leads to poor binding of the bacteria tested.
A puzzling finding is that, whereas the bacteria binding properties of
agglutinin/DMBT1 are destroyed upon reduction, the putative binding
domain (the SRCRP2 peptide) is part of a disulfide bridged loop.
Secondary structure analysis using protein analysis software (Vector
NTI) predicts that the SRCRP2 peptide is composed of two short
The role of the carbohydrate moiety, which comprises 25% of the whole
molecule on a weight basis (22), remains to be elucidated. Glycosylated
regions will be present predominantly in the SIDS, which when compared
with SRCR domains, contain a high density of potential
O-glycosylation sites. The high density of glycans forces
these regions in an extended conformation, thus creating a molecule
with alternating stretched SIDS and globular SRCR domains, promoting
multivalent interaction. In addition, the oligosaccharides may provide
(low affinity) binding sites for microbial receptors, which in the
mature agglutinin/DMBT1 may act in concert with the SRCR peptide domains.
A study on the bacteria binding of the human macrophage MARCO receptor
(38), another member of the SRCR superfamily, has demonstrated that a
peptide encompassing residues 431-441 (RGRAEVYYSGT) was responsible
for binding of MARCO to S. aureus and E. coli (39). This region corresponds to residues 1-11 of SRCRP2 with 55%
homology. Detailed analysis of MARCO variants demonstrated a crucial
role for an arginine-rich segment for this function (40). More
precisely, the motif RXR was identified as an essential element for high-affinity bacterial binding. Strikingly, the consensus sequence SRCRP2 does not contain such an RXR motif but
rather a QXR motif. The present finding that only this
peptide mediates binding to a wide variety of bacteria suggests that
both motifs, RXR and QXR, could play a role in
bacteria binding.
In summary, we propose that the SRCR domains of salivary
agglutinin/DMBT1 are involved in bacteria binding. Furthermore, we find
that only one SRCR domain consensus-based synthetic peptide of 16 amino
acids, which is 100% identical to the actual amino acid sequence of 8 out of 13 SRCR domains, is responsible for the broad ligand binding
behavior. The repeated presence of this peptide in the native molecule
endows agglutinin/DMBT1 with a general bacterial binding feature with a
multivalent character. This lends support for the hypothesis that
numeric variations of the SRCR domains of salivary agglutinin/DMBT1 may
interfere with efficient protection. The crucial next steps will be to
determine which pathogen binding activities are qualitatively or
quantitatively affected by these polymorphisms.
We thank Harold Bark for skillful practical
assistance and Roel van der Schors for mass spectrometric analysis of
the peptides and for excellent Q-TOF MS work.
*
This study was financially supported by The Netherlands
Interuniversity Research School of Dentistry (IOT), the Deutsche
Krebshilfe Grant 10-1835-Mo1 (to J. M.), and the Wilhelm
Sander-Stiftung Grant 99.018.1 (to A. P.).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: Van der Boechorststraat
7, 1081 BT Amsterdam, The Netherlands. Tel.: 0031-(0)20-444- 8674; Fax:
0031-(0)20-444-8685; E-mail: fj.bikker.obc.acta@med.vu.nl.
Published, JBC Papers in Press, June 5, 2002, DOI 10.1074/jbc.M203788200
2
J. A. Bosch, E. C. I. Veerman, M. Turkenburg,
K. Hartog, J. G. M. Bolscher, and A. V. Nieuw Amerongen, manuscript
in preparation.
The abbreviations used are:
DMBT1, deleted in malignant brain tumors 1;
CUB, C1r/C1s Uegf Bmp1;
Lys-C, endoproteinase Lys-C;
PBS, phosphate-buffered saline;
PBS-T, PBS Tween
20;
BSA, bovine serum albumin;
SRCR, scavenger receptor cysteine-rich;
SRCRP, SRCR peptide;
SID, SRCR-interspersed domain;
ZP, zona pellucida;
Q-TOF MS, quadrupole-time of flight mass spectrometry;
HPLC, high
pressure liquid chromatography;
CHAPS, 3-[ lsqb]
(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
MARCO, macrophage receptor with collagenous structure.
Identification of the Bacteria-binding Peptide Domain on
Salivary Agglutinin (gp-340/DMBT1), a Member of the Scavenger Receptor
Cysteine-rich Superfamily*
§,,,,,,, and Department of Dental Basic Sciences,
Section of Oral Biochemistry, Academic Centre for Dentistry
Amsterdam (ACTA), 1081 BT Amsterdam, The Netherlands and the
¶ Division of Molecular Genome Analysis, Deutsches
Krebsforschungszentrum (DKFZ), D-69120 Heidelberg, Germany
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Salivary
agglutinin/DMBT1/gp-340. A, domain
composition of salivary agglutinin. Examples of the SRCR, CUB, and ZP
domains are indicated with arrows. All potential cleavage
sites for endoproteinase Lys-C are indicated (K).
B, alignment of 20 amino acid SIDs. C, alignment
of 22 amino acid SIDs. D, alignment of 13 N-terminal SRCR
domains.
View larger version (11K):
[in a new window]
Fig. 2.
Lys-C digestion of agglutinin and S. mutans binding to the digested fragment. As shown in
A, crude agglutinin was treated with endoproteinase Lys-C,
and proteins were separated on 4-15% SDS-PAGE, transferred to
nitrocellulose, and immunoblotted with monoclonal antibody 213-6 (A and C). Lane 1, crude agglutinin;
lane 2, Lys-C-digested agglutinin. After digestion, a new
band appeared with a lower apparent molecular weight than the parent
protein, representing a fragment that most probably contains
predominantly SRCR domains and SIDs (amino acids 1-1812). As shown in
B, digested agglutinin was blotted onto a nitrocellulose
membrane and subjected to an overlay binding assay with S. mutans. After incubation with S. mutans, bound bacteria
were visualized with an antibody directed against S. mutans.
Bacteria were found at both bands, suggesting that S. mutans
bound to both agglutinin and the Lys-C digested fragment. C,
the digested agglutinin was subjected to a liquid-phase binding assay
with S. mutans. Lane 1, starting material;
lane 2, bound fraction; lane 3, unbound fraction.
Both agglutinin and the digested fragment were found in the bound
fraction but not in the unbound fraction, demonstrating that
S. mutans binds both agglutinin and the Lys-C-digested
fragment. As a molecular marker, we used the high range molecular mass
standards (Bio-Rad): myosin (208 kDa), 
-galactosidase (116 kDa),
bovine serum albumin (84 kDa), and ovalbumin (47 kDa).
SRCR domain and SID consensus sequence based peptides
View larger version (14K):
[in a new window]
Fig. 3.
S. mutans binding to agglutinin-derived
peptides. Peptide-coated microplates were incubated with an
S. mutans suspension. Bound bacteria were detected with a
cell-permeable DNA binding fluorescent stain at 509 nm. S. mutans bound to crude agglutinin ( 
) and a single peptide
tested, namely SRCRP2 (
). All other peptides tested did not show
this behavior. Typical examples of non-binding peptides are
SRCR1 (
), SRCRP4 (
), and SID20 (
). These experiments were
performed at least in triplicate.
View larger version (11K):
[in a new window]
Fig. 4.
Inhibition of S. mutans binding to agglutinin by peptide SRCRP2.
Purified agglutinin-coated microplates (20 µg/ml) were incubated with
an S. mutans suspension that was preincubated with various
concentrations of peptide SRCRP2 (white bar, 50 µg/ml;
gray bar, 100 µg/ml; black bar, 200 µg/ml).
Increased concentrations of peptide SRCRP2 decreased the amount of
bacteria bound to agglutinin, demonstrating a competitive inhibition of
peptide SRCRP2. All other peptides tested did not inhibit S. mutans binding to agglutinin at a concentration of 200 µg/ml;
typical examples are peptides SRCRP1, SRCRP3, SID20, and
SID22.
View larger version (67K):
[in a new window]
Fig. 5.
Calcium-dependent
agglutination of S. mutans by peptide
SRCRP2. Peptide SRCRP2 was added in various concentrations (50, 100, and 200 µg/ml) to S. mutans in the presence of 5 mM EDTA (A), 1 mM calcium
(B), and 10 mM calcium (C). In the
presence of 5 mM EDTA, aggregation was weaker when compared
with aggregation in the presence of 1 mM calcium. In the
presence of 10 mM calcium chloride, aggregation is even
stronger and starts at lower concentrations peptide SRCRP2. All other
peptides tested did not aggregate S. mutans. Typical
examples of non-agglutinating peptides are SRCRP5 (D),
SRCRP6 (E), and SID20 (F) (each 200 µg/ml and
in the presence of 1 mM calcium). These experiments were
performed at least in triplicate.
View larger version (7K):
[in a new window]
Fig. 6.
Turbidometric agglutination of S. mutans by peptide SRCRP2. S. mutans
suspensions were mixed with various concentrations of peptide SRCRP2
(50, 100, and 200 µg/ml), and the OD700 of the remaining
suspension was measured in time. In the presence of 5 mM
EDTA (A), agglutination is much weaker than in the presence
of 10 mM calcium (B). These experiments were
performed at least in triplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheets separated by a -turn (not shown). Strikingly, based on
crystallographic analysis, the same secondary structure has been
proposed for the corresponding sequence in the SRCR domain of the
highly homologous Mac-2-binding protein (23) (Fig.
7). This indicates that formation of the
disulfide bond does not introduce significant constraints on the SRCRP2
structure. Thus, the effect of reduction on the bacteria binding
properties of the SRCRP2 stretch must be communicated by conformational
changes elsewhere in the agglutinin/DMBT1 molecule. For example,
it can be envisaged that upon breaking of disulfide bonds, the SRCR
domains become shielded, thereby rendering them inaccessible to
bacteria and antibodies (29).
View larger version (57K):
[in a new window]
Fig. 7.
Schematic representation of the SRCR domain
of the Mac-2-binding protein, highlighting the putative
bacteria-binding domain of agglutinin/DMBT1. A model of
the SRCR domain of the Mac-2-binding protein (Protein Data Bank
accession code 1BY2) was constructed using RasWin Molecular Graphics.
The stretch that corresponds to SRCRP2 is composed of two short
-sheets joined by a -turn (highlighted in yellow) and
highly homologous (>80%) to the Mac-2-binding protein amino acid
sequence. The QXR motif (blue, Q18;
red, R20) is located at the surface of the SRCR
domain.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Ligtenberg, A. J.,
Veerman, E. C.,
and Nieuw Amerongen, A. V.
(2000)
Antonie Leeuwenhoek
77,
21-30
2.
Ericson, T.,
and Rundegren, J.
(1983)
Eur. J. Biochem.
133,
255-261[Medline]
[Order article via Infotrieve]
3.
Rundegren, J.,
and Ericson, T.
(1981)
J. Oral Pathol.
10,
269-275[CrossRef][Medline]
[Order article via Infotrieve]
4.
Bleiweis, A. S.
(1993)
Adhesion and Cohesion of Plaque Microflora: A Function of Microbial Fimbriae and Fibrils
, pp. 287-299, University of Rochester Press, Rochester, NY
5.
Stenudd, C.,
Nordlund, A.,
Ryberg, M.,
Johansson, I.,
Kallestal, C.,
and Stromberg, N.
(2001)
J. Dent. Res.
80,
2005-2010 6.
Holmskov, U.,
Lawson, P.,
Teisner, B.,
Tornoe, I.,
Willis, A. C.,
Morgan, C.,
Koch, C.,
and Reid, K. B.
(1997)
J. Biol. Chem.
272,
13743-13749 7.
Holmskov, U.,
Mollenhauer, J.,
Madsen, J.,
Vitved, L.,
Gronlund, J.,
Tornoe, I.,
Kliem, A.,
Reid, K. B.,
Poustka, A.,
and Skjodt, K.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
10794-10799 8.
Ligtenberg, T. J.,
Bikker, F. J.,
Groenink, J.,
Tornoe, I.,
Leth-Larsen, R.,
Veerman, E. C.,
Nieuw Amerongen, A. V.,
and Holmskov, U.
(2001)
Biochem. J.
359,
243-248[CrossRef][Medline]
[Order article via Infotrieve]
9.
Mollenhauer, J.,
Wiemann, S.,
Scheurlen, W.,
Korn, B.,
Hayashi, Y.,
Wilgenbus, K. K.,
von Deimling, A.,
and Poustka, A.
(1997)
Nat. Genet.
17,
32-39[CrossRef][Medline]
[Order article via Infotrieve]
10.
Mollenhauer, J.,
Herbertz, S.,
Holmskov, U.,
Tolnay, M.,
Krebs, I.,
Merlo, A.,
Schroder, H. D.,
Maier, D.,
Breitling, F.,
Wiemann, S.,
Grone, H. J.,
and Poustka, A.
(2000)
Cancer Res.
60,
1704-1710 11.
Prakobphol, A., Xu, F.,
Hoang, V. M.,
Larsson, T.,
Bergstrom, J.,
Johansson, I.,
Frangsmyr, L.,
Holmskov, U.,
Leffler, H.,
Nilsson, C.,
Boren, T.,
Wright, J. R.,
Stromberg, N.,
and Fisher, S. J.
(2000)
J. Biol. Chem.
275,
39860-39866 12.
Resnick, D.,
Pearson, A.,
and Krieger, M.
(1994)
Trends Biochem. Sci.
19,
5-8[CrossRef][Medline]
[Order article via Infotrieve]
13.
Aruffo, A.,
Bowen, M. A.,
Patel, D. D.,
Haynes, B. F.,
Starling, G. C.,
Gebe, J. A.,
and Bajorath, J.
(1997)
Immunol. Today
18,
498-504[CrossRef][Medline]
[Order article via Infotrieve]
14.
Freeman, M.,
Ashkenas, J.,
Rees, D. J.,
Kingsley, D. M.,
Copeland, N. G.,
Jenkins, N. A.,
and Krieger, M.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
8810-8814 15.
Gough, P. J.,
and Gordon, S.
(2000)
Microbes Infect.
2,
305-311[CrossRef][Medline]
[Order article via Infotrieve]
16.
Holmskov, U. L.
(2000)
APMIS (Acta Pathol. Microbiol. Immunol. Scand. Suppl.) Suppl.
100,
1-59
17.
Tino, M. J.,
and Wright, J. R.
(1999)
Am. J. Respir. Cell Mol. Biol.
20,
759-768 18.
Crowley, P. J.,
Brady, L. J.,
Piacentini, D. A.,
and Bleiweis, A. S.
(1993)
Infect. Immun.
61,
1547-1552 19.
Demuth, D. R.,
Lammey, M. S.,
Huck, M.,
Lally, E. T.,
and Malamud, D.
(1990)
Microb. Pathog.
9,
199-211[CrossRef][Medline]
[Order article via Infotrieve]
20.
Courtney, H. S.,
and Hasty, D. L.
(1991)
Infect. Immun.
59,
1661-1666 21.
Demuth, D. R.,
Golub, E. E.,
and Malamud, D.
(1990)
J. Biol. Chem.
265,
7120-7126 22.
Oho, T., Yu, H.,
Yamashita, Y.,
and Koga, T.
(1998)
Infect. Immun.
66,
115-121 23.
Hohenester, E.,
Sasaki, T.,
and Timpl, R.
(1999)
Nat. Struct. Biol.
6,
228-232[CrossRef][Medline]
[Order article via Infotrieve]
24.
Bork, P.,
and Beckmann, G.
(1993)
J. Mol. Biol.
231,
539-545[CrossRef][Medline]
[Order article via Infotrieve]
25.
Romero, A.,
Romao, M. J.,
Varela, P. F.,
Kolln, I.,
Dias, J. M.,
Carvalho, A. L.,
Sanz, L.,
Topfer-Petersen, E.,
and Calvete, J. J.
(1997)
Nat. Struct. Biol.
4,
783-788[CrossRef][Medline]
[Order article via Infotrieve]
26.
Sinowatz, F.,
Kolle, S.,
and Topfer-Petersen, E.
(2001)
Cells Tissues Organs
168,
24-35[CrossRef][Medline]
[Order article via Infotrieve]
27.
Mollenhauer, J.,
Herbertz, S.,
Helmke, B.,
Kollender, G.,
Krebs, I.,
Madsen, J.,
Holmskov, U.,
Sorger, K.,
Schmitt, L.,
Wiemann, S.,
Otto, H. F.,
Grone, H. J.,
and Poustka, A.
(2001)
Cancer Res.
61,
8880-8886 28.
Bolscher, J. G.,
Groenink, J.,
van der Kwaak, J. S.,
van den Keijbus, P. A.,
van't Hof, W.,
Veerman, E. C.,
and Nieuw Amerongen, A. V.
(1999)
J. Dent. Res.
78,
1362-1369 29.
Bikker, F. J.,
Ligtenberg, A. J.,
van der Wal, J. E.,
van den Keijbus, P. A.,
Holmskov, U.,
Veerman, E. C.,
and Nieuw Amerongen, A. V.
(2002)
J. Dent. Res.
81,
134-139 30.
Mollenhauer, J., Müller, H., Kollender, G., Lyer, S., Diedrichs,
L., Helmke, B., Holmskov, U., Ligtenberg, T., Herbertz, S., Krebs, I.,
Madsen, J., Bikker, F., Schmitt, L., Wiemann, S., Scheurlen, W., Otto,
H. F., von Deimling, A., and Poustka, A. (2002) Genes
Chromosomes Cancer, in press
31.
de Soet, J. J.,
van Dalen, P. J.,
Pavicic, M. J.,
and de Graaff, J.
(1990)
J. Clin. Microbiol.
28,
2467-2472 32.
van't Hof, W.,
Driedijk, P. C.,
van den Berg, M.,
Beck-Sickinger, A. G.,
Jung, G.,
and Aalberse, R. C.
(1991)
Mol. Immunol.
28,
1225-1232[CrossRef][Medline]
[Order article via Infotrieve]
33.
Nagle, G. T.,
Jong-Brink, M.,
Painter, S. D.,
and Li, K. W.
(2001)
Eur. J. Biochem.
268,
1213-1221[Medline]
[Order article via Infotrieve]
34.
Bank, R. A.,
Crusius, B. C.,
Zwiers, T.,
Meuwissen, S. G.,
Arwert, F.,
and Pronk, J. C.
(1988)
FEBS Lett.
238,
105-108[CrossRef][Medline]
[Order article via Infotrieve]
35.
Coronel, C. E.,
Winnica, D. E.,
Novella, M. L.,
and Lardy, H. A.
(1992)
J. Biol. Chem.
267,
20909-20915 36.
Erickson, P. R.,
and Herzberg, M. C.
(1993)
J. Biol. Chem.
268,
1646-1649 37.
McAlister, M. S.,
Brown, M. H.,
Willis, A. C.,
Rudd, P. M.,
Harvey, D. J.,
Aplin, R.,
Shotton, D. M.,
Dwek, R. A.,
Barclay, A. N.,
and Driscoll, P. C.
(1998)
Eur. J. Biochem.
257,
131-141[Medline]
[Order article via Infotrieve]
38.
Kraal, G.,
van der Laan, L. J.,
Elomaa, O.,
and Tryggvason, K.
(2000)
Microbes Infect.
2,
313-316[CrossRef][Medline]
[Order article via Infotrieve]
39.
Elomaa, O.,
Sankala, M.,
Pikkarainen, T.,
Bergmann, U.,
Tuuttila, A.,
Raatikainen-Ahokas, A.,
Sariola, H.,
and Tryggvason, K.
(1998)
J. Biol. Chem.
273,
4530-4538 40.
Brannstrom, A.,
Sankala, M.,
Tryggvason, K.,
and Pikkarainen, T.
(2002)
Biochem. Biophys. Res. Commun.
290,
1462-1469[CrossRef][Medline]
[Order article via Infotrieve]
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