From the
Departments of Pharmacology and
¶Molecular Genetics, University of Illinois at
Chicago, College of Medicine, Chicago, Illinois 60612 and
||Joseph J. Jacobs Center for Thrombosis and
Vascular Biology, Department of Molecular Cardiology, Cleveland Clinic
Foundation, Cleveland, Ohio 44195
Received for publication, February 12, 2003 , and in revised form, April 10, 2003.
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ABSTRACT |
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INTRODUCTION |
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The expression of CCN1 is essential for normal embryonic development
inasmuch as targeted disruption of the CCN1 gene in mouse results in
embryonic lethality (15).
Interestingly, the majority of the CCN1-null embryos exhibit vascular defects,
consistent with an essential role of CCN1 in normal embryonic angiogenesis. In
adults, CCN1 is present at low levels in the cardiovascular system
(16,
17); however, its expression
is up-regulated in a number of vascular diseases including atherosclerosis and
proliferative restenosis (12,
13,
17,
18). High expression of CCN2
in human advanced atherosclerotic lesions has also been observed
(19,
20). Moreover, both CCN1 and
CCN2 are expressed during cutaneous wound healing
(2123).
It is well established that leukocyte adhesion and emigration play an
important role in inflammation, wound healing, and atherosclerosis
(24). In a recent report, we
showed that human peripheral blood monocytes adhere to CCN1 and CCN2 in an
activation-dependent manner through integrin
M
2
(12), underscoring the
importance of these proteins in the pathophysiologic function of leukocytes.
To establish these CCN proteins as novel ligands of
M
2, we demonstrated direct binding of the I
domain of the integrin
M subunit (
MI) to
immobilized CCN1 and CCN2
(12). It is noteworthy that
monocyte adhesion and
MI domain binding to CCN1 are blocked
by anti-
M monoclonal antibodies as well as by soluble
heparin. These findings suggest that the
M
2
binding site may lie in close proximity to the heparin binding motifs within
the C-terminal domain of CCN1.
To gain further insight into the interaction of
M
2 with CCN proteins, we sought to identify
the
M
2 binding site in CCN1. In the present
study, we demonstrated
M
2-dependent
adhesion of monocytes to SSVKKYRPKYCGS present in the C-terminal domain of
CCN1. Furthermore, the
MI domain binds specifically to this
13-residue sequence of CCN1 with a similar affinity as to the P2
(YSMKKTTMKIIPFNRLTIG) sequence, an
M
2
binding site in the fibrinogen
chain
(25). Our newly identified
M
2 binding sequence in CCN1 bears no
sequence homology to any known
M
2 binding
motif reported to date and may provide a target for blocking
CCN1-
M
2 interaction.
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MATERIALS AND METHODS |
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Peptides sequences were represented by the single letter amino acid code
(26). CCN1-H1
(YSSLKKGKKCSKTKKSPE), CCN1-H2 (SSVKKYRPKYCGS), CCN1-scrH2 (YRSCYSKVKPSGK),
CCN2-H2 (TSVKTYRAKFCGV), and the fibrinogen P2 peptide (YSMKKTTMKIIPFNRLTIG)
were synthesized by ResGen, Inc. For the production of anti-CCN1-H2
antibodies, rabbits were immunized with CCN1-H2 coupled to keyhole limpet
hemocyanin (KLH) using the Imject® maleimide-activated mcKLH kit (Pierce).
Anti-CCN1-H2 antibodies were affinity-purified on protein A-Sepharose and then
on CCN1-H2-coupled Sepharose. Preimmune rabbit IgG was purified on protein
A-Sepharose. Streptavidin (Sigma) was labeled with carrier-free
Na125I (ICN Biomedicals, Inc.) using the Iodobeads iodination
reagent (Pierce) to a specific activity of 0.3 µCi/µg.
Protein PurificationRecombinant mouse CCN1, synthesized in
a baculovirus expression system using Sf9 insect cells, was purified from
serum-free conditioned media by Sepharose S chromatography as previously
described (27). The CCN1
truncation mutant lacking the C-terminal domain (CCN1CT) was produced
as a hexahistidine-tagged fusion protein and purified by nickel-agarose
chromatography as described
(10). Purified CCN1 and
CCN1
CT were analyzed by SDS-polyacrylamide gel electrophoresis followed
by Coomassie Blue staining and immunoblotting.
Recombinant I domain of the integrin M subunit was
expressed as a fusion protein with GST (GST-
MI) and purified
as described previously (28).
Briefly, the coding region for human
MI domain sequence
Asp132Ala318 was amplified and inserted into the
expression vector pGEX-4T-1 (Amersham Biosciences). Escherichia coli
cells were transformed with the above recombinant vector, and protein
expression was induced with 1mM
isopropyl-
-D-thiogalactopyranoside for4hat37 °C. The
GST-
MI fusion protein was affinity-purified from cell
lysates using glutathione-agarose (Sigma).
Isolation of Peripheral Blood MonocytesPeripheral blood monocytes were isolated from human blood as previously described (12). Acid-citrate-dextrose anticoagulated blood was collected from healthy donors and centrifuged to remove platelet-rich plasma. For isolation of mononuclear cells, the remaining packed red blood cells and buffy coat was diluted with phosphate-buffered saline (10 mM sodium phosphate, pH 7.35, 0.15 M NaCl) and centrifuged through a layer of Ficoll-Paque (Amersham Biosciences) at 400 x g for 60 min at 4 °C. The mononuclear cell layer was diluted in an equal volume of phosphate-buffered saline containing 2 mM EDTA, sedimented, and washed twice with modified Tyrode's buffer (10 mM Hepes, pH 7.35, 135 mM NaCl, 2.9 mM KCl, 12 mM NaHCO2,1mM MgCl2,1mM CaCl2, 0.1% dextrose, and 0.2% bovine serum albumin (BSA)) by centrifugation at 130 x g for 10 min. To further separate monocytes from lymphocytes, the mononuclear cells were suspended in modified Tyrode's buffer and subjected to centrifugation through a discontinuous density gradient of Percoll (Amersham Biosciences). Monocytes were isolated between a Percoll density of 1.047 and 1.050 g/ml, washed with modified Tyrode's buffer, and resuspended to a final concentration of 23 x 106 cells/ml. The purity of the monocyte preparation was greater than 80%, as measured by anti-CD14 staining in flow cytometry, and cell viability was more than 95% as judged by trypan blue exclusion.
Cell Adhesion AssayMicrotiter wells (Immulon 2 Removawell
strips, Dynex Technologies, Inc.) were coated with 10 µg/ml CCN1 or
CCN1CT protein or with 4 mg/ml peptide for 20 h at 4 °C. After
protein or peptide coating, the wells were blocked with 0.2% polyvinyl alcohol
(PVA) for 30 min at 37 °C. The amounts of immobilized CCN1 and
CCN1
CT protein were quantified by an ELISA using anti-domain I and
anti-CCN1367381 antibodies as indicated. The amounts of
immobilized peptides were measured by incubation for 2 h at 22 °C with 10
µM PEO-maleimide-activated biotin (Pierce), which reacts with
the free sulfhydryl group in the cysteine residue of the peptides. After
washing, the amounts of coupled biotin were quantified by incubation with 0.5
µM 125I-labeled streptavidin, and bound radioactivity
was measured by
-counting.
In cell adhesion experiments, isolated monocytes were activated with 20 µM ADP, added to the wells (100 µl/well), and incubated for 20 min at 37 °C. After washing to remove non-adherent cells, adherent cells were quantified using the acid phosphatase assay by incubation with the substrate solution (0.1 M sodium acetate, pH 5.5, 10 mM p-nitrophenylphosphate, and 0.1% Triton X-100; 100 µl/well) for 2 h at 37 °C (29). The reaction was stopped by the addition of 15 µlof1 N NaOH/well, and A450 was measured. In inhibition studies, monocytes were preincubated with antibodies for 30 min at 37 °C before addition to microtiter wells.
Binding of GST-MI to Immobilized
CCN1 and CCN-derived PeptidesMicrotiter wells were coated with 30
µg/ml wild type or truncated CCN1 or 4 mg/ml synthetic peptides for 20 h at
4 °C and blocked with 0.2% PVA for 30 min at 37 °C.
GST-
MI fusion protein was added to the wells and incubated
for 12 h at 22 °C. Unbound GST-
MI was removed by
washing with 30 mM Tris-HCl, pH 7.4, 0.2 M NaCl, 1
mM MgCl2, and 0.02% PVA. Bound GST-
MI
was detected by an ELISA using anti-GST followed by an HRP-conjugated
secondary antibody. Bound antibodies were detected using
o-phenylenediamine dihydrochloride (Sigma) as the substrate. The
reaction was stopped by the addition of 25 µl of 6 N HCl, and
A490 was measured. In inhibition experiments using an
anti-
M monoclonal antibody, GST-
MI was
preincubated with the antibody for 30 min at 37 °C before addition to the
wells. In inhibition studies using anti-CCN1-H2 polyclonal antibodies,
CCN1-coated wells were blocked with 1% BSA and preincubated with the
antibodies for 30 min at 37 °C before the addition of the
GST-
MI fusion protein. Binding proceeded for 30 min at 37
°C, and bound GST-
MI was detected as described above. In
binding isotherm studies, input GST-
MI concentrations
required for half-maximal binding of the fusion protein to immobilized
peptides were estimated using the GraphPad Prism software in which the data
was fitted to the one-site binding equation.
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RESULTS |
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The H2 Sequence in the CCN1 C-terminal Domain Supports Monocyte
AdhesionThe inability of CCN1CT to support monocyte
adhesion and
MI domain binding suggests that the recognition
site for
M
2 is located in the C-terminal
domain of CCN1. We focused on the first half of the C-terminal domain, because
this region is highly homologous between CCN1 and CCN2, and both proteins
support monocyte adhesion and
MI domain binding. Also,
heparin has been shown to inhibit monocyte adhesion and
MI
domain binding to CCN1 (12),
and therefore, we examined the possibility that
M
2 binds directly to the heparin binding
motifs in CCN1. In these studies we synthesized two peptides, CCN1-H1 and
CCN1-H2, that correspond to the two heparin binding motifs of CCN1
(Fig. 2A). The
peptides were immobilized onto microtiter wells, and their ability to support
monocyte adhesion was examined. To determine the coating efficiency of CCN1-H1
and CCN1-H2 onto the wells, PEO-maleimide-activated biotin was allowed to
react with the free sulfhydryl group in the single cysteine residue present in
each peptide followed by detection with 125I-labeled streptavidin.
Quantitation of bound radioactivity indicates that similar amounts of both
peptides were immobilized onto the wells
(Fig. 2B). In cell
adhesion studies we found that monocytes adhered to CCN1-H2, but not to
CCN1-H1 (Fig. 2C).
Furthermore, monocyte adhesion to CCN1-H2 was completely blocked by 2LPM19c,
an anti-
M monoclonal antibody, whereas control mouse IgG had
no effect. Microscopic examination of the monocytes adherent to CCN1 protein
and CCN1-H2 peptide revealed that the cells were well spread on both
substrates (data not shown). Together, these results indicate that the H2
sequence in the C-terminal domain of CCN1 specifically mediates monocyte
adhesion in an
M
2-dependent manner.
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We previously reported that monocyte adhesion to CCN1 is markedly enhanced
after cellular activation with ADP and formylmethionylleucylphenylalanine
(12). Enhanced adhesion of
activated monocytes to CCN1 is likely due to inside-out signaling, resulting
in increased M
2 affinity for CCN1 and/or
mobilization of internal
M
2 to the cell
surface. In addition to inside-out signaling, the activation states of
integrins can be modulated by extracellular divalent cations inasmuch as
Mn2+ has been shown to increase the apparent
affinity/avidity of multiple integrins including
M
2
(30). We, therefore, examined
the effect of extracellular Mn2+ on monocyte adhesion to
intact CCN1 protein and to the CCN1-H2 peptide. As expected, the addition of 1
mM Mn2+ to the cell suspension resulted in
enhanced monocyte adhesion to CCN1 (Fig.
3). Likewise, monocyte adhesion to the CCN1-H2 peptide was also
significantly enhanced in the presence of Mn2+. In a
specificity control, monocytes were allowed to adhere to wells coated with a
scrambled CCN1-H2 sequence (CCN1-scrH2). To confirm that CCN1-scrH2 was
adsorbed onto microtiter wells, we performed binding of
PEO-maleimide-activated biotin and 125I-labeled streptavidin as
described above. Even though a higher amount of CCN1-scrH2 (95.6 ± 6.1
fmol/well) was coated onto the wells as compared with CCN1-H2 (43.6 ±
0.9 fmol/well), no significant cell adhesion to CCN1-scrH2-coated wells was
observed in the absence and presence of Mn2+. The
ability of Mn2+ to enhance monocyte adhesion to CCN1 and
CCN1-H2, but not to CCN1-scrH2, indicates that activated
M
2 binds with a higher affinity to intact
CCN1 and to the CCN1-H2 sequence.
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Binding of MI Domain to the H2
Sequences in CCN proteinsThe above cell adhesion data suggest that
the CCN1-H2 sequence, but not CCN1-H1, serves as a binding site for integrin
M
2. To corroborate cell adhesion studies
with receptor binding, we performed
MI domain binding
experiments on immobilized CCN1-H1 and CCN1-H2.
Fig. 4A shows that
GST-
MI bound directly to wells coated with the CCN1 protein
or with the CCN1-H2 peptide, but not to wells coated with the CCN1-H1 peptide
(filled bars). As controls, GST itself did not bind to CCN1, CCN1-H1,
or CCN1-H2 (open bars). To demonstrate binding specificity, we tested
the effect of 2LPM19c on
MI domain binding. As shown in
Fig. 4B, inhibition of
GST-
MI binding to CCN1-H2 was observed with 2LPM19c
(anti-
M) but not with an isotype-matched mouse IgG. These
results indicate that integrin
M
2 binds
directly to the CCN1-H2 sequence to mediate monocyte adhesion to CCN1
protein.
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In addition to CCN1, we previously reported that the MI
domain also binds to CCN2, another CCN family member highly homologous to CCN1
(12). Therefore, we performed
solid phase binding studies to examine whether GST-
MI also
binds to the corresponding H2 sequence in CCN2.
Fig. 5A shows the H2
sequences in CCN1 and CCN2 with the non-conserved residues
underlined. The
MI domain was found to bind dose
dependently to both CCN1-H2 and CCN2-H2 peptides, whereas no binding was
observed with wells coated with the CCN1-scrH2 sequence
(Fig. 5B). Using the
GraphPad Prism software, we estimated that half-maximal binding of
GST-
MI to immobilized CCN1-H2 and CCN2-H2 occurred at input
GST-
MI concentrations of 47.2 ± 6.4 and 128.0
± 26.3 nM (means ± S.E.; n = 5 for CCN1-H2
and n = 3 for CCN2-H2), respectively. Thus, the results of the
binding isotherms indicate that the
MI domain binds with a
higher affinity to CCN1-H2 than to CCN2-H2 (Student's t test,
p < 0.01). Consistently, we previously showed that monocytic THP-1
cells adhere more strongly to CCN1 than to CCN2
(12).
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It has been reported that integrin M
2
binds to fibrinogen in part through interaction with the P2 sequence
(YSMKKTTMKIIPFNRLTIG) at the C-terminal region of the fibrinogen
chain
(25). In the present study, we
compared GST-
MI binding to the H2 sequence of CCN1 and the
P2 sequence of fibrinogen (Fbg-P2). As shown in
Fig. 6A,
GST-
MI bound saturably to both CCN1-H2 and Fbg-P2. The
difference in maximal binding to the two peptides may be due to different
coating efficiencies of these peptides onto microtiter wells. Nonetheless,
half-maximal binding of GST-
MI to CCN1-H2 and Fbg-P2
occurred at 42.6 ± 7.2 and 57.4 ± 9.1 nM
concentrations of the fusion protein (means ± S.E. of triplicate
determinations in one experiment), respectively, indicating that the
MI domain binds with similar affinities to both peptide
sequences. To further investigate the relationship of the CCN1-H2 and Fbg-P2
binding site(s) in the
MI domain, we examined the effect of
soluble Fbg-P2 peptide on GST-
MI binding to intact CCN1
protein and CCN1-H2 peptide. Fig.
6B shows that Fbg-P2 dose-dependently inhibited
MI domain binding to CCN1- and CCN1-H2-coated wells,
indicating mutual exclusive binding of these two peptide sets to the
MI domain. We then compared the potency of soluble CCN1-H2
and Fbg-P2 peptides in blocking GST-
MI binding to
immobilized CCN1 protein. Although CCN1-H2 also significantly inhibited
GST-
MI binding to CCN1-coated wells, much weaker inhibition
was attained with CCN1-H2 (
25%) as compared with Fbg-P2 (
65%) at a
high concentration (1 mM) of these peptides
(Fig. 6C). As a
specificity control, soluble CCN1-scrH2 had no inhibitory effect.
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Effect of Anti-CCN1 Antibodies on
MI Domain Binding to CCN1The above
experiments show that a synthetic peptide corresponding to the CCN1-H2
sequence supports monocyte adhesion and
MI domain binding.
To demonstrate the importance of the H2 sequence in intact CCN1 protein for
interaction with
M
2, we produced an
anti-peptide antibody against CCN1-H2 and tested its effect on
GST-
MI binding to CCN1. Anti-CCN1-H2 antibodies were
affinity-purified by sequential chromatography on protein A-Sepharose and
CCN1-H2-coupled Sepharose. In ELISA, anti-CCN1-H2 antibodies bound with a
higher affinity to CCN1 when protein coating was performed in the presence of
1%
-mercaptoethanol than in the absence of the reducing agent. Also, we
found that GST-
MI bound equally well to non-reduced and
reduced CCN1 (data not shown). Thus, in inhibition studies, CCN1 was
immobilized onto microtiter wells under reducing conditions and preincubated
with the indicated antibodies before the addition of GST-
MI.
As shown in Fig. 7, the
anti-CCN1-H2 antibody effectively inhibited
MI domain
binding to CCN1 by
75%. By contrast, no inhibition was observed with
preimmune rabbit IgG or with two polyclonal antibodies directed against the
C-terminal sequence (Phe367Asp381) or domain II
of CCN1. The lack of inhibition by anti-CCN1367381 and
anti-domain II antibodies was not due to the failure of these antibodies to
bind to CCN1 because they reacted strongly with immobilized CCN1 in ELISA (see
Fig. 1C and
Table I). Thus, these findings
show that anti-CCN1-H2 specifically blocked GST-
MI binding
to intact CCN1 protein, indicating that the H2 sequence is a major binding
site for integrin
M
2 in CCN1.
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DISCUSSION |
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Both H1 and H2 sequences in the CCN1 C-terminal domain contain the
BBXB (B represents a basic amino acid) consensus glycosaminoglycan
binding sequence that interacts with heparin or cell surface HSPGs
(31). Thus, alanine
substitutions of some of the lysine and arginine residues in either H1 or H2
in CCN1 markedly diminish its binding affinity to an heparin-Sepharose column
(7). Moreover, human skin
fibroblasts fail to adhere to a CCN1 mutant (CCN1-DM) with alanine
substitutions of the basic residues in both H1 and H2, which is due to the
inability of CCN1-DM to interact with cell surface HSPGs on fibroblasts
(7). With respect to
M
2-dependent monocyte adhesion to CCN1, we
reported earlier that cell surface HSPGs are involved but not absolutely
required for this adhesion process
(12). Nonetheless, soluble
heparin has been shown to block monocyte adhesion as well as
MI domain binding to wild type CCN1, suggesting that the
M
2 binding site in CCN1 may lie in close
proximity to the H1 and H2 sequences in the C-terminal domain. Consistent with
this speculation, a CCN1 truncation mutant lacking the C-terminal domain does
not support monocyte adhesion and
MI domain binding
(Fig. 1). Using synthetic
peptides that encompass the H1 and H2 sequences of CCN1, we demonstrated that
monocytes adhere and
MI domain binds to immobilized CCN1-H2
but not to CCN1-H1 (Fig. 2).
The specificity of
M
2 interaction with
CCN1-H2 has been further confirmed by the lack of reactivity of a scrambled
CCN1-H2 sequence in both monocyte adhesion and
MI domain
binding assays. In subsequent studies, we showed that an anti-peptide antibody
against CCN1-H2 specifically blocks
MI domain binding to
CCN1, thus affirming the importance of the H2 sequence in intact CCN1 protein
in mediating interaction with
M
2. Based on
these results, we suggest that CCN proteins interact with monocytes through
the cooperative binding of the H1 sequence to cell surface HSPGs and the H2
sequence to integrin
M
2. In support of this
model, there is increasing evidence that certain cell surface HSPGs
(e.g. syndecans) and chondroitin sulfate proteoglycans act as
co-receptors with integrins to mediate cell adhesion and signaling
(32,
33). Moreover, it is
interesting to note that the integrin and proteoglycan binding sites in the
fibronectin heparin III domain and in the laminin
5
N-terminal domain are also spatially close together as in CCN1
(33,
34).
THP-1 cells also adhere to CCN2, and the MI domain also
binds to CCN2 (12); however,
we consistently observed weaker THP-1 cell adhesion to CCN2 as compared with
CCN1. Thus, it is not surprising that the
MI domain binds to
the CCN2-H2 peptide with a lower affinity than to the CCN1-H2 peptide, which
is likely due to the non-conserved Lys to Thr and Pro to Ala substitutions in
the H2 sequences of these two CCN proteins (see
Fig. 5A). Nonetheless,
the ability of the
MI domain to bind to both CCN1-H2 and
CCN2-H2 suggests that the conserved residues in these H2 sequences may provide
coordination sites for interaction with integrin
M
2. The H2 sequence is highly conserved
among other CCN proteins but is missing in CCN5, and therefore, it is tempting
to speculate that
M
2 may also bind to CCN3,
CCN4, and CCN6 through this sequence. Thus, further studies of the H2
sequences in different CCN proteins may help to define the binding specificity
of this novel
M
2 recognition motif.
Among integrin family members, M
2 has
the broadest ligand binding specificity that is not shared by the closely
related
L
2 leukocyte integrin. Recently, we
demonstrated that insertion of the
M-(Lys245Arg261) sequence into
L
2 imparts to the chimeric integrin the
ability to recognize multiple
M
2 ligands,
including CCN1 (35). The broad
ligand binding specificity of
M
2 suggests
that it is capable of interacting with many different recognition sequences
derived from diverse
M
2 ligands. Indeed,
several
M
2 binding sequences have been
identified (Table II),
including the P1 and P2 sequences in the fibrinogen
chain
(25,
36) and a nine-residue
sequence in the coagulation factor X
(37). In addition, a
22-residue sequence in intercellular adhesion molecule-2 binds to both
M
2 and
L
2
(38,
39), and a LLG-C4 biscyclic
nonapeptide derived from phage display screening interacts with
M
2,
L
2,
and
X
2
(40). Our newly identified
CCN1-H2 sequence exhibits no apparent homology with these
M
2 binding motifs other than a pair of
basic amino acid residues is present in both P1 and P2 of fibrinogen, the
factor X sequence, and CCN1-H2. However, it is not likely that the basic amino
acid pair is the sole
M
2 recognition motif
because CCN1-H1, which contains three basic amino acid pairs, does not support
monocyte adhesion and
MI domain binding. Moreover, the
active CCN2-H2 and P2-C (383TMKIIPFNRLTIG395 from the
C-terminal portion of fibrinogen P2) peptides, do not contain a pair of basic
residues. At present, the critical determinants in these
M
2 binding sequences that mediate
receptor-ligand interaction remain to be defined.
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In the solid phase binding assay, we found that the MI
domain binds with a similar affinity to immobilized CCN1-H2 peptide as to the
fibrinogen-P2 peptide. Soluble P2 effectively cross-competes
MI domain binding to CCN1 protein and CCN1-H2 peptides,
suggesting that both peptide sets interact with the same or overlapping
binding sites within the
MI domain. As mentioned earlier,
cells expressing a chimeric receptor in which the
M-(Lys245Arg261) sequence has
been grafted into
L
2 adhere more avidly to
fibrinogen and CCN1 as compared with cells expressing wild type
L
2
(35). Thus,
M-(Lys245Arg261) would likely
play an important role for
M
2 interaction
with both fibrinogen-P2 and CCN1-H2 sequences. Although
MI
domain binds strongly to immobilized CCN1-H2 peptide, soluble CCN1-H2 is a
weak inhibitor for blocking
MI domain binding to CCN1. One
possible explanation is that the
MI domain may bind to
additional sites on CCN1 other than the H2 sequence. Alternatively, soluble
CCN1-H2 peptide may not be able to assume the optimal conformation for high
affinity interaction with the
MI domain. It is noteworthy
that both CCN1-H1 and CCN1-H2 sequences contain a cysteine residue, which may
pair with other cysteines in the CCN1 C-terminal domain
(4), thereby affecting the
affinity of
M
2 interaction with CCN1-H2. At
present, the disulfide bond pattern of CCN1 has not been resolved
experimentally. However, GST-
MI binds similarly to both
non-reduced and reduced CCN1 (results not shown), suggesting that disulfide
bond formation in CCN1 does not play a major role in
M
2 binding. Whether the neighboring regions
of CCN1-H2 would affect its binding affinity to the
MI
domain is under investigation.
Although the pathophysiologic function of CCN proteins remains to be
established, the increased expression of CCN1 and CCN2 in healing wounds,
restenosed blood vessels, and atherosclerotic lesions coupled with their
ability to interact with monocytes suggests that these CCN proteins may play
an important role in inflammatory responses. Thus, both CCN1 and CCN2 are
ligands of integrin M
2 and may induce
integrin-dependent outside-in signaling and gene expression in monocytes. In
this regard, we recently observed that adhesion of peripheral blood monocytes
to CCN1 induces the expression and secretion of proinflammatory mediators
including interleukin 1
and monocyte chemotactic
protein-1.2
Identification of the
M
2 binding site in
CCN proteins would greatly facilitate further mutational studies to
demonstrate the biologic significance of
M
2-CCN protein interaction on monocytes.
Furthermore, it may lead to the development of novel therapeutic agents
targeting the
M
2 binding site on this new
family of matricellular signaling molecules.
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FOOTNOTES |
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Supported by the National Institutes of Health HL-07829 training grant and
by a predoctoral fellowship from the American Heart Association, Midwest
Affiliate.
** An Established Investigator of the American Heart Association.
To whom correspondence should be addressed: Dept. of Pharmacology (M/C 868),
University of Illinois at Chicago, 835 South Wolcott Ave., Chicago, IL 60612.
Tel.: 312-413-5928; Fax: 312-996-1225; E-mail:
sclam{at}uic.edu.
1 The abbreviations used are: CCN, CYR61/CTGF/nephroblastoma-overexpressed;
BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; Fbg,
fibrinogen; GST, glutathione S-transferase; HRP, horseradish
peroxidase; HSPGs, heparan sulfate proteoglycans; PVA, polyvinyl alcohol; HRP,
horseradish peroxidase; PEO, polyethylene oxide.
2 V. M. Hadkar, J. M. Schober, L. F. Lau, and S. C. Lam, unpublished
observations.
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REFERENCES |
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