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INTRODUCTION |
Collagens are a large family of extracellular structural proteins
made up of three
chains that are intracellularly associated and
folded into specific structures including characteristic triple helical
domains (1). The major class, recognized as the fibril-forming collagens, contains molecules with one large uninterrupted triple helical domain (for review, see Refs. 1 and 2). Other members of the
collagen family have one or more non-triple helical domains, which may
constitute the major part of the protein. Most of these collagens do
not form prominent lateral aggregates in a manner similar to that of
the fibril-forming collagens. Instead, they form complex aggregates
together with other matrix macromolecules. Collagen VI is one example,
forming multimolecular filamentous beaded structures after secretion
from the cell (for review, see Ref. 3). This collagen is composed of
three different peptide chains (
1(VI),
2(VI), and
3(VI)),
which form the basic unit consisting of a relatively short triple
helical domain flanked by two large multidomain globular regions (4).
These are composed primarily of repeating units of von Willebrand type
A domains (5). Collagen VI assembles intracellularly into antiparallel, overlapping dimers that then align and form tetramers (6). These
structures are stabilized by disulfide bonds. Secreted tetramers assemble extracellularly in a characteristic end-to-end fashion into
thin (3-10 nm) beaded filaments with a periodicity of about 100 nm
(7-9). Further supramolecular assembly includes lateral associations
of the beaded filaments into microfibrils (8, 9).
Collagen VI is ubiquitous. It can be found intermingled with
fibril-forming collagens and is often enriched in the pericellular matrix (for review, see Refs. 3 and 10). Decreased amounts of secreted
collagen VI resulting from mutations in COL6A1 have been shown in
Bethlem myopathy (11, 12), a dominantly inherited disorder
characterized by progressive muscle weakness and wasting. This suggests
an important role for collagen VI in tissue integrity.
Collagen VI has been shown to interact with several different matrix
constituents. It may have a role in the development of the matrix
supramolecular structure as well as in tissue homeostasis by mediating
interactions of cells with the extracellular matrix. More specifically,
interactions of collagen VI with collagen XIV, collagen IV, the
fibrillar collagens type I and II, decorin, microfibril-associated glycoprotein MAGP-1, and hyaluronan as well as the
1
1 and
2
1 integrins and the cell surface proteoglycan NG2 have been demonstrated (13-20). Collagen VI interacts via its triple helical domain with perlecan and fibronectin (21). Furthermore, a recombinant
3(VI) N-terminal fragment containing domains N9-N2 (5) interacts with both
heparin and hyaluronan (22).
A family of extracellular matrix proteins with characteristic
leucine-rich repeats has several members that show tight binding to collagens. They are present in collagen networks and modulate their
functional properties. One extensively studied example is the small
proteoglycan decorin. Biglycan and decorin represent two distinct but
closely related members of a subgroup within the family of leucine-rich
repeat proteins in the extracellular matrix. They contain 10 leucine-rich repeats of each some 25 amino acids. Decorin and biglycan
are proteoglycans with one and two chondroitin/dermatan sulfate chains,
respectively (for review, see Refs. 23 and 24).
Decorin interacts with fibrillar collagens (25, 26) and intervenes in
collagen fibrillogenesis in vitro (27, 28). Decorin has been
shown also to interact with collagen VI (16) through the
2(VI) chain
(20) and to colocalize with collagen VI in the cornea (29).
Inactivation of the decorin gene leads to alterations of the collagen
fibrillar network, primarily in skin (30).
Decorin is found primarily at a distance from cells, but biglycan
is distributed mainly close to the cells and even pericellularly (31,
32). Mice deficient in biglycan show major alterations in bone (33).
Biglycan can be extracted from, e.g. cartilage and purified
under denaturing conditions. Such preparations have been used in
studies of functional properties indicating that biglycan can
inhibit binding of decorin to collagen VI (16).
In the present study, native biglycan and decorin were shown to
interact tightly via their core protein with the same binding site
close to the N-terminal region of the collagen VI helical domain,
possibly via the
2(VI). Native decorin was shown to bind to collagen
VI with higher (10×) affinity than has been shown previously for the
molecule isolated under denaturing conditions (16).
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EXPERIMENTAL PROCEDURES |
Purified Collagens--
Pepsinized collagen VI was prepared from
human placenta (34). Briefly, the tissue was homogenized in formic acid
and incubated with pepsin for 24 h at room temperature. After a
series of salt precipitations, the collagen VI was purified by gel
filtration, dialyzed into dilute acetic acid, and freeze dried
(34).
Recombinant
1(VI) and
2(VI) chains and the N-terminal fragment
N9-N2 of the
3(VI) chain were purified as described previously (21,
22). The N6-C5 fragment of the
3(VI) chain was produced by excising
the NheI-BamHI fragment from the pCI-neo
3(VI)
N9-C5 cDNA construct (12) and cloning it into pCEP4-BM40-hisEK
(35). The expression construct was transfected into 293-EBNA cells
using FuGENETM6 (Roche Molecular Biochemicals, Mannheim,
Germany), and transfected cells were selected in growth medium
containing 250 µg/ml hygromycin. The recombinant
3(VI) chain
containing an N-terminal His tag, the first six N-terminal domains
(N6-N1), the triple helical domain, and the complete C-terminal domain
(C1-C5) was isolated from serum-free conditioned medium under native
conditions by nickel affinity chromatography on a chelating Sepharose
Fast Flow column (Amersham Pharmacia Biotech) and purified
further by gel filtration chromatography on a Superose 6 HR 10/30
column (Amersham Pharmacia Biotech).
Collagen I was prepared by acid extraction from the fibrous proximal
part of bovine flexor tendon (28, 36).
Purification of Recombinant Proteoglycans--
Recombinant human
biglycan (37) and bovine decorin (38) were produced in human HeLa cells
and Chinese hamster ovary cells, respectively (26). Cells were cultured
in Dulbecco's modified Eagle's medium containing 10% fetal calf
serum. Medium was changed every 2nd day, and the collected medium was
stored frozen at
20 °C until purification. For purification of
proteoglycans, thawed medium (900-1,400 ml) was applied onto a 20-ml
Q-Sepharose (Amersham Pharmacia Biotech) column, equilibrated in 10 mM Tris/HCl, 100 mM NaCl, pH 7.4. The column
was washed with 4 volumes of equilibration buffer, and bound material
was eluted with a gradient of 0.1-1 M NaCl in
equilibration buffer. Fractions containing decorin/biglycan were
identified by SDS-polyacrylamide gel electrophoresis, pooled, and
diluted with 5 mM phosphate buffer, pH 7.4, containing 150 mM NaCl. They were subsequently applied onto a 2-ml column
of Blue Sepharose CL-6B (Amersham Pharmacia Biotech) to remove traces of bovine serum albumin. The flow-through was collected, concentrated in an ultrafiltration chamber (Amicon PM-10), checked for purity by
SDS-polyacrylamide gel electrophoresis, and frozen in aliquots.
Radiolabeling of Small Proteoglycans--
Proteoglycans were
prepared from confluent bovine fibroblast monolayer cultures (25),
which were labeled for 20 h with either 20 µCi/ml
[3H]leucine or 50 µCi/ml [35S]sulfate.
The medium was passed through a 2-ml column of DE52 cellulose
(Whatman), eluted with 10 mM Tris/HCl, 100 mM
NaCl, pH 7.4. Bound proteoglycans were eluted in 100 mM
Tris/HCl, 1.5 M NaCl, pH 7.4, followed by desalting on a 10 ml column of Sephadex G-50 (25) (Amersham Pharmacia Biotech).
Recombinant proteoglycans were purified as described above and
radiolabeled using IODO-BEADS (Pierce) according to the recommendations of the manufacturer. Briefly, 10 µg of protein was added to 0.5 mCi
of Na125I in a vial containing 200 µl of 10 mM sodium phosphate buffer, pH 7.4, and one IODO-BEAD. The
reaction was terminated after 15 min. Free isotope was removed by
desalting on a PD-10 column (Amersham Pharmacia Biotech).
Isolation of Glycosaminoglycans--
Decorin from bovine
articular cartilage was purified as described previously (39, 40) (a
kind gift from Dr. Mark Hickery). The decorin was digested with 0.2 unit of crystalline papain (papaya latex, Sigma)/mg of proteoglycan in
0.1 M sodium phosphate, 0.1 M NaCl, 5 mM cysteine/HCl, 10 mM EDTA, pH 7.4, at
60 °C for 16 h. The digest was then dialyzed against 20 mM Tris/HCl, pH 7.4, and applied onto a 1.7-ml column of
DEAE-Sepharose (Amersham Pharmacia Biotech). To block nonspecific
binding of glycosaminoglycans to the DEAE column two different
solutions, containing 6-sulfated chondroitin sulfate (see below) and
bovine serum albumin (Serva, Heidelberg, Germany) at 1 mg/ml, had been
chromatographed under similar conditions prior to the sample. Dermatan
sulfate was eluted with a linear gradient of 0-1 M NaCl in
20 mM Tris/HCl, pH 7.4. Fractions containing
glycosaminoglycans were identified by the dimethylmethylene blue
assay (41), pooled, dialyzed against water, and lyophilized.
Chondroitin 6-sulfate was isolated from nucleus pulposus as described
previously (42). Hyaluronic acid (Healon) was obtained from Amersham
Pharmacia Biotech.
Coprecipitation of Collagen and Proteoglycan--
Native
radiolabeled proteoglycans were tested for binding to precipitated
collagens. Samples were combined with collagen VI at 20 µg/ml and
bovine serum albumin at 50 µg/ml in 10 mM Tris/HCl, 150 mM NaCl, pH 7.4 (TBS),1 and subsequently
incubated for 18 h at 20 °C. Alternatively, samples were
combined with acid-extracted collagen I at 100 µg/ml and bovine serum
albumin at 500 µg/ml in 30 mM sodium phosphate, 140 mM NaCl, pH 7.4 (25), followed by incubation for 18 h
at 37 °C. Precipitated material was collected by centrifugation at 10,000 × g for 10 min at the respective temperatures.
The pellets and the supernatants were electrophoresed on
SDS-polyacrylamide 4-12% gradient gels (43). The relative amounts of
collagen in the samples were determined by scanning after staining with
Coomassie Blue. The radiolabeled components were detected by
fluorography with sodium salicylate (44). For quantification of the
radiolabeled proteoglycans, fluorographs were scanned using a digital
scanner and evaluated using the Gel-Pro AnalyzerTM software
(Media Cybernetics, Silver Spring, MD). For inhibition experiments,
native radiolabeled proteoglycans were combined with collagen VI as
described above but in the presence of 4 µg of nonlabeled decorin or
5 µg of biglycan, respectively. The quotient of decorin and biglycan
found in the precipitate was calculated after quantification as
described above.
Chondroitinase ABC Digestion--
To remove glycosaminoglycan
chains from recombinant proteoglycans, digestion with chondroitinase
ABC (Seikagaku Corporation, Tokyo, Japan) (1.0 milliunit/µg of
proteoglycan) at a final proteoglycan concentration of 16-48 µg/ml
in TBS was performed. The progress of the digestion was monitored at
232 nm, showing formation of unsaturated disaccharides. Samples were
taken to further analysis immediately after complete digestion,
typically after 0.5-2 h at 37 °C to minimize the risk of core
protein self-aggregation after removal of glycosaminoglycan chains.
Solid Phase Assay of Interactions--
5 µg/ml collagen VI in
TBS was adsorbed overnight onto microtiter plates (Maxisorb plates,
Nunc, Roskilde, Denmark). This and all of the following steps were done
at room temperature.
To avoid nonspecific interactions, wells were blocked for 1 h with
0.03 mg/ml
-casein (Sigma) in TBS. Coated wells were incubated overnight with recombinant biglycan at different concentrations (0-1.2
µg/ml) in TBS containing 0.03 mg/ml
-casein and 0.05% Tween. In
control experiments, only
-casein was coated onto the plate, or the
incubation step with biglycan was omitted. The amount of bound biglycan
was determined by incubation with affinity-purified, polyclonal
anti-biglycan antibodies. Bound IgG was detected with alkaline-phosphatase-conjugated anti-rabbit IgG antibody. Enzyme activity was measured with p-nitrophenyl phosphate as the
substrate at 405 nm. To check the specificity of the interaction,
biglycan was preincubated in inhibition experiments with collagen VI at different concentrations before being added to wells coated with the collagen.
Purified dermatan sulfate chains were tested for their ability to
inhibit the interaction of biglycan with collagen VI. Wells coated with
the collagen were preincubated with 0-200 µg/ml dermatan sulfate for
3 h before the proteoglycan was added as described above.
For studies of interactions of recombinant proteoglycans, collagen VI,
or recombinant fragments (
1(VI),
2(VI) and fragments N9-N2 and
N6-C5 from
3(VI)) were adsorbed to breakable Combiplates (Labsystems, Lund, Sweden) as described above. After blocking with
-casein, radiolabeled proteoglycans or core proteins (100,000 cpm)
were incubated overnight, and individual wells containing bound
radiolabeled protein were then analyzed in an LKB
-counter.
Between steps, wells were rinsed three times with 0.9% NaCl, 0.05%
Tween 20.
Studies of Binding Characteristics by Plasmon
Resonance--
Collagen VI (100-200 µg/ml in 10 mM
sodium citrate, pH 3.2) was immobilized on sensorchips Pioneer B1 or
CM-5 (the latter having a carboxylated dextran matrix with a degree of
carboxylation approximately two times that of Pioneer B1) according to
the recommendations of the manufacturer (BIAcore AB, Uppsala, Sweden)
with a 1:1 mixture of EDAC and N-hydroxysuccinimide
(Sigma). Remaining active groups on the matrix were blocked with 1 M ethanolamine hydrochloride (BDH, Poole, U. K.) at pH
8.5. The running buffer during these immobilization steps was 10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.005% (v/v) surfactant P20 (BIAcore AB). For kinetic studies of core
protein binding, recombinant proteoglycans were taken immediately after
digestion with chondroitinase ABC (described above), diluted with TBS
containing 0.05% (v/v) surfactant P20 to final concentrations of
10-400 nM, and injected over the collagen surfaces at
40-50 µl/min. The sample buffer, which was the same when intact
proteoglycans were studied, was also used as running buffer. Each
kinetic evaluation represents three to seven different protein
concentrations tested.
Electron Microscopy--
Colloidal gold particles of 5 nm ± 15% were prepared by reduction of HAuCl4 with
thiocyanate (45). Colloidal gold was titrated and conjugated (46) to
purified recombinant biglycan or decorin (intact or chondroitinase
ABC-digested) or recombinant collagen VI fragments. Briefly, proteins
were conjugated to colloidal gold for 30 min at room temperature and
then mixed with polyethylene glycol 20000 (molecular weight 20,000) to
a final concentration of 0.025% polyethylene glycol. Gold-labeled or
nonlabeled proteoglycans (biglycan at 1.2 µg/ml, decorin at 0.8 µg/ml, i.e. concentrations ~10 nM) were
incubated in vitro for 1 min on ice with collagen VI at 10 µg/ml (20 nM) in TBS and instantly adsorbed onto a
400-mesh carbon-coated copper grid that was rendered hydrophilic by
glow discharge at low pressure in air. Alternatively, the same amounts of proteoglycans were incubated with recombinant collagen VI fragments. The grid was immediately blotted, washed with two drops of water, and
stained with 0.75% uranyl formate for 15 s. Samples were observed in a Jeol 1200 EX transmission electron microscope operated at 60 kV
accelerating voltage and 75,000 × magnification. Evaluation of
the data from electron micrographs was done as described previously (47).
 |
RESULTS |
Binding of Metabolically Labeled Proteoglycans to Aggregating
Collagens--
Purified, native [35S]sulfate-labeled
proteoglycans were tested for binding to precipitating collagen VI. An
assay for pepsin-extracted collagen VI was developed in which 50-70%
of collagen VI was precipitated. Under these experimental conditions,
16% of the total biglycan was recovered bound to collagen VI, whereas
the proportion of decorin bound was 18% (Fig.
1, a and c). The
binding of radiolabeled proteoglycans was competed for when nonlabeled,
purified proteoglycans were added (Fig. 1a). The addition of
either decorin (panel II) or biglycan (panel III)
resulted in a markedly decreased binding of radiolabeled proteoglycans
of both types (Fig. 1, a and c). In a
corresponding assay with collagen I, more than 95% of the decorin was
coprecipitated with the collagen fibrils. Biglycan was almost totally
recovered in the supernatant, and less than 5% was found precipitated
with the collagen (Fig. 1b).

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Fig. 1.
Binding of metabolically labeled
proteoglycans to aggregating collagens. Medium was collected from
fibroblast monolayer cultures labeled with [35S]sulfate.
After purification of labeled proteoglycans, mixtures of radiolabeled
samples and collagen were incubated for 18 h under conditions that
were chosen to promote precipitation of the collagen. The precipitates
were recovered by centrifugation. Pellets and supernatants were
analyzed by fluorography after SDS-polyacrylamide gel electrophoresis.
In a, the proteoglycans (approximately 50,000 cpm) were
incubated with 10 µg of collagen VI in 500 µl of TBS at 20 °C.
The lanes show the supernatants (S) and the
precipitates (P) recovered in the absence of nonlabeled
proteoglycans (panel I), in the presence of 4 µg of
nonlabeled decorin (panel II), and in the presence of 5 µg
of nonlabeled biglycan (panel III). In b,
radiolabeled proteoglycans (approximately 10,000 cpm) were incubated
with 20 µg of collagen I in 200 µl of phosphate/saline buffer at
37 °C. The lanes show total sample (T),
supernatant (S), and precipitate (P). In
c, the proportion (percent) of total radiolabeled
proteoglycan found in precipitate after incubation with collagen VI in
the absence (I) or presence of nonlabeled decorin
(II) and biglycan (III) is shown. The biglycan
and decorin labels in a and b indicate the
positions of reference proteoglycans.
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The capacity of biglycan, decorin, and glycosaminoglycans to inhibit
binding of proteoglycans in the precipitation assay was examined
further. Almost complete inhibition of decorin binding was achieved
when the molar concentration of nonlabeled proteoglycan exceeded that
of collagen VI (Fig. 2), indicating that
the proteoglycan binding site(s) on collagen VI are saturable and
limited in number. Isolated biglycan was equal or even slightly more
efficient in inhibiting decorin binding to collagen VI than decorin
(Fig. 2). The amounts of collagen VI in the precipitates were not
different in the presence or absence of proteoglycans or
glycosaminoglycans, as revealed by Coomassie staining of the gels prior
to fluorography (not shown). In conclusion, the inhibition experiments
show that the native biglycan and decorin interact close to or at the
same defined site(s) on collagen VI.

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Fig. 2.
Competition for the binding of radiolabeled
decorin to precipitating collagen VI.
[35S]Sulfate-labeled decorin, together with various
amounts of nonlabeled chondroitin sulfate ( ), hyaluronic acid ( ),
intact decorin (×), intact biglycan ( ), or biglycan core protein
( ) were added to 10 µg of pepsin-extracted collagen VI in 500 µl
of TBS and incubated at 20 °C for 18 h. Precipitates were
recovered by centrifugation, and bound radiolabeled decorin was
measured by densitometric scanning of the fluorogram after
SDS-polyacrylamide gel electrophoresis. A preparation of pure,
radiolabeled decorin was used as a reference.
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The isolated biglycan core protein, prepared by treatment of the intact
proteoglycan with chondroitinase ABC, competed for collagen VI binding
to the same extent as the intact proteoglycan (Fig. 2). Neither free
chondroitin sulfate nor hyaluronic acid inhibited the binding of
radiolabeled decorin (Fig. 2).
Interaction of Intact Biglycan with Collagen VI (Microtiter Plate
Assay)--
Incubation of pepsin-extracted collagen VI-coated wells
with biglycan at different concentrations showed saturation of binding sites at 8-10 nM biglycan (Fig.
3). Scatchard plot analysis of the data
gave a KD of 1.1 nM (not shown).
Preincubation of collagen VI-coated wells with excess purified dermatan
sulfate chains did not alter the binding of biglycan. The interaction of biglycan with the surface coat of collagen VI was inhibited by
preincubation of the biglycan with collagen VI in solution (not shown).
Incubation of coated collagen VI fragments with radiolabeled biglycan
only showed binding to
2(VI) but significantly lower compared with
pepsin-extracted, triple helical collagen VI. No binding was observed
to
1(VI) nor to the fragments N9-N2 and N6-C5 of the
3(VI)
chain (Fig. 4).

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Fig. 3.
Binding of intact, recombinant biglycan to
collagen VI in solid phase assay. Pepsin-extracted collagen VI
( ) and -casein ( ) were adsorbed onto microtiter plates.
Biglycan at different concentrations was allowed to interact, and bound
biglycan was quantified using antibodies as described under
"Experimental Procedures." The absorbance reflecting the amount of
bound biglycan is plotted against the concentration added.
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Fig. 4.
Binding of radiolabeled, recombinant biglycan
to collagen VI and recombinant collagen VI fragments in solid phase
assay and in solution. Pepsin-extracted collagen VI, 1(VI),
2(VI), N9-N2 and N6-C5 fragment of 3(VI), or -casein
(control) was adsorbed onto breakable microtiter strips followed by
incubation with radiolabeled recombinant biglycan. Biglycan showed
binding to 2(VI), but it was significantly lower compared with
collagen VI. In solution, as visualized by electron microscopy after
negative staining (inset), biglycan (arrow) could
bee seen to interact moderately only with 2(VI), identified with
gold labeling (arrowhead).
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Interactions Studied by Surface Plasmon
Resonance--
Immobilization of collagen VI resulted in surface
concentrations of 3 ng/mm2 (3,000 relative units) for
sensorchip B1 and about 6 ng/mm2 (6,000 relative units) for
sensorchip CM-5, reflecting the lower degree of carboxylation of the B1
chip. The kinetic data presented in Table
I refer to evaluation with a 1:1 binding
model of interaction studies on sensorchip B1 (intact proteoglycans)
and CM-5 (core proteins). With sensorchip CM-5, reliable data for
intact proteoglycans could not be obtained, probably because of
repulsion effects from remaining, not activated carboxyl groups. By
using sensorchip B1, having a lower degree of carboxylation, and by
increasing the time of activation and deactivation of this surface, we
could minimize the effects of repulsion of molecules with high negative charge density which caused problems in initial experiments with intact
proteoglycans. The interaction kinetics of collagen VI with
proteoglycan core proteins were consistent on both chips, although the
kinetics of core protein binding to collagen VI showed a higher
variability when sensorchip B1 was used. Kinetic evaluation of intact
biglycan and decorin interacting with collagen VI (Fig. 5, a and b)
according to a 1:1 binding model gave a dissociation constants
(KD) close to 30 nM (Table I). The
kinetics of the proteoglycan core protein binding (Fig. 5, c
and d) did not differ significantly from those of the intact
proteoglycans (Table I).
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Table I
Kinetic evaluation of the interaction between biglycan and decorin with
collagen VI
Kinetic data from analyses of binding of biglycan and decorin to
collagen VI immobilized on sensorchip B1 (intact proteoglycans) and
CM-5 (core proteins) are shown. Evaluation was done according to a 1:1
binding model. Data are presented as the mean ± S.D. from three
or four different experiments, each containing three to seven different
analyte concentrations.
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Fig. 5.
Interaction of biglycan and decorin with
collagen VI in the BIAcoreTM2000 system. Intact
recombinant biglycan (a), intact
recombinant decorin (b), chondroitinase
ABC-treated, recombinant biglycan (c),
and chondroitinase ABC-treated recombinant decorin
(d) at different concentrations (20-200
nM) in TBS containing 0.05% surfactant P20 were injected
over immobilized pepsin-extracted collagen VI at flow rates of 40-50
µl/min. The arrows indicate the beginning and the end of
injections.
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Injections of chondroitinase ABC gave no signal, verifying a lack of
binding to immobilized collagen VI.
Electron Microscopy--
Biglycan and decorin were labeled with 5 nm colloidal gold. The proteoglycans were subsequently used to
characterize the interaction with collagen VI. The molecules and their
complexes were visualized by negative staining and electron microscopy.
Both labeled biglycan and decorin were found to bind exclusively at the
small N-terminal globular domain remaining on pepsin-extracted collagen
VI (Fig. 6 a,
arrowheads). These domains appear as small globules
(asterisks) located at the end of collagen VI dimers and
tetramers. Biglycan and decorin treated with chondroitinase ABC and
subsequently tagged by gold labeling showed binding at the N-terminal
part of collagen VI identical to that of the intact proteoglycans.
Similar results were obtained with unlabeled proteoglycans (Fig.
6a, arrow) and core proteins, where the use of
negative staining allowed the identification of the core proteins.

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Fig. 6.
Electron microscopy after negative staining
of recombinant proteoglycans interacting with collagen VI.
a, Gold-labeled and nonlabeled intact recombinant biglycan,
intact recombinant decorin, chondroitinase ABC-treated, recombinant
biglycan and chondroitinase ABC-treated recombinant decorin were
allowed to interact with collagen VI in TBS for 5 min at 4 °C.
Samples were negatively stained with 0.75% uranyl formate. It is
notable that biglycan and decorin interact (arrowheads) with
or close to the remaining N-terminal globular domains. These domains
appear as small globules (asterisks) located at the end of
collagen VI dimers and tetramers. The interaction of nonlabeled intact
proteoglycans and core proteins occurred at the same site as the labeled (arrows).
b, Nonlabeled biglycan (left panel)
and collagen VI (middle panel) as they appear after negative
staining. To the right is a schematic figure of a collagen
VI monomer, dimer, and tetramer. The bars represent 150 nm
(a) and 100 nm
(b).
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Recombinant biglycan and decorin showed moderate binding to both
nonlabeled and gold-labeled
2(VI) (Fig. 4, inset). The
recombinant fragments of collagen VI appeared as globular rather than
linear structures when analyzed with electron microscopy after negative staining.
 |
DISCUSSION |
Interactions of the leucine-rich repeat proteoglycans
biglycan and decorin with collagen VI were characterized. It was
considered important that preparations of extracellular matrix
macromolecules, e.g. decorin, classically obtained via a
number of denaturing steps, sometimes show a weaker binding than
recombinant, native protein expressed in eukaryotic cells, as shown in
the case of decorin binding to collagen I (26). Because denatured
proteins may have an altered binding to their ligands, we used the
native forms of biglycan and decorin, purified under mild conditions, for our binding studies. It was shown that decorin binds 10-fold stronger to collagen VI than in a previous study (16), where decorin
that had been exposed to denaturing conditions was used. In that study,
collagen VI from the same source as ours was used in solid phase assay.
Even if we had used a different method, the BIAcore system, it is
unlikely that this accounts for the differences in the reports because
we do not see any major differences between the values of
KD measured in the enzyme-linked immunosorbent assay
system compared with values measured with the BIAcore system. Instead,
the weaker binding of decorin to collagen VI reported by Bidanset
et al. (16) is likely to depend on exposure of decorin to
denaturing agents. This is consistent with data on decorin binding to
collagen I (26) and suggests that these interactions depend on an
optimal secondary structure of decorin.
The core protein plays the major role in the interaction between
these two small proteoglycans and collagen VI. Treatment of biglycan
and decorin with chondroitinase ABC did not alter the binding kinetics
to collagen VI, showing that the glycosaminoglycan side chains do not
have a significant role in the interaction. This was supported by
experiments demonstrating that the isolated chains were not able to
inhibit the interaction in vitro (data not shown). This is
also consistent with the lack of apparent effects of the
glycosaminoglycan chain observed in studies of decorin binding to
collagen VI (16). In that study, direct binding of biglycan core
protein was not explored. Some uncertainties exist in the literature as
to whether glycosaminoglycan substitutions are necessary for the
interaction. In a recent study (48) with radiolabeled biglycan, no
interaction could be shown in the solid phase assay for biglycan core
protein. In our experimental setup the biglycan core protein interacts
with collagen VI. The difference from previous data can be the result
of blocking of binding sites when collagen VI is adsorbed to a plastic
surface. Indeed, in the enzyme-linked immunosorbent assay we also
observed an abolished interaction after chondroitinase ABC digestion.
Further, in the same system with radiolabeled
proteoglycans we observed a significantly diminished binding. However,
presence of glycosaminoglycan chains appears not to modulate binding
strength of the intact biglycan to collagen VI. Whether the
glycosaminoglycan chains in vivo contribute by interacting
with matrix constituents other than the collagen to which the core
protein is bound remains to be answered.
Our studies of the biglycan-collagen VI interaction show a binding
of equal strength (KD 32 nM) to that of
decorin (KD 27 nM). Biglycan and
decorin apparently share the same binding structure on collagen VI as
is indicated by the coprecipitation experiments, showing that either
proteoglycan inhibited the binding of the other.
In previous studies decorin has been shown to bind to the recombinant
2 chain of collagen VI and to collagen I within the C-terminal CNBr
peptide CB6 of
1(I) (49), whereas the binding site in collagen XIV
is located in the fibronectin type III-repeat in the non-collagenous
N-terminal domain (50). Here, both biglycan and decorin bound to the
N-terminal part that remained on the collagen VI preparation after
pepsin digestion as revealed by electron microscopy, i.e.
either to the N-terminal part of the triple helical domain or to the
first von Willebrand type A-like domains of the N-terminal globular
domain (4). We can also show a weak binding to the recombinant
2(VI)
chain but not to the N6-C5 fragment of
3(VI) containing the
fibronectin type III repeat-like domain C4 (5). Given the weak binding
to the
2(VI) chain in solid phase, the interaction might require a
binding site created by all three chains in combination. A less than
appropriate folding in the absence of the triple helix may also affect
the binding.
In developing murine and adult human cartilage, collagen VI is
localized mainly in the pericellular compartment (51, 52). The same
localization is found for biglycan, whereas decorin primarily colocalizes with collagen fibrils in the interterritorial matrix (31,
53). Thus, in cartilage, based on its localization with collagen VI to
the same compartment, biglycan appears as a more likely ligand for the
collagen VI than decorin.
In skin and tendon, collagen VI seems to form mesh-like structures
adjacent to or in contact with fibrillar collagens (51, 54, 55),
indicating a closer interrelation of the fibrillar collagen and
microfibrillar collagen VI networks. However, studies of the
decorin-deficient mouse showed no apparent disruption of the collagen
VI network (54) but suggested that another member of the family of
small proteoglycans was present. Based on our studies, it is possible
that this proteoglycan is biglycan. Even if biglycan is not
up-regulated at the transcriptional level in the skin in
decorin-deficient mice (30), it should be kept in mind that the protein
level may be altered by a slower catabolism. By analogy, lumican
protein is increased in the fibromodulin-deficient mouse at the same
time as levels of mRNA are decreased (56).
This study demonstrates the specific interaction of native biglycan and
decorin with a domain localized close to the N-terminal part of the
triple helical region of collagen VI suggests a role for these small
leucine-rich repeat proteoglycans in the modification of collagen VI
supramolecular structure and functionality.