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INTRODUCTION |
Decorin is a member of the small leucine-rich proteoglycan
(SLRP)1 family of related
glycoproteins found in mammalian extracellular matrices (ECMs) (2). The
core proteins of SLRPs are similar in structural organization and size,
ranging from 35 to 42 kDa. The dominant structural feature is a central
domain containing 6-10 leucine-rich repeats (LRRs) that is flanked by
N-terminal and C-terminal regions with conserved Cys residues (3). The LRR motif has been found in a number of proteins of diverse origin and
function and varies in length from 20 to 29 amino acids (4). Protein
crystal structure analysis of the RNase inhibitor, internalin B, and
glycoprotein 1B suggests that each LRR motif forms a
-strand/turn/helix, giving protein segments composed of tandem
repeats of LRRs an arch-like shape (5-7). The SLRPs contain 24-amino
acid long LRRs and may also assume an arch-shaped structure as proposed
by molecular modeling studies of decorin (8).
SLRPs may be subgrouped based on gene organization, amino acid sequence
similarity, the number of LRRs in the central domain, and the spacing
of Cys residues in the N-terminal and C-terminal segments (3). Members
of subgroup I (10 LRRs) include decorin, biglycan, and a recently
described molecule, asporin (9, 10). The former two may contain 1 or 2 dermatan sulfate/chondroitin sulfate chains in the N terminus,
respectively, whereas the latter appears to occur exclusively as a
glycoprotein with a N-terminal stretch of Asp residues. Fibromodulin,
lumican, keratocan, and osteoadherin, which may be substituted with
keratan sulfate polysaccharides in the LRR domain, as well as the
glycoprotein PRELP, are the members of subgroup II (10 LRRs). Subgroup
III (6 LRRs) includes epiphycan and osteoglycin, which may be
substituted with dermatan sulfate/chondroitin sulfate and keratan
sulfate polysaccharides, respectively, and the glycoprotein opticin
(11). The structure of a 12th member, chondroadherin, differs
sufficiently from those of other SLRPs and perhaps should be assigned
to a separate subgroup. The spacing of 4 Cys residues in the N-terminal
sequence of the members of subgroups I, II, and III is
CX3CXCX6C,
CX3CXCX9C, and CX2CXCX6C, respectively.
One theme in SLRP function appears to be the regulation of
extracellular matrix architecture. Decorin, biglycan, fibromodulin, and
lumican reportedly interact with collagens and influence collagen fibrillogenesis in in vitro assays (12-16). Analyses of
transgenic mice generated with targeted inactivation of individual SLRP
genes has revealed that the loss of function of each SLRP resulted in a
mild phenotype characterized by the abnormal morphology of specific ECMs. Decorin or fibromodulin-deficient mice exhibit collagen fiber
defects in tendon, whereas decorin or lumican-deficient mice have
fragile skin, possibly because of abnormal collagen fibers in the
dermis (17-19). Lumican-deficient mice also display abnormally
thickened collagen fibers in the cornea. Recent studies suggest that
the phenotype of mice deficient in both decorin and biglycan is more
severe than those observed in cases where decorin or biglycan genes
were individually inactivated (20). Taken together, these results
indicate a certain degree of functional redundancy among the different
SLRP members.
SLRPs may differentially affect cell behavior by modulating growth
factor activity or by influencing the interactions of cell surface
receptors with matrix components. Transfection of the decorin cDNA
into Chinese hamster ovary cells results in the overexpression of the
proteoglycan and correlates with reduced proliferation, presumably
because of an inhibition of transforming growth factor-
signaling (21). Decorin has also been reported to inhibit the growth of
certain cancer cell types by a mechanism thought to involve an
interaction between decorin and the epidermal growth factor receptor
(22). The ability of SLRPs to affect cell-matrix interactions can
involve SLRP-ECM or SLRP-integrin interactions. Decorin has been shown
to interfere with the adhesion of mammalian cells to substrates
composed of fibronectin and thrombospondin, and in a
glycosaminoglycan-dependent manner, decorin inhibits cell
migration on fibronectin and collagen matrices (23-26). On the other
hand, osteoadherin and chondroadherin were shown to support osteoblast
and chondrocyte adhesion through interactions with integrins
v
3 and
2
1,
respectively (27, 28).
We recently demonstrated that decorin and biglycan are Zn2+
metalloproteins (1). The results of equilibrium dialysis experiments indicated that both decorin and biglycan bind 2 Zn2+ ions
with a KD of ~2 × 10
6
M2. Utilizing Zn2+-chelate
chromatography, the Zn2+-binding sites on decorin were
localized to the N-terminal sequence of the core protein. Furthermore,
a Zn2+-induced change in the circular dichroism spectrum
for a peptide mimicking the N-terminal domain suggests that
Zn2+ may affect the conformation of this segment of the
core protein. These observations lead us to search for a possible
functional consequence of the putative structural changes arising from
Zn2+ binding to decorin. We now report that
Zn2+ influences the binding of decorin to several matrix
molecules, including collagens, fibronectin, and fibrinogen. Because
the interaction of decorin with fibrinogen to our knowledge has not been previously reported, we decided to characterize the
Zn2+-enhanced binding of decorin to fibrinogen.
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EXPERIMENTAL PROCEDURES |
Expression and Purification of Recombinant Decorin
Glycoforms--
Recombinant decorin with an N-terminal
(His)6 tag was expressed in the mammalian cell-line HT1080
utilizing the vaccinia virus T7 bacteriophage expression system as
previously described (29). A mixture consisting of recombinant decorin
proteoglycan and decorin core protein (lacking the chondroitin sulfate
polysaccharide) was isolated from the cell culture media using
Ni2+-chelate chromatography. A HiTrap chelating column
(Amersham Biosciences) was charged with Ni2+ as per
the manufacturer's instructions and equilibrated with 20 mM Tris, 0.5 M NaCl, 30 mM
imidazole, 0.2% (w/v) CHAPS, pH 8.0. After loading the cell culture
media onto the column, it was washed with 10 column volumes of
equilibration buffer to remove unbound material. Decorin was eluted
with 3 column volumes of 20 mM Tris, 0.5 M
NaCl, 150 mM imidazole, 0.2% (w/v) CHAPS, pH 8.0.
Anion exchange chromatography was utilized to separate decorin
proteoglycan from decorin core protein. Following
Ni2+-chelate chromatography, the decorin preparation was
diluted with 1 volume of 16.8 mM
Na2HPO4, 3.2 mM
NaH2PO4, 0.2% (w/v) CHAPS, pH 8.0, and loaded
onto a Mono-Q column (Amersham Biosciences) equilibrated in 16.8 mM Na2HPO4, 3.2 mM
NaH2PO4, 0.3 M NaCl, 0.2% (w/v)
CHAPS, pH 8.0. Decorin core protein was recovered during washing with
equilibration buffer, whereas the proteoglycan eluted in 16.8 mM Na2HPO4, 3.2 mM
NaH2PO4, 1.5 M NaCl, 0.2% (w/v)
CHAPS, pH 8.0. The purity of these final decorin preparations was
determined by SDS-PAGE followed by Coomassie Brilliant Blue-R
staining and Western blotting using rabbit anti-human decorin
antiserum, PR2. In accord with a previous report, decorin proteoglycan
appeared as a diffuse band centered at an apparent molecular weight of ~90,000; whereas the decorin core protein substituted with
either 2 or 3 N-linked oligosaccharides was observed with
apparent molecular weights of 49,000 or 53,000, respectively (30).
Purification of MBPDcnNTD and DcnNTD--
The maltose-binding
protein (MBP) and a fusion protein consisting of amino acid
residues Asp31-Pro71 of the murine
decorin sequence linked to the C terminus of MBP (MBPDcnNTD) were
expressed and purified from Escherichia coli strain TB1 as
previously described (1). The purity of these recombinant proteins was
examined by SDS-PAGE with Coomassie Brilliant Blue-R staining.
A 45-amino acid residue decorin peptide (DcnNTD), which
includes the murine decorin sequence
Asp31-Pro71 preceded by a GSNG sequence
originating from the vector, was expressed as a glutathione
S-transferase (GST) fusion protein, digested with thrombin,
and isolated by high performance liquid chromatography under acidic,
denaturing conditions as previously described (1). Following the first
high performance liquid chromatography step, the DcnNTD preparation was
diluted with 3 volumes of 20 mM Hepes, 150 mM
NaCl, pH 7.4 (HBS), containing 0.1 mM ZnCl2, to
raise the pH and promote Zn2+ binding. This preparation was
concentrated, loaded onto a C18 column equilibrated in TEAP buffer
(aqueous 0.11% (v/v) phosphoric acid, 0.28% (v/v) triethylamine, pH
6.5) and eluted with a gradient of 0 to 85% acetonitrile in TEAP
buffer. The identity of the final decorin peptide preparation was
confirmed by MALDI-TOF mass spectrometry (Tufts University, Boston, MA).
The results of preliminary experiments suggested that a subpopulation
of the fusion protein (MBPDcnNTD) and the decorin peptide (DcnNTD) was
"inactive" (i.e. incapable of binding to fibrinogen). Because some Zn2+ metalloproteins reportedly are
susceptible to oxidation during purification from an E. coli
lysate, we adapted a strategy to reactivate or charge the decorin
N-terminal domain with Zn2+ ions (31). Following
purification, MBPDcnNTD or DcnNTD was preincubated with excess DTT and
0.1 mM ZnCl2. The reducing agent was added to
break disulfide bonds and make available cysteinyl residues that may
participate in Zn2+ ion coordination. Subsequently, the
reduced protein was dialyzed against HBS, 0.1 mM
ZnCl2 to promote Zn2+ binding to the decorin
N-terminal domain with the removal of the reducing agent.
Biotinylation of Recombinant Proteins--
The decorin
proteoglycan, fusion protein (MBPDcnNTD), or the decorin peptide
(DcnNTD) were labeled with biotin utilizing sulfo-NHS-LC-biotin (Pierce) (32). Next, biotin-labeled proteoglycan was dialyzed against
HBS containing 0.2% (w/v) CHAPS and 0.1 mM
ZnCl2, whereas MBPDcnNTD and DcnNTD were dialyzed similarly
with the omission of CHAPS. Prior to use in experiments, the
concentration of recombinant proteins was calculated from the
absorbance at 280 nm using the calculated extinction coefficients of
19,862 M
1 cm
1 for decorin
proteoglycan, 69,080 M
1 cm
1 for
MBPDcnNTD, and 3,280 M
1 cm
1 for
DcnNTD (32).
Solid-phase Binding Assays--
The binding of decorin to
selected proteins was examined using an enzyme-linked immunosorbent
assay-type binding assay. Microtiter plate wells (Immulon 1B, Dynatech
Labs) were coated overnight at 4 °C with 50 µl of HBS, pH 7.4, containing 1 µg of human fibronectin (kindly provided by Jung Hwa
Kim, Center for Extracellular Matrix Biology, Institute of Bioscience
and Technology, Houston, TX), human fibrinogen (Enzyme Research Labs),
fragment D or fragment E of human fibrinogen (Calbiochem), or chicken
ovalbumin (Sigma), or 10 µg of bovine type I collagen (Cohesion),
type II collagen from bovine nasal septum (Sigma), type IV collagen
from human placenta (Sigma), or type V collagen from human placenta
(Sigma). The next day, wells were washed twice with 150 µl of HBS
containing 0.5% (v/v) Tween 20 (HBST) and in between each of the
following steps to remove unbound proteins from the wells. To block
residual protein-binding sites in the wells after coating, 100 µl of
blocking buffer (1% (w/v) ovalbumin in HBS) was added to each well and incubated for 1 h at ambient temperature. Subsequently, 50 µl of
either 0.45 µM biotin-labeled decorin proteoglycan or
0.25 µM biotin-labeled MBPDcnNTD in HBS, 0.4 mM ZnCl2 was added to each well and allowed to
incubate for 4 h at ambient temperature. For the detection of
biotin-labeled proteins retained in the wells, a streptavidin-alkaline
phosphatase conjugate (Roche Molecular Biochemicals) was diluted
5000-fold in HBS, containing 0.1% (w/v) ovalbumin and 50 µl was
dispensed into each well. After a 30-min incubation at ambient
temperature, the wells were washed with 150 µl of HBST followed by
150 µl of HBS. Finally, the alkaline phosphatase reaction was
initiated by the addition of 100 µl of a freshly prepared 1 mg/ml
p-nitrophenyl phosphate (Sigma) solution in 1.3 M diethanolamine, 1 mM MgCl2, pH
9.8. After 30 min at 37 °C, the absorbance at 405 nm was measured
using a Thermomax microplate reader (Molecular Devices Corp., Menlo
Park, CA). The mean value from triplicate wells was plotted utilizing
Kaleidagraph (Synergy Software, Reading, PA).
Similarly, time-dependent binding experiments were carried
out with the following modifications. At the specified times, the blocking solution was washed from triplicate wells with 150 µl of
HBST, and either 0.45 µM biotin-labeled decorin
proteoglycan, or 0.25, 0.75, or 1.8 µM biotin-labeled
MBPDcnNTD in 50 µl with HBS, 0.1 mM ZnCl2
was added. The detection procedure was conducted as previously mentioned.
To monitor the time-dependent binding of fibrinogen to
immobilized MBPDcnNTD, microtiter plate wells were coated overnight at
4 °C with 1 µg of the fusion protein or MBP in HBS containing 0.1 mM ZnCl2. The wells were blocked and washed
between steps as previously described. At the indicated times, 0.25 µM fibrinogen in HBS was added to each well. Retained
fibrinogen was detected by adding anti-fibrinogen polyclonal antibodies
(Dako) diluted 1:3000 in HBS, 0.1% (w/v) ovalbumin to each well. After
1 h, goat anti-rabbit alkaline phosphatase-conjugated
polyclonal antibodies (Bio-Rad) at a dilution of 1:4000 was added to
each well for 30 min. Color development with p-nitrophenyl
phosphate and data processing were performed as previously mentioned.
To obtain concentration-dependent binding data and an
apparent KD describing the interaction of each
decorin construct with intact fibrinogen or fibrinogen fragment D, a
related protocol was followed. Specifically, increasing concentrations
of biotin-labeled protein in HBS, 0.1 mM ZnCl2
were allowed to interact with immobilized molecules for a period of
time sufficient to reach equilibrium, as determined from the plateau of
each time-dependent binding curve. The mean value of the
absorbance at 405 nm was graphed as a function of protein concentration
and analyzed utilizing the program DynaFit (33). Given that MBPDcnNTD
recognized fragment D in the presence of Zn2+ (Fig. 3), we
analyzed the binding data by fitting it to a single binding site model.
The curve obtained from this approach coincided well with the actual
data points. Because fibrinogen contains two D regions, we assume that
there are 2 identical, independent binding sites for decorin per
fibrinogen molecule, resulting in a KD of
M2 dimension. The best-fit results from DynaFit
were imported into Kaleidagraph and displayed by lines superimposed on
the binding curves.
The concentration-dependent binding of biotin-labeled
decorin peptide to immobilized fusion protein (MBPDcnNTD) or MBP was similarly assessed. To estimate the number of molecules per oligomer and the KD describing decorin self-association, the
program DynaFit along with binding models describing the formation of dimers, tetramers, or hexamers was employed. The results of fitting to
each model appear in the plot of mean absorbance values
versus peptide concentration. We report the
KD with M3 dimension because
a model for tetramer formation appears to best describe the binding.
Inhibition solid-phase binding experiments were also conducted to
compare the binding of different decorin constructs to fibrinogen. After coating and blocking the wells as previously described, immobilized fibrinogen or ovalbumin was preincubated with increasing concentrations of unlabeled decorin proteoglycan, MBPDcnNTD, DcnNTD, or
MBP for 4 h. Next, biotin-labeled decorin proteoglycan or
MBPDcnNTD from a concentrated stock solution was added directly to the
wells to reach a final concentration of 0.45 or 0.25 µM,
respectively, and incubated for 2 h. This period was chosen
because no significant difference in the extent of inhibition was
observed with increased time. The detection steps and data analysis
were performed as described.
Size Exclusion Chromatography--
A Superdex-75 column
(Amersham Biosciences) with a reported separation range of 3 to 70 kDa
was connected to a fast protein liquid chromatography system,
pre-equilibrated in HBS, and calibrated with blue dextran (2000 kDa),
albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa),
ribonuclease (13.7 kDa) (Amersham Biosciences), a 19-amino acid peptide
(1.9 kDa) kindly provided by Sivashankarappa Gurusiddappa, or
tryptophan (204 Da) at a flow rate of 0.5 ml/min. The
Zn2+-charged decorin peptide (DcnNTD) at a concentration
of 47 µM in HBS, 100 µM ZnCl2
was applied in a 100-µl volume to the column pre-equilibrated in HBS,
1 µM ZnCl2. In another experiment, 47 µM DcnNTD was preincubated with 10 mM EDTA,
0.1 mM DTT for 3 h at ambient temperature. Next, the
peptide was dialyzed overnight into HBS, 1 mM EDTA, 0.1 mM DTT and run on a column equilibrated in the same buffer.
Decorin peptide was detected in the eluate by Western blotting, and
asterisks mark these peaks. During a run where buffer lacking the
decorin peptide was injected onto the column, additional peaks were
present and appear to be because of buffer additives.
Fluorescence Polarization--
To observe the interaction of the
decorin peptide (DcnNTD) with fibrinogen in solution, a fluorescence
polarization assay was conducted. To this end, DcnNTD was labeled with
fluorescein as described earlier (34). After purification, the
molecular weight of the fluorescein-tagged peptide, determined by
MALDI-TOF mass spectrometry (Tufts University), was consistent with
that of a singly labeled version of DcnNTD. Next, fluorescein-labeled DcnNTD was resuspended to a final concentration of 20 µM
in HBS, 40 µM ZnCl2. Subsequently,
fluorescein-labeled DcnNTD was diluted to a final concentration of 10 nM and incubated with increasing concentrations of fragment
D or ovalbumin in HBS containing 20 µM ZnCl2
for 2 h in the dark at ambient temperature. For each sample, the
fluorescence polarization signal produced by the excitation of the
fluorescein label at 491 nM was measured at a wavelength of
520 nm utilizing an LS50B luminescence spectrometer (PerkinElmer Life
Sciences) with FL-WinLab software (PerkinElmer Life Sciences). Kaleidagraph was employed to plot binding data as a function of protein
concentration and to fit data by nonlinear regression to Equation 1,
|
(Eq. 1)
|
where
P refers to the fluorescence polarization
signal change,
Pmax denotes the maximum
fluorescence polarization signal change, and KD
corresponds to the equilibrium dissociation constant describing the
binding of fibrinogen fragment D to fluorescein-labeled DcnNTD.
 |
RESULTS |
Zn2+ Enhances Decorin Binding to Matrix
Macromolecules--
In a previous study, we discovered that
Zn2+ binding to decorin could alter the conformation of the
N-terminal segment of the protein. To explore the possibility that the
presence of Zn2+ affects the interaction of decorin with
other ECM molecules, a solid phase binding assay was conducted in the
presence of excess Zn2+ or EDTA (Fig.
1). In these studies, we used a
recombinant form of the decorin proteoglycan presumably retaining its
native conformation because it was secreted by a mammalian cell line
infected with recombinant vaccinia viruses and isolated without the use
of chaotropic or denaturing agents. Decorin was labeled with biotin and
incubated in microtiter wells coated with types I, II, IV, or V
collagen, fibronectin, fibrinogen, or ovalbumin. The results of these
experiments show that in the presence of the chelating agent EDTA, the
amounts of decorin bound to the different ECM proteins were marginally higher than the amounts bound to ovalbumin. However, the addition of
Zn2+ appears to greatly stimulate the binding of decorin to
these ECM proteins, with the possible exception of type II collagen. The inclusion of Zn2+ did not result in a substantial
increase in decorin binding to the control protein ovalbumin. Because
microtiter plate wells coated with fibrinogen bound the highest amount
of decorin in the presence of Zn2+ and because this
interaction previously has not been reported, we chose to further
characterize the binding of decorin to fibrinogen as an example of
Zn2+-dependent decorin binding to ECM
molecules.

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Fig. 1.
Screen for Zn2+-enhanced binding
of decorin to extracellular matrix components. Microtiter plate
wells were coated with 1 µg of fibronectin, fibrinogen, or ovalbumin
(negative control), or 10 µg of types I, II, IV, or V collagen
overnight at 4 °C in HBS. Biotin-labeled decorin proteoglycan
was added to the wells at a final concentration of 0.45 µM in HBS supplemented with either 0.4 mM
ZnCl2 or EDTA for a 4-h incubation. After washing the wells
to remove unbound proteoglycan, bound decorin was detected utilizing a
streptavidin-alkaline phosphatase conjugate and
p-nitrophenyl phosphate as a substrate. Similar data were
obtained in replicate experiments.
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Localization of the Fibrinogen-binding Site on Decorin--
To
identify the domain in decorin that mediates binding to fibrinogen, we
examined different components of decorin for their ability to inhibit
the binding of biotin-labeled decorin proteoglycan to fibrinogen-coated
microtiter wells. Initial experiments showed that the intact
proteoglycan and the core protein were equally efficient inhibitors in
this experiment (data not shown) suggesting that the fibrinogen-binding
site is located to the core protein.
The binding of decorin to fibrinogen shows a strong Zn2+
dependence. Previously, we localized the Zn2+-binding site
to the N-terminal segment of the decorin core protein. Therefore, we
explored the possibility that this domain also contains the
fibrinogen-binding site (Fig.
2A). Initially, we examined a
recombinant form of the N-terminal domain of decorin expressed in
E. coli and presented as a maltose-binding protein fusion
(MBPDcnNTD) for the ability to inhibit the binding of biotin-labeled
decorin proteoglycan to adsorbed fibrinogen. Indeed, MBPDcnNTD inhibits the binding of biotin-labeled decorin proteoglycan in a
concentration-dependent manner. Essentially complete
inhibition was seen at concentrations of MBPDcnNTD greater than or
equal to 2 µM, with half-maximal inhibition occurring at
0.31 µM of the recombinant fusion protein. This
inhibitory activity is comparable with that observed with the unlabeled
proteoglycan, where half-maximal inhibition is achieved with 0.58 µM decorin. In contrast, MBP alone did not interfere with
decorin binding to fibrinogen, suggesting that this inhibitory activity
is specific to the N-terminal domain of decorin.

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Fig. 2.
Location of the fibrinogen-binding site on
decorin. A, wells containing adsorbed fibrinogen or
ovalbumin ( ) were preincubated with increasing concentrations of
unlabeled decorin proteoglycan, DcnPg ( ), MBPDcnNTD ( ), or MBP
( ) in HBS, 100 µM ZnCl2, pH 7.4. B, microtiter plate wells coated with fibrinogen ( ) or
ovalbumin ( ) were preincubated with increasing concentrations of
unlabeled decorin peptide (DcnNTD) in HBS, 100 µM
ZnCl2, pH 7.4. In both A and B,
biotin-labeled decorin proteoglycan was added to a final concentration
of 0.45 µM. The binding of biotinylated molecules was
detected as previously described. These data are representative of
replicate experiments.
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|
To confirm that the fibrinogen binding activity is because of the
decorin-derived component of MBPDcnNTD, we examined the inhibitory
activity of the decorin N-terminal domain (DcnNTD) in the absence
of a fusion partner (Fig. 2B). We expressed the 45-amino
acid residue DcnNTD as a recombinant GST fusion protein with a thrombin
cleavage site located in a linker peptide between GST and DcnNTD. After
thrombin digestion of GST-DcnNTD, DcnNTD was initially purified under
reducing conditions and subsequently isolated in the presence of
Zn2+. This Zn2+-charged peptide was then
examined for the ability to inhibit the binding of biotin-labeled
proteoglycan to fibrinogen. We observed dose-dependent,
complete inhibition of decorin proteoglycan binding to fibrinogen by
the isolated decorin peptide, with half-maximal inhibition observed
above 1 µM DcnNTD and complete inhibition achieved
at 5 µM DcnNTD. From these experiments, we conclude
that the fibrinogen-binding site on decorin is located in the
N-terminal domain of the core protein. In fact, we subsequently showed
in direct binding assays that DcnNTD binds to fibrinogen (see below).
The Decorin-binding Site on Fibrinogen--
To localize the
decorin-binding site on fibrinogen, we first conducted a solid-phase
binding assay to determine whether biotin-labeled decorin proteoglycan
or MBPDcnNTD recognizes the plasmin generated fibrinogen fragments D or
E (Fig. 3). We found that in the presence of Zn2+, both decorin proteins bound to microtiter wells
coated with intact fibrinogen or fragment D but not to the E fragment
or the control protein ovalbumin. In the presence of EDTA, the binding of decorin to all four proteins was minimal. Hence, in a
Zn2+-dependent fashion, decorin appears to
specifically recognize the globular D regions of fibrinogen, mainly
consisting of amino acid residues 111-197 of the
chain, 134-461
of the
chain, and 88-406 of the
chain (35, 36).

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Fig. 3.
Location of the decorin-binding domain of
fibrinogen. Intact fibrinogen, fragment D, fragment E, or
ovalbumin was coated on microtiter plate wells and allowed to incubate
with either 0.45 µM biotin-labeled decorin proteoglycan,
DcnPg, or 0.25 µM biotin-labeled MBPDcnNTD in HBS, pH
7.4, containing either 0.4 mM ZnCl2 or EDTA.
Biotin-labeled protein was detected as previously described. These data
are congruent with replicate experiments.
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|
Characterization of the Decorin-Fibrinogen Interaction--
To
gain insight on the mechanism of decorin-fibrinogen recognition, we
initially wanted to determine the KD for the interaction of decorin proteoglycan with fibrinogen (Fig.
4A). In the presence of
Zn2+, fibrinogen- or ovalbumin-coated microtiter wells were
incubated with increasing concentrations of the biotin-labeled
proteoglycan until equilibrium was reached. Minimal amounts of decorin
were retained in the ovalbumin containing wells. However, decorin bound fibrinogen in a concentration-dependent, saturable manner.
Assuming that there are two identical, independent decorin-binding
sites on fibrinogen, we analyzed these data utilizing the program
DynaFit and obtained an apparent KD of 6.8 × 10
7 M2.

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Fig. 4.
Concentration-dependent binding
of decorin to immobilized fibrinogen. A, increasing
concentrations of biotin-labeled decorin proteoglycan were incubated
with microtiter plate wells coated with fibrinogen ( ) or ovalbumin
( ). B, increasing concentrations of a biotin-labeled
decorin peptide (DcnNTD) were incubated with adsorbed fibrinogen ( )
or ovalbumin ( ). C, increasing concentrations of
biotin-labeled MBPDcnNTD were incubated with adsorbed fibrinogen ( ),
fragment D ( ), or ovalbumin ( ). Biotin-labeled proteins were
detected as previously described. Values of nonspecific binding to the
negative control, ovalbumin, were subtracted from the binding data
prior to analysis. Superimposed on each data set is the fit generated
utilizing the program DynaFit (33) to estimate KD
values for the interactions taking place in HBS, 100 µM
ZnCl2, pH 7.4. Similar results were observed in replicate
experiments.
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|
In a previous experiment, we showed that the decorin peptide, DcnNTD,
completely inhibits decorin proteoglycan binding to fibrinogen. To
directly observe the interaction of DcnNTD with fibrinogen, increasing
concentrations of biotin-labeled decorin peptide were allowed to
equilibrate with fibrinogen- or ovalbumin-coated wells in the presence
of Zn2+ (Fig. 4B). Low levels of DcnNTD were
detected in the ovalbumin containing wells. In contrast, the decorin
peptide binding to fibrinogen was concentration-dependent
and saturable with an apparent KD of 3.0 × 10
7 M2.
We also demonstrated that MBPDcnNTD recognizes both fibrinogen and
fragment D in a Zn2+-dependent fashion. In this
experiment, the concentration-dependent binding of
biotin-labeled MBPDcnNTD to adsorbed fibrinogen, fragment D, or
ovalbumin was observed under equilibrium conditions with Zn2+ present (Fig. 4C). MBPDcnNTD was minimally
retained by wells coated with ovalbumin. However, MBPDcnNTD
incrementally bound to both fibrinogen and fragment D in accord with
increasing MBPDcnNTD concentration until a plateau was reached. Based
on the analysis of these data utilizing DynaFit, an apparent
KD of 1.2 × 10
7
M2 describes MBPDcnNTD binding to
fibrinogen and an apparent KD of 9.0 × 10
8 M characterizes MBPDcnNTD recognition
of fragment D.
To further characterize the binding of the decorin N-terminal domain to
fibrinogen fragment D, we conducted fluorescence polarization experiments. A fluorescein-labeled version of the decorin peptide was
mixed with increasing concentrations of fragment D or ovalbumin in the
presence of Zn2+ (Fig. 5).
Little or no change in the fluorescence polarization signal was
observed for the peptide in the presence of ovalbumin. Instead, we
observed an increase in the fluorescence polarization signal of the
fluorescein-peptide conjugate with increasing concentrations of
fragment D until a maximum was approached. An estimated
KD of 1.7 × 10
6 M
describes the interaction of the decorin N-terminal domain with
fibrinogen fragment D in solution.

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Fig. 5.
Concentration-dependent
interaction of fibrinogen fragment D with DcnNTD in solution.
Increasing concentrations of fibrinogen fragment D ( ) or ovalbumin
( ) were allowed to incubate with the Zn2+-charged,
fluorescein-labeled decorin peptide in HBS, 20 µM
ZnCl2, pH 7.4. The best fit generated utilizing the program
Kaleidagraph overlays the D fragment-peptide binding data.
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To further characterize the interaction of decorin with fibrinogen, we
examined the time-dependent binding of decorin proteoglycan to immobilized fibrinogen in the presence of Zn2+ (Fig.
6A). Biotin-labeled
proteoglycan at a concentration of 0.45 µM was incubated
with microtiter plate wells coated with fibrinogen for increasing
lengths of time and required ~4 h to reach saturation. Although the
binding was slow, decorin does appear to specifically recognize
fibrinogen because minimal amounts of the proteoglycan were retained in
the ovalbumin containing wells.

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Fig. 6.
Time-dependent binding
experiments. A, biotin-labeled decorin proteoglycan at
a concentration of 0.45 µM was incubated in microtiter
plate wells coated with fibrinogen ( ) or ovalbumin ( ) for
increasing lengths of time in HBS, 100 µM
ZnCl2, pH 7.4. B, biotin-labeled MBPDcnNTD at a
concentration of 0.25 ( ), 0.75 ( ), or 1.8 µM ( )
was incubated with microtiter wells coated with fibrinogen or ovalbumin
( ) for different lengths of time in HBS, 100 µM
ZnCl2, pH 7.4. In both A and B,
biotin-labeled protein was detected as previously described.
C, fibrinogen at a concentration of 0.25 µM
was incubated in microtiter wells coated with either MBPDcnNTD ( ) or
MBP ( ) for various lengths of time in HBS, 20 µM
ZnCl2, pH 7.4. Bound fibrinogen was detected utilizing
anti-fibrinogen polyclonal antibodies followed by goat anti-rabbit
alkaline phosphatase-conjugated polyclonal antibodies.
Antibody-fibrinogen complexes bound to the immobilized molecules were
detected using p-nitrophenyl phosphate as a substrate. These
data are representative of replicate experiments.
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Next, the time-dependent binding of MBPDcnNTD to
fibrinogen- or ovalbumin-coated wells was explored utilizing several
concentrations of the recombinant fusion protein (Fig. 6B).
Alternatively, the time required for MBPDcnNTD to saturate the
decorin-binding sites depended on the concentration of the fusion
protein. For example, a maximum level of binding was observed about
1.5 h after the addition of 0.25 µM MBPDcnNTD to
fibrinogen containing wells. However, at concentrations of 0.75 and 1.8 µM MBPDcnNTD, the decorin-binding sites became saturated
after only 30 min. Binding to ovalbumin over time was minimal at all
three concentrations of MBPDcnNTD tested.
In an enzyme-linked immunosorbent assay-type experiment, we also
monitored the binding of 0.25 µM fibrinogen to wells
coated with either MBPDcnNTD or MBP over time (Fig. 6C).
Fibrinogen slowly accumulated on the MBP-coated surface in what we
think is a nonspecific interaction. On the other hand, fibrinogen
binding to MBPDcnNTD reached a plateau already after ~30 min. The
results of these time-dependent binding experiments lead us
to hypothesize that decorin-fibrinogen interaction involves more than
one step.
Zn2+ Promotes the Self-association of
Decorin--
Earlier studies by Liu et al. (37) suggested
that biglycan proteoglycan forms dimers in the presence of EDTA and
hexamers in the presence of Zn2+. Previously, we reported
that both biglycan and decorin proteoglycans are Zn2+
metalloproteins capable of binding 2 Zn2+ ions with similar
affinity at the N-terminal region of the core protein (1). Initially,
we examined the possibility that the 45-amino acid decorin peptide,
DcnNTD, self-associates in a Zn2+-dependent
fashion by gel filtration chromatography (Fig.
7). The peptide at a concentration of 47 µM in the presence of either Zn2+ or EDTA
with DTT was applied to a Superdex-75 gel filtration column with a
reported separation range of 3 to 70 kDa. These chromatograms show that
the decorin peptide elutes in a significantly lower volume in the
presence of Zn2+ than in the presence of EDTA with DTT. The
Zn2+-charged peptide elutes in a broad peak located at 8.5 ml, the same volume required to elute albumin (Table
I). However, in the presence of EDTA with
DTT, DcnNTD (4.88 kDa) elutes in 13.5 ml, which is intermediate of the
elution volume of ribonuclease (13.7 kDa) and for a 19-amino acid
peptide standard (1.9 kDa). These results demonstrate that
Zn2+ promotes the self-association of the decorin
peptide.

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Fig. 7.
Gel filtration chromatography of DcnNTD in
the presence of either Zn2+ or EDTA with
DTT. A, Zn2+-charged decorin peptide
(DcnNTD) at an initial concentration of 47 µM was loaded
onto a Superdex-75 column and eluted in HBS, 1 µM
ZnCl2, pH 7.4. B, the Zn2+-charged
decorin peptide was preincubated with 10 mM EDTA, 0.1 mM DTT, pH 7.4, and dialyzed overnight against the running
buffer consisting of HBS, 1 mM EDTA, 0.1 mM
DTT, pH 7.4, prior to chromatography. C, gel filtration
column calibration standards were loaded and run individually or
pairwise as per the manufacturer's instructions. The characteristics
of each standard are reported in Table I.
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To quantitatively evaluate the Zn2+-dependent
self-association of the decorin N-terminal peptide, increasing
concentrations of biotin-labeled DcnNTD were incubated with MBPDcnNTD,
MBP, or ovalbumin adsorbed to microtiter plate wells in the presence of Zn2+ (Fig. 8). The decorin
N-terminal domain appears to specifically recognize itself when
presented as a fusion to MBP, as the peptide was only minimally
retained in MBP- or ovalbumin-coated wells. The amount of decorin
peptide bound to the MBPDcnNTD-coated wells increased with
concentration, reached a maximum at ~10 µM peptide and
could not be increased by the addition of more peptide. To estimate the
KD for the decorin N-terminal domain binding to
MBPDcnNTD as well as the number of molecules per oligomer, the data
were analyzed utilizing the program DynaFit to test models for the
formation of dimers, tetramers, or hexamers. The curve that appears
most coincident with the binding data is that describing tetramer
formation with an apparent KD of 6.5 × 10
7 M3. These results suggest
that the decorin N-terminal domain self-associates to form an oligomer
containing a defined number of monomeric components.

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Fig. 8.
Concentration-dependent binding
of DcnNTD to MBPDcnNTD. Increasing concentrations of the
biotin-labeled decorin peptide (DcnNTD) in HBS, 100 µM
ZnCl2, pH 7.4, were incubated for 2 h with microtiter
plate wells coated with MBPDcnNTD ( ), MBP ( ), or ovalbumin ( ).
Retained biotin-labeled peptide was detected as described earlier. The
curves superimposed on the data represent the best-fit
results utilizing the program DynaFit with a dimer (- - -),
tetramer ( ) or hexamer (- - -) model. Replicate experiments yielded
similar data.
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DISCUSSION |
We show here that Zn2+ promotes the interaction of
decorin proteoglycan with fibrinogen. Fibrinogen circulates in blood as
a soluble 340-kDa glycoprotein at a concentration of ~9
µM (38). During the final stage of the clotting cascade,
thrombin cleaves fibrinopeptides from the central E region of
fibrinogen to yield fibrin that rapidly assembles into a provisional
matrix. When vascular damage causes the extravasation of blood,
fibrinogen comes in contact with extracellular matrix components,
including decorin exhibiting a broad tissue distribution (39, 40). The possibility that decorin and fibrinogen may co-localize in the extracellular milieu during wound repair coupled with our interest in
the role of Zn2+ as a regulator of decorin structure and
function lead us to further characterize the decorin-fibrinogen interaction.
The domains that mediate decorin-fibrinogen binding were deduced from
our analysis of the results of solid phase and fluorescence polarization binding assays. We determined that recombinant decorin proteoglycan, expressed by mammalian cells using vaccinia virus as well
as recombinant forms of the N-terminal domain produced in E. coli exhibit equivalent apparent KD values for binding to fibrinogen-coated wells. Thus, additional decorin core protein segments beyond the N-terminal domain do not appear to be
required for stable binding to fibrinogen or fragment D. MBPDcnNTD bound to fragment D-coated wells with an apparent KD of 9.0 × 10
8 M. However, fragment D
binding to DcnNTD in solution approached saturation with an estimated
KD of 1.7 × 10
6 M.
These divergent observations could be a consequence of the complexity
of the molecular interactions under study. For instance, the
possibility that several multimeric forms of the decorin peptide are
present and that they may differ in affinity for the D domain of
fibrinogen has not been incorporated into our analyses. In addition,
the conformation of the fibrinogen D domain reportedly may be altered
by immobilization (41). Hence, the apparent KD values depend on the experimental conditions of the assay.
Nevertheless, we show here that the N-terminal Zn2+-binding
domain of decorin interacts with the D region of fibrinogen with a
reasonable affinity.
Decorin and biglycan are self-associating Zn2+
metalloproteins. A previous report showed that biglycan proteoglycan is
hexameric in the presence of Zn2+ and dimeric in the
presence of EDTA (37). Subsequently, we discovered that both decorin
and biglycan bind 2 Zn2+ ions with the N-terminal segment
of the core protein (1). We now propose that the N-terminal domain of
decorin core protein mediates the
Zn2+-dependent formation of oligomers. Our gel
filtration chromatography experiments show that
Zn2+-charged DcnNTD elutes as a broad peak, possibly
indicating the coexistence of several oligomeric forms in the presence
of Zn2+. Similarly, we observed several overlapping peaks
during gel filtration chromatography of MBPDcnNTD in the presence of
Zn2+ (data not shown). We also noticed that the peak
elution volume of DcnNTD shifts to slightly higher volume as we
decrease the concentration of peptide loaded, suggesting a dissociation
of oligomers (data not shown). Moreover, DcnNTD oligomers are
effectively dissociated by incubation with excess EDTA. Liu and
colleagues (37) showed that the biglycan core protein, lacking the 2 chondroitin sulfate polysaccharides normally attached to the N-terminal
domain, formed aggregates in the presence of Zn2+. We did
not detect DcnNTD or MBPDcnNTD in the column void volume and observed
saturation binding of DcnNTD to MBPDcnNTD-coated wells. Taken together,
these results indicate that the N-terminal domain can form oligomeric
structures with defined compositions.
The mechanism(s) whereby Zn2+ promotes the binding of
decorin to a variety of other matrix components including types I, IV, and V collagen, fibronectin, and fibrinogen remains to be determined. In some cases, perhaps increased decorin binding in the presence of
Zn2+ is attributable to the formation of oligomeric forms
that bind to the adsorbed matrix proteins; whereas reduced apparent
binding in the presence of EDTA may reflect bound monomeric forms.
Alternatively, Zn2+ may play a role in the folding and
structure of the decorin N-terminal domain. In this case, the
conformation of the N-terminal domain formed in the presence of
Zn2+ exhibits a higher affinity for some matrix molecules,
such as fibrinogen, than the conformation of the N-terminal domain in the presence of EDTA. Our study illustrates the significance of Zn2+ as a modulator of decorin structure and function. This
relationship should be considered in future efforts to elucidate the
biological function(s) of decorin and perhaps other SLRPs.
The biological consequence(s) of a putative decorin-fibrinogen
interaction is unclear at this time. To determine whether these molecules can interact in a physiological setting, we passed human plasma over a decorin matrix composed of MBPDcnNTD covalently attached
to Sepharose 4B. SDS-PAGE and Western blots showed that both fibrinogen
and fibronectin were retained by the decorin matrix during extensive
washing and eluted with EDTA (data not shown). Retention of fibronectin
by the decorin N-terminal domain is interesting because a previous
study postulated a second fibronectin-binding site on decorin, in
addition to the previously reported NKISK site of LRR 1 (42).
Fibrinogen, in the form of fibrin, and fibronectin represent dominant
protein components of the provisional matrix formed during the initial
stage of wound repair (43). When present at sites of wound repair,
decorin might participate in organizing the structure of the
provisional matrix, thus one could speculate that decorin plays a role
in the wound healing process.