Download Bacteria Binding by DMBT1/SAG/gp-340 Is Confined to

THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 279, No. 46, Issue of November 12, pp. 47699 –47703, 2004
Printed in U.S.A.
Bacteria Binding by DMBT1/SAG/gp-340 Is Confined to the
VEVLXXXXW Motif in Its Scavenger Receptor
Cysteine-rich Domains*
Received for publication, June 2, 2004, and in revised form, September 7, 2004
Published, JBC Papers in Press, September 7, 2004, DOI 10.1074/jbc.M406095200
Floris J. Bikker‡, Antoon J. M. Ligtenberg‡§, Caroline End¶储, Marcus Renner ¶,
Stephanie Blaich¶, Stefan Lyer ¶, Rainer Wittig ¶, Wim van ’t Hof ‡, Enno C. I. Veerman‡,
Kamran Nazmi‡, Jolanda M. A. de Blieck-Hogervorst‡, Petra Kioschis储,
Arie V. Nieuw Amerongen‡, Annemarie Poustka¶, and Jan Mollenhauer ¶
From the ‡Department of Oral Biochemistry, Academic Centre for Dentistry Amsterdam (ACTA), Vrije Universiteit en
Universiteit van Amsterdam, The Netherlands, the ¶Department of Molecular Genome Analysis, German Cancer Research
Center (DKFZ), 69120 Heidelberg, Germany, and the 储Institute of Molecular Biology and Cell Culture Technology,
University of Applied Sciences Mannheim, 68163 Mannheim, Germany
The scavenger receptor cysteine-rich (SRCR) proteins
form an archaic group of metazoan proteins characterized by the presence of SRCR domains. These proteins
are classified in group A and B based on the number of
conserved cysteine residues in their SRCR domains, i.e.
six for group A and eight for group B. The protein
DMBT1 (deleted in malignant brain tumors 1), which is
identical to salivary agglutinin and lung gp-340, belongs
to the group B SRCR proteins and is considered to be
involved in tumor suppression and host defense by
pathogen binding. In a previous study we used nonoverlapping synthetic peptides covering the SRCR consensus sequence to identify a 16-amino acid bacteriabinding protein loop (peptide SRCRP2; QGRVEVLYRGSWGTVC) within the SRCR domains. In this study, using
overlapping peptides, we pinpointed the minimal bacteria-binding site on SRCRP2, and thus DMBT1, to an
11-amino acid motif (DMBT1 pathogen-binding site 1 or
DMBT1pbs1; GRVEVLYRGSW). An alanine substitution
scan revealed that VEVL and Trp are critical residues in
this motif. Bacteria binding by DMBT1pbs1 was different from the bacteria binding by the macrophage receptor MARCO in which an RXR motif was critical. In addition, the homologous consensus sequences of a
number of SRCR proteins were synthesized and tested
for bacteria binding. Only consensus sequences of
DMBT1 orthologues bound bacteria by this motif.
The scavenger receptor cysteine-rich (SRCR)1 proteins form
an archaic group of metazoan proteins (1–5). This group of
glycoproteins comprises cell surface molecules as well as se* This work was supported by the Netherlands Institute for Dental
Sciences (IOT), the European Molecular Biology Organization (EMBO)
Grant ASTF 115-02, the Netherlands Organization for Scientific Research (NWO) Grant ER 90-184, the Deutsche Krebshilfe Grant 835-Mo
I, and the Wilhelm Sander-Stiftung Grant 99.018.2. The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Oral Biochemistry,
ACTA, Van der Boechorststr. 7, 1081 BT Amsterdam, The Netherlands.
Tel.: 31-20-4448674; Fax: 31-20-4448685; E-mail: ajm.ligtenberg@
vumc.nl.
1
The abbreviations used are: SRCR, scavenger receptor cysteinerich; DMBT1, deleted in malignant brain tumors 1; DMBT1pbs1,
DMBT1 pathogen binding site 1; DMBT1SAG, DMTB1 isoform secreted
in the saliva; MSR1, macrophage scavenger receptor 1.
This paper is available on line at http://www.jbc.org
creted proteins that are characterized by the presence of one or
more SRCR domains. SRCR domains consist of ⬃110 amino
acids and are divided into groups A and B based on the number
of conserved cysteine residues, namely six for group A and
eight for group B.
The best studied members of the group A SRCR proteins are
the macrophage scavenger receptor (MSR1), the Mac 2-binding
protein (Mac-2bp), and MARCO. Both MSR1 and MARCO are
known to interact with bacteria (6, 7). In contrast to MARCO
(8), the SRCR domain of MSR1 does not seem to be involved in
bacteria binding (9, 10). Bacteria binding by MARCO involves
an RXR motif within the SRCR domain, indicating that ionic
interactions play a crucial role in the interaction with its negatively charged ligands (6).
Group B SRCR proteins are generally involved in the regulation of cellular immune responses. In vertebrates, the group
B SRCR proteins can be divided, on the basis of their structure
and sequence homology, into three subgroups (11). The first
subgroup includes CD5 (12), CD6 (13), and SP␣ (14). CD5 and
CD6 are composed of an extracellular region of three SRCR
domains, a transmembrane region, and a cytoplasmic region.
SP␣ lacks the latter two regions but contains three SRCR
domains that are highly homologous to those of CD5 and CD6.
These three proteins are mainly expressed by T-cells and Bcells (12, 13). The second subgroup of SRCR group B molecules
is the workshop cluster 1 (WC1) family, which includes WC1,
CD163, and M160 (11, 15). These molecules are primarily related to monocyte and macrophage cell lineages. CD163 scavenges haptoglobin-hemoglobin complexes (16). The third subgroup of group B SRCR proteins includes human DMBT1
(deleted in malignant brain tumors 1) (17–22) with homologues
in the rat (Ebnerin) (23), mouse (CRP-ductin) (24), rabbit (Hensin) (25), and cow (bovine gallbladder mucin) (26). Pema-SRCR
from the sea lamprey Pertromyzon marinus (27) is also included in this subgroup. Molecules of this subgroup are expressed by epithelial cells in the gastrointestinal tract and the
ducts of the exocrine glands and are commonly secreted in
mucosal fluids (28). They are associated with host defense, e.g.
by pathogen binding, but also have been suggested as playing
a role in epithelial differentiation (27).
Salivary agglutinin and lung gp-340 are encoded by the gene
DMBT1 on chromosome 10q25.3-q26.1 (18, 20, 22, 29, 30).
These proteins represent the DMBT1 isoforms secreted in the
saliva (DMBT1SAG) and the lung fluid (DMBT1GP340), respectively. For about two decades DMBT1SAG, which manifests a
47699
47700
Pathogen Recognition Site of DMBT1
FIG. 1. Determination of the minimal binding sequence within the SRCR consensus sequence of DMBT1. A, based on the SRCR
consensus sequence of DMBT1, overlapping synthetic 16-mer peptides were designed and synthesized, extending six residues amino- and
carboxyl-terminal of SRCRP2. *, peptides possessing bacterial agglutination activity. B, bacteria binding properties of peptides tested in adhesion
and agglutination assays. Peptides SRCRP2 ⫹ 4N to SRCRP2 ⫹ 1C bound S. mutans in the adhesion assay. Fluorescence is a measure for bacterial
binding. Error bars represent the S.E. The bacterial agglutination assay confirmed the results of the adhesion assay (black bars, agglutination;
white bars, no agglutination). C, the homologous sequence shared by the bacteria binding peptides (GRVEVLYRGSW) was synthesized and tested
for bacteria binding in the adhesion and aggregation assays. This peptide is the smallest peptide showing bacteria binding. Amino-terminal and
carboxy-terminal truncated peptides did not show bacteria binding.
broad bacteria binding spectrum, has been intensively investigated with regard to its role in caries prevention by binding and
agglutination of cariogenic bacteria in the oral cavity (31, 32).
DMBT1GP340 is putatively involved in respiratory tract protection because it interacts with the defense collectin surfactant
proteins D (SP-D) and A (SP-A) and is able to stimulate alveolar macrophage migration (18, 33).
By initial protein digestion and utilization of non-overlapping synthetic peptides, we recently identified a protein loop in
the SRCR domains of DMBT1 that was able to bind to various
bacteria (16). The use of synthetic peptides thus offered us a
simple in vitro system to explore fundamental aspects of
DMBT1- and SRCR-mediated bacteria binding in general.
In this study we have defined the minimal bacteria-binding
motif on DMBT1 as an 11-mer peptide designated DMBT1pbs1
(DMBT1 pathogen-binding site 1). By an alanine substitution
scan, critical amino acid residues within this motif were identified. Consensus peptides were derived from the DMBT1pbs1corresponding regions of other SRCR proteins. Only the peptides derived from DMBT1 and its orthologues showed
significant bacterial binding.
EXPERIMENTAL PROCEDURES
Purification of DMBT1SAG—DMBT1SAG was purified by gel filtration
as described previously (29). DMBT1SAG was eluted from a Sephacryl
S-400 HR column (Amersham Biosciences) with either phosphate-buffered saline or Tris-buffered saline (10 mM Tris-HCl and 150 mM NaCl,
pH 7.4).
Bacteria—Streptococcus mutans (Ingbritt), Streptococcus gordonii
(HG222), and Escherichia coli (OM36 –1) were cultured on blood agar
plates under anaerobic conditions with 5% CO2 at 37 °C for 48 h.
Subsequently, single colonies of S. mutans and S. gordonii were cultured in Todd Hewitt medium (Oxoid, Hampshire, United Kingdom).
Single colonies of E. coli were cultured in Luria Broth (Oxoid) at 37 °C
for 24 h in air/CO2 (19:1). Cells were harvested and washed twice in
Tris-buffered saline supplemented with 0.1% (v/v) Tween 20 and 1 mM
CaCl2 (TTC). Helicobacter pylori (NCTC 11637) was cultured on selective Dent plates (Oxoid) at 37 °C for 72 h. H. pylori was harvested by
wiping off the plates and washed twice in 100 mM sodium acetate, pH
4,2, supplemented with 0.1% (v/v) Tween 20 and 1 mM CaCl2. Bacteria
were diluted in buffer to a final OD700 of 0.5, corresponding with ⬃5 ⫻
108 cells/ml.
Peptide Design, Synthesis, and Purification—All synthetic peptides
were SRCR domain consensus-based and designed as described previously using alignment software (DNASTAR, Lasergene Inc., Madison,
WI) (17). For determination of the minimal bacteria binding site we
synthesized overlapping 16-mer peptides covering the SRCR consensus
sequence ranging from six amino acids toward the amino terminus
(peptide SRCRP ⫹ 6N) to six amino acids toward the carboxyl-terminus
(SRCRP2 ⫹ 6C), relative to SRCRP2. To study the role of the individual
amino acids in pathogen binding, we synthesized a set of peptides in
which each residue, in its turn, was substituted for alanine (alanine
substitution scan). To identify corresponding sequences in other SRCR
proteins, the Pfam data base (www.sanger.ac.uk/Software/Pfam/index.
shtml) was searched using “SRCR” or the Protein Data Bank accession
code “1BY2” as the key word, which led us to the SRCR superfamily
data base. Consensus sequences of the SRCR domains in these proteins
were calculated with DNAstar (17).
Solid phase synthesis by Fmoc (N-(9-fluorenyl)methoxycarbonyl)
Pathogen Recognition Site of DMBT1
chemistry was performed with a MilliGen 9050 peptide synthesizer
(MilliGen/Biosearch, Bedford, MA), as described previously. After purification, the purity of these peptides was ⬎90% (17). The authenticity
of the peptides was confirmed by quadrupole-time of flight mass spectrometry on a tandem mass spectrometer (Micromass Inc., Manchester,
United Kingdom) as described previously (34). The peptides used in the
experiments of Table I were obtained from Eurogentec S.A. (Seraing,
Belgium) and had a purity of at least 70%.
Adhesion Assays—Bacterial adhesion was examined using a microtiter plate method based on the labeling of microorganisms with cellpermeable DNA binding probes, as reported earlier (17, 35). Fluotrac
600 microtiter plates (Greiner, Recklinghausen, Germany) were coated
with various amounts of either synthetic peptides or purified
DMBT1SAG. The peptides were dissolved in coating buffer (100 mM
sodium carbonate, pH 9.6) to a starting concentration of 40 ␮g/ml and
2-fold serially diluted. After incubation at 4 °C for 16 h, the plates were
washed twice with the relevant buffers. Subsequently, 100 ␮l of a
bacterial suspension (5 ⫻ 108 bacteria/ml TTC) were added to each well
and incubated at 37 °C for 2 h. Plates were washed three times with the
same buffer using a plate washer (Mikrotek EL 403, Winooski, VT).
Bound bacteria were detected using 100 ␮l/well SYTO-9 solution (1 mM)
(Molecular Probes), a cell permeating and DNA-binding fluorescent
probe. Plates were incubated in the dark at the ambient temperature
for 15 min and washed three times with the same buffer. Fluorescence
was measured in a Fluostar Galaxy microtiter plate fluorescence reader
(BMG Laboratories, Offenburg, Germany) at 488-nm excitation and
509-nm emission wavelengths. These experiments were repeated at
least three times.
Agglutination Assays—100 ␮l of a bacterial suspension (5 ⫻ 108
bacteria/ml) were mixed with 20 ␮l of peptide solution at final peptide
concentrations of 0 –200 ␮g/ml in 48-well microtiter plates (Falcon,
Piscataway, NJ) and incubated at 37 °C for 5–15 min. Agglutination
was scored visually. Turbidimetric analysis of the agglutination process
was carried out with a UVICON 930 spectrophotometer (Kontron Instruments, Watford, United Kingdom) using 150 ␮M peptide as described earlier (17). The optical densities of the bacterial suspensions
were monitored at 700 nm at 37 °C for 60 min. These experiments were
repeated at least three times.
RESULTS
Determination of the Minimal Pathogen-binding Site of
DMBT1—We have defined the precise binding site within
SRCRP2 by testing the bacteria binding properties of a set of
sequentially overlapping 16-mer peptides extending from residues 12–27 to 24 –39 (SRCRP2 ⫹ 6N to SRCRP2 ⫹ 6C) of the
SRCR domain consensus sequence (Fig. 1A). Only peptides
14 –29 and 19 –34 (SRCRP2 ⫹ 4N to SRCRP2 ⫹ C1) bound
bacteria in the adhesion assay (Fig. 1B). Peptide 19 –29, representing the shared motif within these peptides (19GRVEVLYRGSW29), also bound to bacteria and was further referred to
as DMBT1pbs1. Further truncation of this motif, either at the
amino or the carboxyl terminus, yielded peptides without bacteria binding properties (Fig. 1C). Like SRCRP2 and the native
DMBT1, DMBT1pbs1 bound to Gram-positive (S. mutans and
S. gordonii) as well as to Gram-negative bacteria (E. coli and
H. pylori) (Fig. 2B). With SRCRP2, the agglutination of
S. mutans was observed after ⬃30 min, whereas with the
corresponding concentrations of DMBT1pbs1 the agglutination
of S. mutans was already observed after 2–3 min (Fig. 2).
Determination of Essential Amino Acid Residues in
DMBT1pbs1—To determine key residues in DMBT1pbs1 required for bacteria binding, a full alanine substitution scan was
performed. A set of peptides was synthesized in which each
residue across the DMBT1pbs1 peptide, in its turn, was substituted for alanine. This peptide set was tested in the adhesion
assay (Fig. 3). Binding to S. mutans was strongly reduced by
substitution of Val-3, Glu-4, Val-5, Leu-6, and Trp-11 (Fig. 3).
Agglutination assays (not shown) confirmed the results of the
adhesion assays.
The Motif in the Carboxyl-terminal SRCR Domain of DMBT1
Is Functionally Distinct—The DMBT1pbs1 peptide is present
in 10 of the 14 SRCR domains of DMBT1. To investigate the
47701
FIG. 2. Comparison of SRCRP2- and DMBT1bps1-mediated
bacterial agglutination. A, S. mutans suspension was mixed with
SRCRP2, DMBT1pbs1 (both 150 ␮M) or control (Tris-buffered saline),
and agglutination was recorded turbidimetrically at 700 nm.
DMBT1pbs1-mediated agglutination was detected at 2–3 min, whereas
SRCRP2-mediated agglutination was observed no sooner than after
20 –30 min. B, DMBT1pbs1 mediated bacterial agglutination of S. gordonii (Gram-positive), and E. coli and H. pylori (Gram-negative) bacteria. DMBT1pbs1 agglutinates all three bacteria.
bacteria binding of the motifs present within the four remaining SRCR domains, the corresponding peptides were synthesized and tested. Only the SRCR14 peptide, in which five
residues are different from the consensus sequence, did not
bind to bacteria (Table I).
Analysis of the Bacteria Binding Features of Motifs Present
within Other Members of the SRCR Superfamily—Because
SRCR domains are highly conserved across species, we investigated whether DMBT1pbs1-corresponding sequences in other
SRCR proteins also exhibited bacteria binding. Using the Pfam
(protein family) data base, 85 SRCR proteins were found containing altogether 301 SRCR domains. For each protein, the
consensus sequence of its SRCR domains was determined, and
the motif corresponding to DMBT1pbs1 was synthesized. In
this way a representative number of 11-mer peptides covering
the consensus sequences of 20 different SRCR proteins were
tested for bacteria binding. Only the peptides derived from
DMBT1 and DMBT1 orthologues such as mouse CRP-ductin,
the rat Ebnerin, the rabbit Hensin, and the bovine gallbladder
mucin, bound bacteria (Table I).
DISCUSSION
In the present study we determined the minimal bacteriabinding site of DMBT1 and examined whether corresponding
domains in other SRCR proteins also exhibit bacteria binding.
By testing a set of overlapping peptides, the minimal bacteriabinding site was pinpointed as an 11-amino acid sequence,
GRVEVLYRGSW, designated DMBT1pbs1. Alanine substitution of Val-3, Glu-4, Val-5, Leu-6, and Trp-11 resulted in a
drastic decrease of bacteria binding. These residues are highly
conserved within the 14 SRCR domains of DMBT1, the only
difference being the presence of Ile-5 in domains 1 and 14. In
previous studies, we have found that DMBT1SAG agglutinated
bacteria in a calcium-dependent fashion (17). We postulate that
the critical role of Glu-4 and Trp-11 in bacteria binding reflects
the Ca2⫹ chelation by their side chains. The sensitivity to Ala
47702
Pathogen Recognition Site of DMBT1
FIG. 3. Determination of critical
residues in DMBT1pbs1. The table on
the left-hand side shows the amino acid
sequences originating from an alanine
substitution scan of DMBT1pbs1. The
plot at the right displays the binding activity of each peptide expressed as a percentage compared with DMBT1pbs1,
which served as positive control. SRCRP1
was included as negative control (neg.
contr.). Error bars are S.E. Substitution of
the amino acids Val-3, Glu-4, Val-5,
Leu-6, and Trp-11 by Ala in DMBT1pbs1
almost eliminated bacteria binding completely. N-term, amino terminus; C-term,
carboxyl terminus.
TABLE I
Bacteria binding by DMBT1pbs1 homologous peptides of other SRCR domains
The three DMBT1pbs1 homologues that were not identical with the consensus sequence were synthesized. For a number of SRCR proteins the
consensus sequence of its SRCR domains was calculated as described under “Results,” and DMBT1pbs1 homologous peptides were synthesized. A
and B denote group A and group B SRCR proteins. Binding and agglutination activity of these peptides was tested with S. mutans and E. coli. Only
the DMBT1pbs1 peptide of DMBT1 orthologues showed significant bacterial binding and agglutination capacity. Of the SRCR domains of DMBT1,
the peptide of domain 14 exerted no bacteria binding.
Type
DMBT1pbs1
DMBT1 human(SRCR domain 2–7,9–11,13/bovine
DMBT1 human (SRCR domain 1)
DMBT1 human (SRCR domain 8 ⫹ 12),
Hensin rabbit (DMBT1 orthologue)
DMBT1 (SRCR domain 14)
CRPD mouse (DMBT1 orthologue)
Ebnerin rat (DMBT1 orthologue)
CD6 humana
PEMA-SRCR Petromyzon marinus
WC11 bovine
CD5 human
Mac-2 binding protein human/hamster
AR Geodia cydoniuma
Plo2 Perca flavescens
Sp22D Anopheles gambiae
MARCO human
Sp85 Arabica punctulataa
Tequila Drosophila melanogaster
Hemicentrotis pulcherrimus
C06b8.7 Caenorhabditis elegans
Tramp Polyandrocarpqa misakiensis
PRSS7 pig/bovine
IF Xenopus laevis
a
Sequence
Binding/agglutination
⫹
B
B
B
GRVEVLYRGSW
GRVEILYRGSW
GRVEVLYQGSW
⫹
⫹
B
B
GRVEIYHGGTW
GRVEILYQGSW
⫺
⫹
B
B
B
B
A
A
A
A
A
A
A
A
A
A
A
A
GRVEVLFRGSW
GRVEVLYGGSW
GRVEVLDQGSW
GQVEVYQGDLW
GRVEIFYRGQW
GRVEVFYNGVW
GRVEVLHEGRW
GRVEINYHGTW
GRAEVYYSGTW
GRVEVYHGGEW
GRLEVKHNGQW
GRVEIFHDGAW
GRLQVQFRDQW
GHVEVYKGREW
GLVQFRIQSIW
GIIKVKLTFEQ
⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
Consensus sequences only; other sequences are both consensus sequences and actual sequences within the SRCR proteins.
substitution of the three hydrophobic residues surrounding
Glu-4 is less obvious in this model. As these substitutions have
little effect on overall hydrophobicity, this sensitivity indicates
that a proper fit is required for bacteria binding. This is corroborated by the finding that the replacement of Val-5 by the
more similar Ile, as in the SRCR domain 1 peptide, has little if
any effect (Table I).
Analysis of the SRCR consensus domain using sequentially
overlapping 16-mer peptides showed that peptides lacking
Gly-1 were completely incapable of bacteria binding. Together
with the conservation of this residue throughout the SRCR
superfamily (Table I), this finding points to an essential role for
this residue. Surprisingly, the substitution of Gly-1 by Ala had
no effect. In terms of the above model, this result is explained
by assuming that the introduction of a positively charged N
terminus at Arg-2 by the omission of Gly-1 would lead to
detrimental electronic repulsion.
The absence of binding by the DMBT1pbs1 motif of SRCR
domain 14 is not surprising, because this domain shows much
less homology (60%) with the other 13 SRCR domains (87–
100%) of DMBT1. Also, its location in the molecule within two
CUB domains does not support a role in multivalent bacteria
binding, which is probably fulfilled by the other SRCR
domains.
The DMBT1pbs1 11-mer peptide induced agglutination more
rapidly than did the 16-mer SRCRP2 peptide (Fig. 2). This may
be explained by the fact that SRCRP2 has a lower isoelectric
point (pI ⫽ 8.22) than DMBT1pbs1 (pI ⫽ 8.75), resulting in a
lower positive charge and thus a decreased affinity for the
negatively charged bacterial surface.
The first three amino acids (QGR) of the SRCRP2 16-mer
peptide were homologous with the bacteria binding site on the
SRCR domain of MARCO (RGR) (6). Although both bacteria
binding sites are located on the SRCR domains of the proteins,
the results in this paper suggest different bacteria binding
mechanisms. DMBT1pbs1 does not contain the QGR motif, and
substitution of Arg-2 in DMBT1pbs1 did not result in the
disappearance of binding. The positively charged RXR motif of
MARCO suggests an electrostatic interaction with the negatively charged bacterial surface. In DMBT1pbs1, a negatively
charged residue (E4) and the hydrophobic residues (Val-3,
Val-5, Leu-6, and Trp-11) are important for bacteria binding.
Pathogen Recognition Site of DMBT1
Binding studies with corresponding peptides of other SRCR
consensus sequences showed that only highly homologous sequences, such as those present in DMBT1 orthologues, displayed bacteria binding (Table I). Strikingly, SRCR consensus
sequences of some other proteins, including P. marinus PemaSRCR, bovine WC1, and Plo2 of Perca flavescens, possessed the
minimal bacteria binding motif but did not bind to bacteria.
These results suggest that, in addition to the presence of the
VEVLXXXXW motif, a proper three-dimensional folding of
DMBT1pbs1 is quintessential for bacteria binding. For example,
the replacement of Arg-8 by Ala will not influence the secondary
structure, whereas replacement by Gly as in Pema-SRCR, resulting in a di-glycine motif, may favor the introduction of a ␤-turn.
As Pema-SRCR still contains the critical VEVLXXXXW sequence, this observation suggests that Trp must have a proper
orientation with respect to the VEVL stretch for bacteria binding.
This sensitivity of DMBT1pbs1 to the substitution of seemingly
non-critical residues underlines the need for whole proteins to
be tested for conclusive results of bacteria binding.
SRCR domains are present in proteins of all Metazoa. We
studied the degree of conservation of the amino acids in SRCR
domains by using hidden Markov model logos (37) (at logos.
molgen.mpg.de/cgi-bin/logomat-m.cgi?pfamid) in combination
with the Pfam (protein family) data base (www.sanger.ac.
uk/cgi-bin/Pfam/getacc?PF00530). This study revealed that
Gly-1, Val-3, Glu-4, Val-5, and Trp-11 of DMBT1pbs1 are highly
conserved along the whole SRCR superfamily, suggesting that
these residues are important for the structure and function of
SRCR domains. Despite the high degree of conservation, bacteria
binding was only found for DMBT1 and its orthologues by this
motif (Table I). These results suggest that less conserved residues, which seemed to be non-critical in the alanine substitution
scan, were also important for bacteria binding. Because SRCR
proteins are usually involved in different kinds of ligand binding
(38), the conserved residues in DMBT1pbs1 may form a general
ligand binding motif of SRCR proteins that, in DMBT1 orthologues, has developed into a bacteria binding motif.
After studying the phylogenetic tree of the SRCR superfamily, Holmskov et al. identified P. marinus as the most primitive
species with a DMBT1 orthologue (30). The consensus sequence
of this protein possesses the DMBT1pbs1 binding motif, although no binding for this peptide was demonstrated (Table I).
Next to the Pema-SRCR protein, DMBT1 orthologues have only
been characterized in Mammalia, for example CRP-ductin/
Vomeroglandin in the mouse, Ebnerin in the rat, Hensin in the
rabbit, and bovine gallbladder mucin. It remains to be investigated whether DMBT1 orthologues are present in non-mammalian vertebrates and whether these proteins are involved in
bacteria binding in vivo.
In conclusion, we have narrowed down the minimal bacteriabinding site on the SRCR domains of DMBT1 to an 11-mer
peptide, GRVEVLYRGSW, and we have identified a number of
critical residues within this stretch. Despite considerable sequence similarities in this motif throughout the SRCR superfamily, bacteria binding by this motif is thus far restricted to
DMBT1 orthologues.
47703
REFERENCES
1. Resnick, D., Pearson, A., and Krieger, M. (1994) Trends Biochem. Sci. 19, 5– 8
2. Pahler, S., Blumbach, B., Müller, I., and Müller, W. E. (1998) J. Exp. Zool. 282,
332–343
3. Müller, W. E. (1997) Cell Tissue Res. 289, 383–395
4. 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
5. 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
6. Brännström, A., Sankala, M., Tryggvason, K., and Pikkarainen, T. (2002)
Biochem. Biophys. Res. Commun. 290, 1462–1469
7. Dunne, D. W., Resnick, D., Greenberg, J., Krieger, M., and Joiner, K. A. (1994)
Proc. Natl. Acad. Sci. U. S. A. 91, 1863–1867
8. 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
9. Doi, T., Higashino, K., Kurihara, Y., Wada, Y., Miyazaki, T., Nakamura, H.,
Uesugi, S., Imanishi, T., Kawabe, Y., Itakura, H., Yazaki, Y., Matsumoto,
A., and Kodama, T. (1993) J. Biol. Chem. 268, 2126 –2133
10. Platt, N., and Gordon, S. (2001) J. Clin. Investig. 108, 649 – 654
11. Grønlund, J., Vitved, L., Lausen, M., Skjødt, K., and Holmskov, U. (2000)
J. Immunol. 165, 6406 – 6415
12. Jones, N. H., Clabby, M. L., Dialynas, D. P., Huang, H. J., Herzenberg, L. A.,
and Strominger, J. L. (1986) Nature 323, 346 –349
13. Aruffo, A., Melnick, M. B., Linsley, P. S., and Seed, B. (1991) J. Exp. Med. 174,
949 –952
14. Gebe, J. A., Kiener, P. A., Ring, H. Z., Li, X., Francke, U., and Aruffo, A. (1997)
J. Biol. Chem. 272, 6151– 6158
15. Law, S. K., Micklem, K. J., Shaw, J. M., Zhang, X. P., Dong, Y., Willis, A. C.,
and Mason, D. Y. (1993) Eur. J. Immunol. 23, 2320 –2325
16. Kristiansen, M., Graversen, J. H., Jacobsen, C., Sonne, O., Hoffman, H.-J.,
Law, S. K. A., and Moestrop, S. K. (2001) Nature 409, 198 –201
17. Bikker, F. J., Ligtenberg, A. J. M., Nazmi, K., Veerman, E. C. I., Van ’t Hof, W.,
Bolscher, J.G.M., Poustka, A., Nieuw Amerongen, A. V., and Mollenhauer,
J. (2002) J. Biol. Chem. 277, 32109 –32115
18. Holmskov, U., Lawson, P., Teisner, B., Tornøe, I., Willis, A. C., Morgan, C.,
Koch, C., and Reid, K. B. (1997) J. Biol. Chem. 272, 13743–13749
19. Kang, W., and Reid, K. B. (2003) FEBS Lett. 540, 21–25
20. 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
21. Mollenhauer, J., Holmskov, U., Wiemann, S., Krebs, I., Herbertz, S., Madsen,
J., Kioschis, P., Coy, J. F., and Poustka, A. (1999) Oncogene 18, 6233– 6240
22. Prakobphol, A., Xu, F., Hoang, V. M., Larsson, T., Bergstrom, J., Johansson, I.,
Frängsmyr, L., Holmskov, U., Leffler, H., Nilsson, C., Borén, T., Wright,
J. R., Strömberg, N., and Fisher, S. J. (2000) J. Biol. Chem. 275,
39860 –39866
23. Li, X. J., and Snyder, S. H. (1995) J. Biol. Chem. 270, 17674 –17679
24. Cheng, H., Bjerknes, M., and Chen, H. (1996) Anat. Rec. 244, 327–343
25. Takito, J., Yan, L., Ma, J., Hikita, C., Vijayakumar, S., Warburton, D., and Al
Awqati, Q. (1999) Am. J. Physiol. 277, F277–F289
26. Nunes, D. P., Keates, A. C., Afdhal, N. H., and Offner, G. D. (1995) Biochem.
J. 310, 41– 48
27. Mayer, W. E., and Tichy, H. (1995) Gene 164, 267–271
28. Bikker, F. J., Ligtenberg, A. J. M., van der Wal, J. E., van den Keijbus,
P. A. M., Holmskov, U., Veerman, E. C. I., and Nieuw Amerongen, A. V.
(2002) J. Dent. Res. 81, 134 –139
29. Ligtenberg, T. J. M., Bikker, F. J., Groenink, J., Tornøe, I., Leth-Larsen, R.,
Veerman, E. C. I., Nieuw Amerongen, A. V., and Holmskov, U. (2001)
Biochem. J. 359, 243–248
30. Holmskov, U., Mollenhauer, J., Madsen, J., Vitved, L., Grønlund, J., Tornøe, I.,
Kliem, A., Reid, K. B., Poustka, A., and Skjødt, K. (1999) Proc. Natl. Acad.
Sci. U. S. A. 96, 10794 –10799
31. Carlén, A., Bratt, P., Stenudd, C., Olsson, J., and Strömberg, N. (1998) J. Dent.
Res. 77, 81–90
32. Ericson, T., and Rundegren, J. (1983) Eur. J. Biochem. 133, 255–261
33. Tino, M. J., and Wright, J. R. (1999) Am. J. Respir. Cell Mol. Biol. 20, 759 –768
34. Nagle, G. T., Jong-Brink, M., Painter, S. D., and Li, K. W. (2001) Eur. J. Biochem. 268, 1213–1221
35. Bosch, J. A., Veerman, E. C. I., Turkenburg, M., Hartog, K., Bolscher, J. G. M.,
and Nieuw Amerongen, A. V. (2003) J. Microbiol. Methods 53, 51–56
36. Deleted in proof
37. Schuster-Böckler, B., Schultz, J., and Rahmann, S. (2004) BMC Bioinformatics
http://www.biomedcentral.com/1471–2105/5/7
38. Sarrias, M.R., Grønlund, J., Padilla, O., Madsen, J., Holmskov, U., and
Lozano, F. (2004) Crit. Rev. Immunol. 24, 1–37