Decorin Is a Zn2+ Metalloprotein*
Vivian W-C
Yang
§,
Steven R.
LaBrenz
,
Lawrence C.
Rosenberg¶,
David
McQuillan§, and
Magnus
Höök§
**
From the
Graduate School of Biomedical Sciences,
University of Texas, Houston Health Science Center, Houston, Texas
77030, the § Center for Extracellular Matrix Biology, Albert
B. Alkek Institute of Biosciences and Technology, the
Department
of Biochemistry and Biophysics, Texas A&M University, Houston, Texas
77030, and the ¶ Orthopaedic Research Laboratory, Montefiore
Medical Center, Bronx, New York 10467
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ABSTRACT |
Decorin is ubiquitously distributed in the
extracellular matrix of mammals and a member of the proteoglycan family
characterized by a core protein dominated by leucine-rich repeat
motifs. We show here that decorin extracted from bovine tissues under
denaturing conditions or produced in recombinant "native" form by
cultured mammalian cells has a high affinity for Zn2+
as demonstrated by equilibrium dialyses. The Zn2+-binding
sites are localized to the N-terminal domain of the core protein that
contains 4 Cys residues in a spacing reminiscent of a zinc finger. A
recombinant 41-amino acid long peptide representing the N-terminal
domain of decorin has full Zn2+ binding activity and binds
two Zn2+ ions with an average KD of
3 × 10
7 M. Binding of Zn2+
to this peptide results in a change in secondary structure as shown by
circular dichroism spectroscopy. Biglycan, a proteoglycan that is
structurally closely related to decorin contains a similar high
affinity Zn2+-binding segment, whereas the structurally
more distantly related proteoglycans, epiphycan and osteoglycin, do not
bind Zn2+ with high affinity.
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INTRODUCTION |
Decorin, a small chondroitin/dermatan sulfate proteoglycan, is
found in the extracellular matrix of a variety of tissues such as skin
(1-3), cartilage (4, 5), and bone (6, 7). This proteoglycan is
composed of a 40-kDa core protein and one glycosaminoglycan chain
attached to a serine residue in the N-terminal part of the protein. The
decorin core protein is dominated by a central region composed of 10 leucine-rich repeat units. Each unit contains 21-26 amino acid
residues and is proposed to adopt a characteristic
-helix/
-sheet
folding pattern (8, 9). The C- and N-terminal regions of the core
protein are believed to form globular structures stabilized by
disulfide bonds between sets of cysteine residues. Several
proteoglycans have core proteins of similar size and structural
organization. These related molecules are considered to form a family
called small leucine-rich proteoglycans (SLRP)1 (9, 10). The family
of SLRPs include decorin, biglycan, and epiphycan, all of which contain
chondroitin/dermatan sulfate chains attached to the N-terminal domain
of the core protein and fibromodulin, lumican, keratocan, PRELP, and
osteoglycin which often have keratan sulfate linked to asparagine
residues in the central region of the core protein. The extracellular
matrix glycoprotein chondroadherin has a structural organization
similar to the core proteins of the SLRPs but has not been shown to be
substituted with glycosaminoglycan chains (11). The biological
importance of the different SLRPs is unclear. In vitro
binding studies have shown that decorin, biglycan, and fibromodulin can
interact with several types of collagen (12-16) and different SLRPs
are believed to be important regulators of collagen fibrillogenesis. In
support of this hypothesis, a decorin-deficient mouse was found to have fragile skin with an abnormal organization of collagen fibers (17). The
phenotype appears to be largely restricted to the skin, perhaps
suggesting that other SLRPs have similar functions and may fulfill this
role in other collagenous tissues. In fact, a lumican-deficient mouse
also exhibited abnormal collagen fibers both in the skin and cornea
(18).
Decorin may also affect the production of other extracellular matrix
components by regulating the activity of transforming growth factor-
(19, 20). Additionally, decorin can modulate the interactions of matrix
molecules such as fibronectin with cells (21-23). These observations
suggest that decorin and perhaps other SLRPs regulate at several levels
the production and assembly of the extracellular matrix and hence the
remodeling of connective tissue.
Zinc, a divalent cation, is one of the essential trace elements for
eukaryotic organisms. Zinc ions play a key role in biological processes
by being directly involved in enzyme catalysis or by binding to
specific sites in a protein to stabilize the conformations which are of
importance to the function of the protein (24, 25). In the
extracellular matrix, zinc is required for the activity of matrix
metalloproteases which are responsible for the degradation of
structural extracellular matrix components (26). Breakdown and
remodeling of the ECM occur in normal embryo development, wound
healing, and many pathological processes such as cancer, arthritis, and
osteoporosis (26, 27). Structural extracellular matrix molecules such
as laminin (29), link protein (30), nidogen (31), and COMP (32) have
been shown to bind zinc ions. The Zn2+-binding sites have
been located to subdomains of some of these proteins (31, 33).
In a previous study, biglycan was shown to self-associate in the
presence of zinc ions and shown to bind Zn2+,
Ni2+, and Cu2+ in metal chelate affinity
chromatography studies (34). The binding affinity and binding domain,
however, have not been reported. In this study, we demonstrate that
decorin is a metalloprotein and binds two zinc ions per core protein
with an average KD of ~1 µM. The
Zn2+-binding domain is mapped to the N-terminal region in
the decorin core protein and a short (41 amino acid long) peptide is
shown to have full Zn2+ binding activity. Furthermore, the
Zn2+ binding activity of the corresponding segments in the
structurally related core proteins of biglycan, epiphycan, and
osteoglycin are analyzed.
 |
EXPERIMENTAL PROCEDURES |
Preparation of Decorin Proteoglycans--
Decorin was extracted
and purified from bovine skin under denaturing conditions as described
previously (3). Intact recombinant decorin was produced in HT1080 cells
using a vaccinia virus-based expression system. The recombinant virus
construct contains a segment encoding a N-terminal polyhistidine tag, a
Factor Xa cleavage site, and the mature human decorin core protein
starting with an Asp residue found at position 31 of the full-length
human sequence (35). The polyhistidine tag in the recombinant protein
allows for its efficient purification by Ni2+ chelating
chromatography. The resulting decorin preparation contains a mixture of
proteoglycan and core protein forms. The details of this system have
been described earlier (35, 36). For the studies described here, the
polyhistidine tag was removed from the recombinant decorin by digestion
with Factor Xa (Pierce, Rockford, IL) followed by reapplication of the
digestion mixture to a Ni2+ charged column. The final
product of cleaved recombinant decorin was collected in the
"flow-through" fractions whereas the polyhistidine tag and
uncleaved protein were retained on the column.
Recombinant Decorin Core Protein Fragments--
A series of
recombinant decorin core protein fragments were produced in
Escherichia coli. A mouse embryo cDNA library
(CLONTECH, Palo Alto, CA) was used as a template
along with appropriate primers (Table I)
to PCR amplify the decorin cDNA segments. Conditions for PCR were
94 °C for 7 min, followed by 35 cycles involving 94 °C for 1 min,
50 °C for 2 min, and 72 °C for 3 min. The individual segments
were purified, cleaved with appropriate restriction enzymes, and
ligated into the pMAL-p2 expression vector (New England Biolabs, Beverly, MA) which contain a segment encoding the maltose-binding protein (MBP) at the 5' end of the expression cassette. The cDNA fragments (MD and MD3) generated with the DCNR2 primer which contains a
EcoRI site at 3' end were first subcloned into the
pBluscript SK± vector (Stratagene, La Jolla, CA) through
BamHI and EcoRI sites. Subsequently, the
MD/pBluscript and MD3/pBluscript constructs were digested with
BamHI and HindIII and then ligated into the pMAL-p2 expression vector. To produce MBP as a control protein without
any segment of decorin fused to it, a modified pMAL-p2 vector
containing a stop codon and a short polylinker was constructed. The
complementary oligonucleotides, MBPF (5'-gatcctgatctagagcatgcctgca-3') and MBPR (5'-ggcatgctctagatcag-3') were obtained and allowed to form a
double-stranded oligonucleotide containing a cohesive BamHI site at the 5'-end followed by a stop codon, XbaI,
SphI, and PstI sites. The original pMAL-p2 vector
digested with BamHI and PstI was then ligated
with that double-stranded oligonucleotide. The newly introduced
SphI site which is not present in the original pMAL-p2
vector was used in a restriction enzyme digestion screen to isolate
clones carrying the engineered plasmid. The sequences of all
decorin/pMAL-p2 constructs isolated from selected clones were confirmed by DNA sequencing (Department of Veterinary
Pathobiology, Texas A&M University, College Station, TX).
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Table I
Primers for PCR amplification of different decorin and biglycan
fragments (A) and the corresponding primer pairs for expressed protein
subdomains (B)
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Expression and Purification of Recombinant Decorin Core Protein
Subdomains--
Previously, a method developed for the production of
full-length bovine decorin core protein using a pMAL expression system has been reported (37). This procedure involved solubilization and
refolding of protein recovered from inclusion bodies. In our case, we
obtained significant amounts of soluble recombinant protein from all
constructs and we did not attempt to solubilize protein from inclusion bodies.
The expression vector constructs were used to transform E. coli strain TB1. The procedure adopted for the expression and
purification of the MBP-decorin fusion protein was based on the manual
provided by New England Biolabs. In brief, a flask containing 950 ml of Lennox LB (Sigma) was inoculated with 50 ml of a TB1/MBP-decorin overnight culture and grown on a platform shaker for 3~4 h at 37 °C when the culture reached an OD600 of 0.6-0.8.
Expression of fusion protein was induced by adding
isopropyl-1-thio-
-galactopyranoside (Life Technologies, Inc.,
Gaithersburg, MD) to the culture at a final concentration of 0.2 mM and incubation was continued for another 3 h. The
bacterial cells were harvested by centrifugation at 4500 rpm for 20 min. The cell pellet was resuspended in Buffer A (20 mM
Tris, 200 mM NaCl, pH 8.0) to a final volume of 10 ml/liter of culture and frozen at
80 °C for a minimum of 18 h. To
purify the fusion proteins, the cells were thawed and lysed using a
French press. The lysate was cleared by centrifugation at 40,000 rpm for 20 min and the supernatant was applied to a 5-ml amylose affinity column (New England Biolabs, Beverly, MA) equilibrated with 25 ml of
Buffer A. The column was washed with 50 ml of Buffer A and the
MBP-decorin fusion protein was eluted by Buffer A containing 10 mM maltose. The purity of the fusion protein was >70% as
judged by SDS-PAGE and the yield was 5-10 mg/liter of culture. To
improve the purity of fusion proteins, an additional purification step was required. Ion exchange chromatography was used for further purifying MBP-MD2 and MBP-MD3 protein. The proteins were dialyzed into
a buffer composed of 25 mM NaCl, 20 mM Tris, pH
8.0, and applied to a column of Q-Sepharose (Pharmacia Biotech,
Piscataway, NJ) equilibrated with the same buffer. The protein was
subsequently eluted with a NaCl gradient (25-500 mM) in 20 mM Tris, pH 8.0. Metal ion chelating chromatography on
iminodiacetic acid immobilized Sepharose 6B resin (Sigma) charged with
Zn2+ was used to further purify MBP-MD, MBP-MD1, and
MBP-MD4. The procedure used for charging the column with
Zn2+ ions is described in the following section. The
purified recombinant proteins were analyzed on SDS-PAGE and Western
blot using two polyclonal antibodies that were raised against synthetic
peptides corresponding to amino acid residues 31-47 and 309-326 of
the mouse decorin core proteins,
respectively.2 The protein
concentrations were determined based on the absorbance at
A280 and the calculated molar extinction
coefficient of the different protein constructs (38).
Production and Purification of a N-terminal Decorin Peptide
(MD4)--
To produce a large quantity of MD4 peptide, we used a
pGEX-2T expression vector (Pharmacia Biotech Inc., Piscataway, NJ). This MD4/pGEX-2T construct encodes glutathione S-transferase
(GST) followed a thrombin cleavage site and the MD4 fragment. The
cDNA fragment encoding MD4 was obtained by PCR using the mouse
embryo cDNA library and primers DCNF9 and DCNR19 as described above
(Table IA). The resulting PCR fragment was cleaved by BamHI
and PstI, purified, and subcloned into pBluscript. The
isolated MD4/pBluscript plasmid was digested with BamHI and
EcoRI and ligated into the pGEX-2T expression vector. The
recombinant MD4/pGEX-2T plasmid was then transformed into E. coli strain BL-21 (Stratagene, La Jolla, CA). An isolated clone
containing the MD4/pGEX-2T plasmid was used after the plasmid had been
analyzed by restriction enzyme digestion and sequencing. To express the
GST-MD4 fusion protein, flasks containing 950 ml of Lennox LB were
inoculated with 15 ml of a BL21/GST-MD4 overnight culture and incubated
for 3-4 h when the culture reached an A600 of
0.4-0.5. Recombinant protein expression was induced by adding
isopropyl-1-thio-
-galactopyranoside to the culture at a final
concentration of 0.2 mM. Bacterial cells were harvested
3 h later by centrifugation at 4500 rpm for 20 min. The cell
pellet was resuspended in phosphate-buffered saline (8.4 mM
Na2HPO4, 1.9 mM
NaH2PO4, 150 mM NaCl, pH 7.4),
supplemented with 5 mM EDTA and adjusting the pH 7.5 to a
final volume of 10 ml/liter of culture and frozen at
80 °C for a
minimum of 18 h. The cells were thawed and lysed using a French
press. One ml of 10% Triton X-100 (Sigma) was added to the cell lysate
and mixed until homogeneous. The cell homogenate was centrifuged and
filtered through a 0.45-µm membrane to remove cell debris.
The GST-MD4 fusion protein present in the filtered supernatant was
purified by affinity chromatography on a 10-ml column of glutathione-agarose (Sigma) equilibrated with 50 ml of Buffer B
(phosphate-buffered saline with 1 mM EDTA, pH 7.5). The
column was washed with 75 ml of Buffer B and the GST-MD4 protein was eluted from the column with 20 ml of Buffer C (50 mM Tris,
10 mM glutathione, pH 7.5). The purity of GST-MD4 in the
eluate was >90%, as judged by SDS-PAGE and the yield was 25-30
mg/liter of culture.
To isolate the MD4 peptide, the fusion protein was cleaved with
thrombin. The pH and the concentration of NaCl of the eluant containing
GST-MD4 was adjusted to pH 8.3, 0.15 M NaCl using 1 M NaOH and 3 M NaCl stock solutions. Bovine
thrombin (Sigma) was added to give a 1/40 (w/w) enzyme/substrate ratio.
The digestion was allowed to proceed overnight at room temperature.
Subsequently,
-mercaptoethanol was added to the incubation mixture
to a final concentration of 1% (v/v) and the solution was adjusted to
pH 9.5 by addition of 1 M NaOH. The solution was incubated
at 37 °C for 30 min and filtered through a 0.45-µm membrane to
remove any particulate material. The MD4 peptide was purified on a
Waters 25 × 200-mm RCM semi-preparative C18 HPLC column and
eluted with a gradient of 28 to 36% solvent B (95% acetonitrile, 5%
H2O, 0.1% trifluoroacetic acid) in solvent A (95%
H2O, 5% acetonitrile, 0.1% trifluoroacetic acid) over 10 min at a flow rate of 25 ml min
1.
The purity of the peptide was monitored by running a three-layer
Tricine-SDS-PAGE gel consisting of a 15% acrylamide slab, 10%
acrylamide spacer, and a 3.5% acrylamide stacker (39). The identity of
the peptide was confirmed by matrix-assisted laser desorption
ionization-mass spectroscopy (The Center for Analytical Chemistry,
University of Texas in Houston, Houston, TX).
Biglycan Preparations--
Biglycan isolated from bovine
articular cartilage was extracted and purified under denaturing
conditions as described previously (3). Recombinant biglycan containing
a N-terminal polyhistidine tag was produced in HT1080 cells using the
vaccinia virus expression system as described (36). The polyhistidine
tag was removed and the recombinant proteoglycan was re-isolated as
described above. The preparation of recombinant biglycan contains a
mixture of proteoglycan and core protein forms (36). The N-terminal segment of the biglycan core protein was produced as a recombinant MBP
fusion protein in E. coli. The encoded fusion protein
MBP-MB-N is composed of the maltose-binding protein followed by a
biglycan peptide, corresponding to amino acid residues 38 to 77. The
primers BGNF11 and BGNR13 (Table I) and the mouse embryo cDNA
library were used to PCR amplify an appropriate cDNA fragment. The
resulting PCR product was cleaved, purified, and ligated into the
pMAL-p2 vector. Protein expression and purification protocols were the same as those described above in the preparation of MBP-decorin fusion proteins.
Dialysis of the Protein--
In preparation for Zn2+
binding experiments, all proteins were dialyzed against Buffer D (20 mM Tris-HCl, 150 mM NaCl, pH 7.0) supplemented
with 5 mM EDTA (Sigma) to remove possibly contaminating divalent metal ions and subsequently dialyzed against Buffer D (without
EDTA) with three changes. Buffer D was shown to contain less than
5 × 10
8 M Zn2+ ions as
analyzed by 4-(2-pyridylazo)resorcinol (PAR) (Sigma) complex formation
(40, 41).
Zn2+-chelating Affinity Column Chromatography--
A
resin composed of iminodiacetic acid-immobilized Sepharose 6B (Sigma)
was charged with 10 volumes of 2 mg/ml ZnCl2 (Sigma) in
deionized water. The resin was then washed with 5 volumes of Buffer E
(20 mM Tris-HCl, 150 mM NaCl, pH 8). The
charged Sepharose 6B was packed on top of an equal amount of uncharged
iminodiacetic acid-Sepharose 6B that has been pre-equilibrated in
Buffer E. The purpose of having the uncharged resin on the bottom of
the column is to trap zinc ions released from the charged resin during the experiment. The dialyzed test sample was applied to the column. The
column was washed with excess amount of Buffer E and the bound protein
was eluted by Buffer F in which the pH of Buffer E was titered to 4.0 by addition of glacial acetic acid.
Zn2+ Equilibrium Dialysis--
The equilibrium
dialysis experiments were carried out in a double acrylic microdialysis
module (Hoffer, San Francisco, CA). A dialysis membrane was assembled
between the two modules to separate each of the eight chambers into two
compartments. The molecular weight cut-off of the dialysis membrane was
chosen depending upon the molecular size of the test protein. The
12,000-14,000 Da cut-off membrane (Hoffer) was used for the
proteoglycan preparations and the MBP fusion proteins. The 2,000 Da
cut-off membrane (Spectra/Pro, Spectrum Medical Industries, Inc.,
Houston, TX) was used for the MD4 peptide. Aliquots of 150 µl of test
protein in Buffer D were added to the inner compartments. The same
volume of Buffer D containing increasing concentrations of
ZnCl2 (0, 2, 4, 8, 16, 24, 32, and 40 µM) was
added to the outer compartments. The assembled modules were fitted on a
rotating axis and incubated at 4 °C for at least 40 h to reach equilibrium.
After incubation, the concentration of Zn2+ in the outer
compartments was determined using the metallochromic indicator PAR (41). An aliquot of 120 µl from each outer compartment was added to
glass tubes containing 2.4 µl of a 5 mM PAR. The
components were mixed and incubated for 30 min at room temperature to
allow the red PAR·Zn2+ complex to form. The absorbance at
A500 from each sample was determined using a
DU-70 spectrophotometer (Beckman, Fullerton, CA). A standard curve of
ZnCl2 (0-40 µM) in Buffer D was generated which showed a linear relationship by plotting
A500 versus the increasing
concentration of Zn2+. The concentration of free
Zn2+ in solution from each outer compartment was determined
by inserting the observed absorbance into the standard curve equation.
The concentration of Zn2+ bound to protein was determined
using Equation 1,
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(Eq. 1)
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[Zn2+]Total is the total concentration
of Zn2+ added to the outside compartment of the equilibrium
dialysis chamber at the beginning of the experiment. The
[Zn2+]free term is multiplied by a factor of
2 to take into account the dilution of
[Zn2+]Total at equilibrium in the absence of protein.
An equation derived for multiple, independent binding sites (Equation 2) was used to analyze the equilibrium binding data (42),
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(Eq. 2)
|
Total protein is the concentration of protein used in the
equilibrium dialysis experiment, n is the number of
Zn2+ molecules binding to each protein molecule, and
KD is the observed dissociation constant for the
Zn2+-protein complex. The reported
KD ± S.E. and Zn2+ binding
curves represent the average of at least three independent experiments.
Circular Dichroism Studies--
CD spectra were collected on a
Jasco J-720 spectropolarimeter calibrated with
d-10-camphorsulfonic acid, using a round, 0.5-mm quartz
cell. The CD data were collected at a scan speed of 20 nm/min at 0.5-nm
intervals, a time constant of 1 s, and a band width of 1 nm,
averaging 4 scans for the final data. All spectra were corrected for
buffer contributions and are presented in units of mean residue
ellipticity, [
]MRW, calculated with Equation 3,
|
(Eq. 3)
|
where
is the ellipticity in millidegrees, MRW is
the mean residue weight of MD4, c is the concentration of
MD4 in mg/ml, and d is the cell path length in centimeters.
The samples were prepared by first reducing the MD4 peptide in 100 mM EDTA and 1%
-mercaptoethanol at 100 °C for 1 min.
The reduced sample was cooled to room temperature and the buffer
exchanged to 50 mM Tricine, 0.1 mM EDTA at pH
6.6 on a PD-10 de-salting column (Pharmacia, Piscataway, NJ). The
concentration of fractions from the PD-10 column were calibrated at 280 nm using
280 = 2600. Samples for CD spectroscopy were
prepared by diluting the stock MD4 samples with 50 mM
Tricine and 0.1 mM EDTA for the sample without zinc salt or
adding an aliquot of a 100 mM ZnCl2 or
ZnSO4 stock to the dilution buffer before the addition of
peptide. All samples were prepared to a final volume of 200 µl at
concentrations of 200 µg/ml MD4 peptide and 2 mM zinc
salt where indicated.
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RESULTS |
The Zn2+ Binding Activity of Decorin Is Located to the
Core Protein--
We recently reported that a Zn2+ charged
column of iminodiacetate-Sepharose could be used to separate decorin
and epiphycan isolated from fetal bovine epiphyseal cartilage (43).
Decorin was retained on the column suggesting that the proteoglycan can bind Zn2+. Equilibrium dialysis has now been used to
demonstrate a saturable binding of Zn2+ to decorin (Fig.
1). The proteoglycan isolated from bovine
skin (BDCN) and recombinant human decorin produced in mammalian cells (vvHDCN) bound a maximum approaching two Zn2+ ions per
decorin molecule with an average KD of 1.0 ± 0.3 µM and 3.9 ± 1.8 µM, respectively
(Fig. 1, A and B).

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Fig. 1.
Zn2+ binding to decorin;
equilibrium dialysis. Bovine decorin extracted from fetal skin
(BDCN), human decorin produced as a recombinant using a vaccinia virus
vector (vvHDCN), mouse decorin core protein expressed as a prokaryotic
recombinant as a fusion protein with maltose-binding proteins (MBP-MD),
or MBP were examined for their Zn2+ binding activity using
equilibrium dialysis. For further details see "Experimental
Procedures." The KD and number of binding sites
(n) ± S.E. represents the average of three
experiments.
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The recombinant mammalian decorin appears as a mixture of molecular
species including proteoglycans and core proteins without glycosaminoglycan chains. The observation that the recombinant proteoglycan/core protein mixture could bind similar amounts of Zn2+ suggests that the primary Zn2+-binding
sites in decorin are located in the core protein. This hypothesis was
further examined by analyzing the Zn2+ binding properties
of prokaryotic recombinants. Recombinant mouse decorin (MD) core
protein produced in E. coli as a fusion protein where the
core protein is linked at its N terminus to the MBP also bound
Zn2+ when examined by equilibrium dialyses whereas no
saturable binding of Zn2+ to MBP alone could be detected
(Fig. 1, C and D). A maximum of 1.8 Zn2+ ions could bind per molecule of MBP-MD fusion protein
with a KD of 3 µM under these
conditions (Fig. 1C). Isolated dermatan sulfate or
chondroitin sulfate polysaccharides were not retained on a
Zn2+ charged iminodiacetate-Sepharose (data not shown),
suggesting that the core protein alone is responsible for
Zn2+ binding activity of decorin.
The Zn2+ Binding Activity Is Located to the N-terminal
Domain of the Decorin Core Protein--
To further locate the domain
responsible for the Zn2+ binding activity in the core
protein, we analyzed recombinant segments of the mouse decorin core
protein produced in E. coli as fusion proteins linked to the
C terminus of MBP. The different constructs made are shown in Fig.
2. All the fusion proteins which were at least partially soluble when produced as described under
"Experimental Procedures," were purified by a combination of
affinity chromatography on amylose-Sepharose and ion exchange
chromatography. In the presence of reducing agent, a major component in
the different preparations of core protein fragments migrated as
expected when analyzed by SDS-PAGE (data not shown).

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Fig. 2.
Constructs of decorin core protein
fragments. The diagram shows the recombinant decorin constructs
expressed in E. coli, their molecular weight
(Mw), and summarizes the results of the different fusion
protein's ability to bind to a Zn2+ charged iminodiacetic
acid column.
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Chromatography of the different fusion proteins on a Zn2+
charged matrix demonstrated that MBP-MD, MBP-MD1, and MBP-MD4 bound Zn2+ whereas MBP-MD2 and MBP-MD3 did not bind the metal
ions (summarized in Fig. 2). The Zn2+-binding recombinant
proteins all contained the N-terminal segment of the decorin core
protein whereas this segment was not present in the non-binders.
Equilibrium dialyses showed that MBP-MD4 could bind Zn2+ in
a process that exhibits saturation kinetics approaching a maximum of
two Zn2+ ions bound per protein molecule (Fig.
3A). The average
KD for these interactions was 2.4 ± 0.6 µM. Binding of Zn2+ to MBP-MD2 and MBP-MD3,
respectively, could not be demonstrated using equilibrium dialyses
(data not shown). These results suggest that the 41-amino acid long N
terminus of decorin retain full zinc binding activity.

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Fig. 3.
Zn2+-binding affinity of MBP-MD4
and MD4 peptide. Equilibrium dialysis was used to analyze the
binding of decorin N-terminal domain as the MBP-MD4 or as isolated MD4
peptide to Zn2+. Each data point was the average of
triplicate experiments. The correlation coefficients of the curve was
0.98 and 0.99, respectively.
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Characterization of Zn2+ Binding to the MD4
Peptide--
We decided to examine the Zn2+ binding
behavior of the MD4 peptide in the absence of a fusion protein partner.
A new MD4 expressing plasmid using the pGEX-2T vector was constructed.
The resulting recombinant fusion protein is composed of GST followed by
a linker containing a thrombin cleavage site and MD4. This fusion
protein was expressed, purified, and cleaved. The released MD4 peptide, which contains 4 extra amino acid residues (Gly-Ser-Asn-Gly) at the N
terminus followed by residue 31-71 of mouse decorin, was purified by
RP-HPLC. The expected size of the peptide was 4878.56 Da as calculated
from the amino acid sequence and the molecular mass of the isolated
peptide was 4878.37 Da as determined by matrix-assisted laser
desorption ionization-mass spectroscopy.
The purified MD4 peptide bound to a Zn2+ charged
iminodiacetate-Sepharose column (data not shown) and equilibrium
dialyses experiments showed that the MD4 peptide bound Zn2+
ions in a concentration dependent manner that approached a maximum of
two Zn2+ ions bound per peptide molecule (Fig.
3B). Analyses of the binding data assuming the presence of
two independent Zn2+-binding sites in the MD4 peptide
suggest an average KD of 0.28 ± 0.03 µM for these sites. Analyses of the binding data in a
Hill plot did not reveal any pronounced cooperativity between the two
binding sites.
Zn2+ Binding Induces a Conformational Change in the MD4
Peptide--
Analyses by circular dichroism spectroscopy (Fig.
4) showed that the MD4 peptide has a
different conformation in the presence of Zn2+ ions. In the
presence of EDTA (but absence of Zn2+ ion), the spectrums
exhibit a minimum of 200 nm. When Zn2+ ions are added
either in the form of ZnSO4 or ZnCl2, the
minima has a reduced amplitude and is shifted to a slightly lower
wavelength. In addition, a shoulder appears at 215-220 mm. These
observations demonstrate a change in secondary structure in the peptide
on Zn2+ binding.

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Fig. 4.
Far-UV CD of reduced MD4 in the absence and
presence of Zn2+. The spectra are: , reduced MD4
peptide in 50 mM Tricine and 0.1 mM EDTA; ,
reduced MD4 peptide in 50 mM Tricine, 0.1 mM
EDTA, and 2 mM ZnCl2; , reduced MD4 peptide
in 50 mM Tricine, 0.1 mM EDTA, and 2 mM ZnSO4.
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The Zn2+ Binding Activity of Biglycan--
The amino
acid sequence of the Zn2+ binding MD4 decorin peptide is
partially conserved in biglycan but less conserved in epiphycan and
osteoglycin. In previous studies, tissue extracted biglycan from bovine
articular cartilage have been shown to form multimers in the presence
of Zn2+ (34). We therefore examined the Zn2+
binding activity of different forms of biglycan. Binding to a Zn2+ charged column was demonstrated for biglycan isolated
under denaturing conditions from bovine articular cartilage (BBGN) or
produced as a recombinant proteoglycan in HT1080 (vvHBGN) (data not
shown). Equilibrium dialyses showed that BBGN and vvHBGN bound a
maximum of two Zn2+ ions per molecule with an average
KD of 3.3 ± 2.4 and 5.4 ± 1.4 µM, respectively (Fig. 5,
A and B). The N-terminal segment of mouse
biglycan corresponding to amino acid residues 38-77 was expressed in
E. coli as a recombinant MBP fusion protein (MBP-MB-N) and
shown to bind to a Zn2+ charged column of
iminodiacetate-Sepharose (data not shown). Analyses by equilibrium
dialyses showed that MBP-MB-N could bind a maximum of two molecules of
Zn2+ with an average KD of 2.7 ± 1.6 µM (Fig. 5C). These results suggest that
biglycan is also a Zn2+ binding molecule and that the N
terminus of its core protein has zinc binding activity.

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|
Fig. 5.
Zn2+ binding affinity of
different forms of biglycan. The Zn2+ binding of
biglycan extracted from bovine articular cartilage (BBGN), produced in
culture mammalian cells using a vaccinia virus vector (vvHBGN), or the
N-terminal peptide expressed as a maltose-binding protein fusion
(MBP-MB-N) were analyzed by equilibrium dialysis as described under
"Experimental Procedures." Each binding curve was plotted from the
average of triplicate experiments.
|
|
Segments of mouse epiphycan and osteoglycin core proteins containing
the N-terminal domains were expressed as fusion proteins (MBP-ME-N and
MBP-MO-N, respectively), purified, and analyzed for Zn2+
binding activity. Both fusion proteins bound to the Zn2+
charged column (data not shown). In equilibrium dialysis experiments, however, the binding affinity of Zn2+ to MBP-ME-N and
MBP-MO-N proteins was too weak (KD
>10
5 M) to be detected. These results
suggest that residues specifically found in decorin and biglycan but
not in epiphycan or osteoglycin are required for forming the high
affinity Zn2+-binding sites.
 |
DISCUSSION |
In this article, we report that decorin and biglycan are
Zn2+ metalloproteins. These macromolecules are capable of
binding Zn2+ regardless of whether they are purified under
denaturing conditions from tissues or are produced and isolated under
nondenaturing conditions from cultured cells infected by recombinant
viruses. Furthermore, the ability to bind Zn2+ is not
restricted to decorin from one particular species since in this study,
we demonstrate a binding of Zn2+ to decorin derived from
human, mouse, or bovine tissue. Decorin and biglycan are members of a
growing family of ECM proteins that exhibit high affinity for
Zn2+. In addition to matrix metalloproteases (28), this
family includes, laminin (29), link protein (30), nidogen (31), and
COMP (32).
The Zn2+ binding activity of decorin is localized to a
segment in the N-terminal part of the core protein. A GAG attachment site is also located in this region and involves a Ser residue at
position 34 in the mouse decorin sequence. The carbohydrate components
of the proteoglycans, however, are not directly involved in
Zn2+ binding. In fact, a recombinant peptide corresponding
to the decorin N-terminal domain and produced in E. coli has
full Zn2+ binding activity. The amino acid sequence of this
peptide which contains four Cys residues is reminiscent of a zinc
finger but the spacing of the Cys residues is different from previously
reported Zn2+-binding motifs (24, 25). This apparently
novel Zn2+ binding sequence must adopt a conformation that
allows the binding of two Zn2+ ions per peptide. Cys
residues appear to be involved in coordinating the Zn2+
ions since in preliminary experiments we have shown that reduction and
alkylation of the MBP-MD4 protein resulted in loss of Zn2+
binding activity. Earlier analyses of tryptic peptides obtained from
bovine biglycan showed that the first and the fourth Cys residues in
the N-terminal domain are linked through a disulfide bond (44). This
observation leaves the second and third Cys residues as potential
Zn2+ coordinators. If in fact the Cys residues are involved
in Zn2+ binding, this could help explain the different
affinities we see for Zn2+ among the different forms of
decorin tested. The isolated MD4 peptide which was isolated as a
homogenous form by RP-HPLC has the highest apparent affinity for
Zn2+.
The Zn2+ binding MBP fusion proteins all occurred as
mixtures of several molecular forms where some Cys residues appear to
be engaged in forming disulfied linked multimers. The measured
KD values for Zn2+ binding to these
proteins were higher compared with that recorded for the isolated MD4
peptide. Perhaps Cys residues engaged in coordinating Zn2+
in the MD4 peptides in the multimers are involved in disulfide linkages. Alternatively, this observation may suggest that structures outside the actual binding site may influence the Zn2+
binding activity of this domain. Thus the isolated sequence present in
the peptide appears to have a higher affinity for Zn2+ than
when the sequence is part of a larger structure as in an intact core
protein or a proteoglycan. Decorin contains in addition to a
traditional N-terminal signal, a 13-amino acid long propeptide, which
also often has been removed from the proteoglycan isolated from
tissues. The decorin MD4 peptide and the proteoglycan forms here shown
to bind Zn2+ with a high affinity do not include the
propeptide sequence. It is unclear if the presence of a propeptide may
affect Zn2+ binding to decorin or biglycan.
Recombinant N-terminal segments of epiphycan and osteoglycin core
protein made as MBP fusions, which contain sequences similar to the
Zn2+ binding sequences present in decorin and biglycan, are
also retained on a Zn2+-charged iminodiacetate-Sepharose.
However, the affinity of the MBP-fusion proteins for Zn2+
is too low to measure reliable binding constants by equilibrium dialyses. In an earlier study, we found that Zn2+ chelating
chromatography could be used to fractionate tissue-extracted epiphycan
(which did not bind to the column) from decorin (which bound to the
column). This result is in contrast to the chromatography data obtained
in the current study and could be explained if the full-length
epiphycan proteoglycan has a lower affinity for Zn2+
compared with the recombinant N-terminal peptide. Such a pattern was
established for decorin. In the N-terminal segments of epiphycan and
osteoglycin, the Cys residues are spaced differently than in the
Zn2+-binding peptides of decorin and biglycan; there are
two compared with 3 amino acid residues between the first and second
Cys in epiphycan osteoglycin, and decorin/biglycan, respectively. It is
possible but not yet demonstrated that the spacing of Cys residues determines the Zn2+ binding activity of these segments.
The concentration of Zn2+ in body fluids such as blood
plasma is approximately 15 µM, which suggests that both
decorin and biglycan in vivo occur in complex with
Zn2+. The significance of this complex is unclear. Decorin
and biglycan which are abundant molecules in the tissues could serve as
Zn2+ storage pools where the metal ions could be released
to proteins which have a higher affinity for and need Zn2+
ions for their activity. In addition, Zn2+ may stabilize a
conformation in the proteoglycan core proteins which are important for
their functions. In fact, analyses of the MD4 peptides using circular
dichroism spectroscopy demonstrate that the conformation of the peptide
is altered in the presence of Zn2+. This observation is
consistent with the observation of Liu et al. (34) who found
that biglycan had a tendency to aggregate in the presence of
Zn2+, which could be caused by a conformational change in
the protein induced by Zn2+ binding. Future studies of the
biology of decorin and biglycan must take into account the fact that
these molecules are Zn2+ metalloproteins.
 |
ACKNOWLEDGEMENTS |
We thank Alice Woodworth for assistance in
the preparation of this manuscript. We also thank Dr. John Putkey for
critical readings and comments on this manuscript.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants AR42919 (to M. H.) and AR42826 (to D. M.) and
NASA/Texas Medical Center Grant NCC9-36 (to M. H. and D. M.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence should be addressed. Tel.: 713-677-7551;
Fax: 713-677-7576; E-mail: mhook{at}ibt.tamu.edu.
2
V. W-C. Yang, W. Zhou, J. Johnson, and M. Höök, manuscript in preparation.
 |
ABBREVIATIONS |
The abbreviations used are:
SLRP, small
leucine-rich proteoglycan;
PRELP, proline-arginine-rich and
leucine-rich repeat protein;
COMP, cartilage oligomeric matrix protein;
MBP, maltose-binding protein;
GST, glutathione
S-transferase;
HPLC, high pressure liquid chromatography;
PAR, 4-(2-pyridylazo) resorcinol;
DTT, dithiothreitol;
PAGE, polyacrylamide gel electrophoresis;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
PCR, polymerase chain reaction;
RP-HPLC, reverse transcriptase-polymerase
chain reaction;
MD, mouse decorin.
 |
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