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INTRODUCTION |
The large aggregating proteoglycans referred to as lecticans (1)
or hyalectans (2) are ubiquitous extracellular matrix components. The
family consists of aggrecan, versican, brevican, and neurocan. Aggrecan
is expressed by chondrocytes in cartilage. Versican is found in many
tissues, e.g. blood vessels and dermis expressed by smooth
muscle cells and fibroblasts. Brevican and neurocan are found in the
central nervous system, expressed by astrocytes and neuronal cells,
respectively. These proteoglycans have important functions in many
tissues. Aggrecan, for example, with its many glycosaminoglycan side
chains and thus high fixed charge density gives rise to a pronounced
osmotic swelling pressure that is crucial for the biomechanical
properties of cartilage (3, 4).
The core proteins of these proteoglycans are organized with a central
elongated glycosaminoglycan-carrying part of variable length, flanked
by globular domains mediating interactions with other matrix molecules
(5-9). The amino-terminal globular G1 domains bind to hyaluronan in
interactions stabilized by link protein (10-13). The carboxyl-terminal
globular G3 domain consists of one or two EGF repeats, a C-type
lectin-like domain (CLD),1
and a sushi repeat. It has recently been shown that the CLDs function
in binding. So far four ligands have been identified as follows: the
brain-specific extracellular matrix protein tenascin-R (14, 15),
tenascin-C (16), sulfated glycolipids (17), and fibulin-1 (18).
The CLDs of the lecticans are highly conserved; the amino acid sequence
of the chick versican CLD, for example, is 96% identical to the human
homologue. This high degree of conservation suggests that the CLD motif
of the G3 domain has important functions. We propose that one of these
functions is to organize the forming hyaluronan-lectican
complexes in the assembly of the extracellular matrix. This can be
achieved through binding other matrix components with multiple lectican
CLD-binding sites. Several of the CLD ligands identified so far are
multimeric proteins as follows: tenascin-R is a dimer or trimer (19),
tenascin-C is a hexamer (20, 21), and fibulin-1 may self-assemble (22,
23). In all these cases the lectican CLD binding is directed to
extended rod-like stretches of the multimeric extracellular matrix
molecules. These proteins are thus excellent candidates for
cross-linking the hyaluronan-lectican complexes. Due to the very large
size of the link protein-stabilized hyaluronan-lectican complexes, such
cross-linking is probably not critical to retain the lecticans in the
mature extracellular matrix. Indeed, previous studies have shown that
although proteolytic cleavage of aggrecan leads to a progressive loss
of the G3 domain with aging (9), the hyaluronan-aggrecan complexes are
still retained in the tissue. Cross-linking may, however, be of
importance in the extracellular matrix formation during development and
in response to damage. Support for this notion is found in heart development. In the heart defect (Hdf) mice, truncation of
the versican gene by insertion of a transgene resulted in heart
malformation and embryonic lethality (24, 25). Interestingly, fibulin-1 and -2 are produced together with versican and hyaluronan in the endocardial cushion tissue (26), suggesting involvement of the fibulins
in organization of the hyaluronan-versican complexes of the developing
heart extracellular matrix.
The fibulins are a growing family of extracellular matrix proteins
(27). Like fibulin-1, fibulin-2 is an extended protein containing two
globular domains. The carboxyl-terminal domain III corresponds to the
novel FBLC (fibulin/fibrillin carboxyl-terminal domain) motif (27). The
other globular domain, I, consists of three anaphylatoxin-like repeats.
The rod-like domain II, which is composed of EGF-like repeats, connect
the two globular domains. Fibulin-2 contains an additional rod-like
amino-terminal domain (28, 29). Fibulin-1 can form dimers but is
predominantly found as in the monomeric form. Fibulin-2, on the other
hand, forms disulfide-linked dimers (30). Interactions between domains
N and II give the fibulin-2 dimers a polymorph shape with X-, Y-, or
rod-shaped dimers (30). During development, fibulins are broadly
expressed (31-33) with particularly high levels in the developing
heart valves (26, 34). In the adult, both fibulin-1 and -2 are found in
microfibrils and in many other tissue structures, notably in basement
membranes and vessel walls (28, 35, 36).
We here show that fibulin-2 also is a strong ligand for several
lecticans. They interact through their lectin domains with two
different binding sites in domain II of fibulin-2. The dimeric nature
of fibulin-2 allows for efficient cross-linking of hyaluronan-lectican complexes, which could play an essential role in the supramolecular organization of cartilaginous and other extracellular matrices.
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EXPERIMENTAL PROCEDURES |
Production of Recombinant Alkaline Phosphatase-tagged Lectin
Domains--
The construction of His-tagged rCLD mammalian expression
pCEP4 plasmids have previously been described (15, 18). A cDNA coding for alkaline phosphatase (AP) was inserted into the pCEP4 plasmid at the SalI site between the lectin domains and the
histidine tags. The AP cDNA was amplified from the AP-tag-1 plasmid
(37) using the following primers: 5'-GACCGTCGACCATCATCCCAGTTGAGGAG-3' and 5'-CAGCGTCGACCCAGGGTGCGCGG-3'. All expression constructions were
sequenced before use. The resulting expression vectors code for fusion
proteins containing an immunoglobulin signal peptide, a C-type
lectin-like domain, soluble alkaline phosphatase, and a hexahistidine
tag. The recombinant proteins were produced in human embryonal kidney
293-EBNA cells and purified from the cell culture medium through metal
affinity chromatography as described previously (18).
Recombinant Fibulin-2 Proteins--
The production of most of
the recombinant fibulin-2 proteins has been described previously(30).
The novel fragments EG + III (position 1036-1195), (EG)2 + III (position 993-1195), IIb-e (position 730-912), and Iid-g
(position 821-993) were produced by amplifying the corresponding
regions of the cDNA. Primers were used in the polymerase chain
reaction that introduced an NheI site immediately upstream
and an XbaI site immediately downstream the fibulin-2 fragment. The resulting products were then introduced into the pCEP-Pu
mammalian expression vector containing the BM-40 signal peptide (38).
The recombinant proteins all contain the vector-derived amino acid
sequence APLA preceding the first amino acid residue of the fibulin-2
fragment. The recombinant proteins were produced in 293-EBNA cells,
purified, and characterized as described previously (30).
Surface Plasmon Resonance Binding Studies--
The different
rCLDs were diluted with 10 mM sodium acetate, pH 4.0, and
immobilized in different flow cells of a CM5 sensorchip (BIAcore,
Uppsala, Sweden). Immobilization levels were between 1500 and 2000 resonance units, as described previously (18). For affinity
measurements, binding and dissociation were monitored in a BIAcore 2000 instrument. The recombinant proteins were injected at different
concentrations over the rCLD-coated surfaces at 35 µl/min (in running
buffer: 10 mM Hepes, pH 7.5, 150 mM NaCl,
0.005% surfactant P20, and 1 mM CaCl2,
25 °C). A flow cell subjected to the coupling reaction without
protein was used as a control for bulk resonance changes. The lectin
surfaces were regenerated by injection of a 200-µl pulse of running
buffer containing 20 mM EDTA followed by 50 µl of 20 mM CaCl2 between each experiment. In control
experiments with the same concentrations of recombinant proteins, but
with 5 mM EDTA instead of CaCl2 in the running
buffer, no binding was seen. Bacterially expressed fibronectin type III repeats 3-5 from rat tenascin-R were used as a positive control (18).
After X and Y normalization the blank curves from
the control flow cell were subtracted, and association
(ka) and dissociation (kd) rate
constants were determined using a global simultaneous Langmuir fit in
the BiaEvaluation 3.0 program. The equilibrium dissociation constants
(KD) were calculated from these values.
Solid Phase Competition Binding Studies--
Microtiter wells
(NUNC Maxisorp, Nalge, Denmark) were coated at room temperature
overnight with 1.5 µg/ml tenascin R, fibulin-1C, and fibulin-2 II + III, respectively. The wells were washed with TTBS-Ca (5 mM
CaCl2, 150 mM NaCl, 0.05% Tween 20, 50 mM Tris-HCl, pH 7.4), incubated 1-2 h with blocking
solution (3% BSA in TTBS), and washed with BSA/TTBS-Ca (0.3% BSA in
TTBS-Ca). Potential inhibitors, i.e. the proteins used for
coating, were loaded into the wells in triplicates starting at a
dilution of 400 nM together with 1 µg/ml (14 nM) of AP-tagged lectin domain (AcL-AP-his, NcL-AP-his, and
VcL-AP-his, respectively) or AP alone in BSA/TTBS-Ca and incubated 1 h. After washing extensively with TTBS-Ca, substrate (1 mg/ml
-nitrophenyl phosphate in 5 mM MgCl2, 100 mM NaCl, 100 mM Tris-HCl, pH 9.5) was added,
and the absorbance at 405 nm was measured after a 1-h incubation.
Extraction of Aggrecan in the Native State--
Swarm rat
chondrosarcoma tumor tissue was dissected free of surrounding fascia
and sliced with a razor blade, and 200 g were briefly homogenized
(Polytron homogenizer, Kinematica GmbH, Switzerland) in 1 liter (5 ml/g
tissue) of prechilled TBS-Ca (5 mM CaCl2, 150 mM NaCl, 50 mM Tris-HCl, pH 7.4) containing 5 mM benzamidine hydrochloride, 5 mM
N-ethylmaleimide, and 0.5 mM
phenylmethylsulfonyl fluoride (39). The resulting tissue dispersion was
extracted for 45 min at 4 °C with stirring after which residues were
collected by centrifugation for 30 min at 20,000 rpm (4 °C, Beckman
J2-21, JA-20 rotor, 48, 400 × gav). This
extraction cycle was repeated twice. The residual tissue was then
extracted 3 times, 2 times for 60 min, and finally overnight, as
described above in TBS-EDTA (10 mM EDTA) with protease inhibitors. For anion exchange a DE52 cellulose (Whatman) 10 × 5-cm column was equilibrated with 20 mM NaCl, 50 mM Tris-HCl, pH 7.4, loaded with supernatants from the
Ca2 + and EDTA extractions, washed, and eluted using a
gradient of 20 mM to 2 M NaCl over 2 bed
volumes. The flow rate was 25 ml/h, and fractions of 4 ml were
collected. Aliquots of the fractions were analyzed for contents of
specific proteins by SDS-polyacrylamide gel electrophoresis (PAGE) and
sequential Alcian Blue and Coomassie Brilliant Blue R-250 staining.
Fractions with proteoglycan retained in the stacking gel were pooled
(140 ml) and taken to gel filtration on a Sepharose CL-2B (Amersham
Pharmacia Biotech) 100 × 5-cm column equilibrated and eluted with
20 mM NaCl, 50 mM Tris-HCl, pH 7.4. The flow
rate was 40 ml/h, and 10-ml fractions were collected. As a final
concentration step, size-excluded material of interest was loaded onto
another DE52 cellulose 13 × 1-cm column and chromatographed as
described above. The flow rate was 10 ml/h, and fractions of 5 ml were collected.
Isolation of Versican from Cell Cultures--
MG63 osteosarcoma
cells (CRL 1427; American Type Culture Collection, Manassas, VA) were
grown in Dulbecco's modified Eagle's medium (Life Technologies,
Inc.). Versican was prepared by anion exchange chromatography of
conditioned culture medium. For batch binding, 10 ml of Q-Sepharose
Fast Flow (Amersham Pharmacia Biotech) was added to 1 liter of medium
(1% v/v) containing 5 mM benzamidine hydrochloride and 0.5 mM phenylmethylsulfonyl fluoride and incubated overnight at
4 °C. The resin was packed into a column, washed with 150 mM NaCl, 50 mM Tris-HCl, pH 8, and versican was
eluted with a 0-2 M NaCl gradient over 20 column bed
volumes. Versican containing fractions were identified by dot blotting
with monoclonal antibody 12C5 (40), pooled, and concentrated.
Digestion with Chondroitinase ABC--
Aggrecan and versican
were dialyzed against 100 mM sodium acetate, 100 mM Tris-HCl, pH 7.3. They were digested with 10 milliunits of chondroitinase ABC (Sigma) per mg of proteoglycan for 4 h at 37 °C in the presence of 10 µg/ml ovomucoid as protease inhibitor. The aggrecan digest was chromatographed on a Superose 6 (Amersham Pharmacia Biotech) 140 × 1-cm column equilibrated and eluted with 150 mM NaCl, 50 mM Tris-HCl, pH 7.4. Flow rates
were 10 ml/h, and 1.5-ml fractions were collected. The versican digest
was loaded onto a Mono-Q (Amersham Pharmacia Biotech) 1-ml column and
eluted using a gradient of 0-2 M NaCl over 10 column bed volumes.
Radioiodination and Affinity Purification--
Chondroitinase
ABC-treated aggrecan and versican were radiolabeled using
125I and IODO-BEADS (Pierce) and were recovered in TTBS-Ca
(5 mM CaCl2, 150 mM NaCl, 0.05%
Tween 20, 50 mM Tris-HCl, pH 7.4). Labeled material was
affinity-purified on recombinant tenascin-R fibronectin type III
repeats 3-5 (15) coupled to CNBr-activated Sepharose 4B (Amersham
Pharmacia Biotech). After an extensive wash with TTBS-Ca, bound
material was eluted with TTBS-EDTA (10 mM EDTA).
Solid Phase Binding Assays with Native Full-length
Proteoglycan--
Microtiter wells (Breakable Combiplate-Enhanced
Binding, Labsystems Oy, Finland) were coated with 1.5 µg/ml
tenascin-R, fibulin-1C, or fibulin-2 in 10 mM sodium
carbonate buffer, pH 9.5, overnight at room temperature in a humid
chamber. After washing the wells with TTBS-Ca or TTBS-EDTA,
radiolabeled and affinity-purified aggrecan or versican at different
concentrations was added at a starting dilution of 100,000 and 40,000 cpm/well, respectively, in triplicates in either TTBS-Ca or TTBS-EDTA.
After overnight incubation the wells were extensively washed and
counted in a gamma counter (Packard Cobra II Quantum, Packard
Instrument Co.).
Electron Microscopy--
Glycerol spraying/rotary shadowing,
negative staining, and evaluation of the data from electron micrographs
were carried out as described previously (41). For rotary shadowing
20-µl aggrecan samples (typical concentrations 10-20 µg/ml) were
dialyzed overnight at 4 °C against 0.2 M ammonium
hydrogen carbonate, pH 7.9. They were subsequently mixed with equal
volumes of 80% glycerol and sprayed onto freshly cleaved mica with a
nebulizer designed for small volumes. They were dried in vacuum and
shadowed under rotation with 2 nm platinum/carbon at 9°, followed by
coating with a stabilizing 10 nm carbon film. For negative staining
5-µl samples of complexes between fibulins and native aggrecan or
fibulins and rCLDs (typical concentrations 5-10 µg/ml in TBS/5
mM CaCl2) were adsorbed to 400-mesh
carbon-coated copper grids, washed briefly with water, and stained with
0.75% uranyl formate. Since the rCLDs alone were too small to be
visualized, we used AP-tagged proteins. After mixing the proteins in
equimolar amounts, they were incubated 1 h before adsorption of
the formed complexes to grids. Aggrecan complexes, affinity purified as
above, were also mixed with an affinity-purified antibody against the
aggrecan lectin domain to distinguish the G3 domain of the core
protein. The grids were rendered hydrophilic by glow discharge at low
pressure in air. In some experiments the fibulins or rCLDs were labeled
with colloidal thiocyanate gold (42). Specimens were observed in a Jeol
1200 EX transmission electron microscope operated at 60-kV accelerating voltage. Images were recorded on Kodak SO-163 plates without
preirradiation at a dose of typically 2000 electrons/square nm.
Antibodies and Immunohistochemistry--
Mouse tissues were
fixed in 95% ethanol, 1% acetic acid, embedded in paraffin, cut into
4-µm sections, and immunostained as described previously (18).
Antibodies used were directed against rat aggrecan (18), mouse
fibulin-1 (affinity purified) (43), and mouse fibulin-2 (affinity
purified) (43). An antiserum was also raised against recombinant rat
aggrecan lectin domain and will be described in detail elsewhere.
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RESULTS |
Fibulin-2 Binds Proteoglycan Lectin Domains--
Fibulin-2 bound
strongly to the rCLDs of aggrecan, versican, and brevican lectin
domains in BIAcore experiments (Fig. 1). In contrast, fibulin-2 showed no binding to the neurocan rCLD, whereas
this protein was functional and bound tenascin-R (not shown). There was
only a very slow dissociation of fibulin-2 bound to aggrecan (Fig.
1A) or versican (Fig. 1C) rCLD, whereas the fibulin-brevican rCLD complex showed more pronounced dissociation (Fig.
1B). All interactions depended completely on calcium ions as
expected for C-type lectins (not shown). The KD
values of the proteoglycan rCLD interactions with fibulin-2 were in the subnanomolar to nanomolar range (Table
I).

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Fig. 1.
Fibulin-2 binds proteoglycan C-type
lectins. Recombinant full-length fibulin-2 was injected over
recombinant proteoglycan C-type lectin domains in a BIAcore 2000. A, aggrecan rCLD. B, brevican rCLD. C,
versican rCLD. Injection started at 115 s and ended at 235 s
(arrows). No binding was seen to neurocan rCLD (not shown).
Bound fibulin-2 was rapidly and completely removed through injection of
EDTA (not shown).
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Table I
Dissociation constants for the interactions between the different
lectican rCLDs and fibulin-2, fibulin-1C, fibulin-1D, and tenascin-R
Data were obtained by surface plasmon resonance analysis. NB indicates
no binding.
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The Different Proteoglycan Lectin Domain Ligands Compete for the
Same Binding Site--
In solid phase assays using a surface coated
with fibulin-2, we allowed alkaline phosphatase-tagged aggrecan or
versican rCLD to bind to the coated protein in the presence of varying
concentrations of the different ligands (tenascin-R FnIII-repeat 3-5,
fibulin-1C, and fibulin-2). Binding of versican rCLD to the immobilized
ligand protein could be inhibited by any of the other proteins (Fig. 2B). Aggrecan rCLD binding to
fibulin-2 could only be inhibited by tenascin-R and fibulin-2 but not
by fibulin-1C at a 20-fold molar excess (Fig. 2A).

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Fig. 2.
Fibulins and tenascin-R compete for the same
binding site on aggrecan and versican C-type lectin domains.
Microtiter plate wells were coated with recombinant fibulin-2;
unspecific binding sites were blocked with albumin, and alkaline
phosphatase-tagged aggrecan (A) or versican (B)
rCLDs (14 nM) was added in the presence of varying
concentrations of tenascin-R fibronectin type III repeats 3-5
( ), fibulin-1C ( ), or fibulin-2 ( ). After incubation
and washing, substrate was added, and the phosphatase reaction product
was measured at 405 nm.
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In assays using surfaces coated with the ligands tenascin-R FnIII 3-5
and fibulin-1C (not shown), binding of versican rCLD could be equally
inhibited by all three competitors. Aggrecan rCLD binding to tenascin-R
was not inhibited by fibulin-1C in solution, and this rCLD did not bind
to a fibulin-1C surface (not shown). Neurocan rCLD and AP alone did
not bind to the coated fibulin surfaces (not shown).
Native Full-length Proteoglycans Can Be Affinity-purified on
Tenascin-R--
To obtain full-length aggrecan, we extracted and
purified rat chondrosarcoma under native conditions. The associatively
extracted aggrecan is present in its ternary complex together with
hyaluronan and link protein (44). After digestion with chondroitinase
ABC, the core protein was radioiodinated and affinity-purified on the rCLD-binding tenascin-R FnIII 3-5 fragment (Fig.
3A). Bound aggrecan was eluted
with EDTA, taking advantage of the calcium dependence of CLD binding.
SDS-PAGE analysis of the eluted material (Fig. 3B) shows
accumulation of aggrecan core protein in the full-length molecular
weight range, fragments, and copurified link protein. We confirmed that
the eluted material consisted of full-length aggrecan core protein rich
in globular domains G1, G2, and G3 (9) by glycerol spraying/rotary
shadowing electron microscopy (Fig. 3D). The nonbinding
fraction, however, contained the pair of G1 and G2 domains at the amino
terminus but lacked in most cases the carboxyl-terminal domain G3 (Fig.
3C).

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Fig. 3.
Affinity purification of full-length
aggrecan. Aggrecan isolated from rat chondrosarcoma under native
conditions was chondroitinase ABC-digested, radioiodinated, bound to
tenascin-R fibronectin type III repeats 3-5 immobilized on Sepharose,
washed, and eluted with EDTA (A, arrow). SDS-PAGE
(B) of the eluted fractions show dominant full-length
aggrecan core protein and link protein (39 kDa) bands. The proteins of
intermediate molecular weight may represent aggrecan fragments. As
visualized using rotary shadowing electron microscopy, full-length
aggrecan rich in globular G3 domain is found in the eluate
(D, G3 denoted by arrowheads), and the
flow-through contained aggrecan lacking G3 (C).
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Affinity-purified Proteoglycans Bind Lectin Domain Ligand in Solid
Phase Assays--
In the proteoglycan G3 domain the CLD is flanked by
EGF and sushi repeats. To determine whether these motifs influence the CLD ligand binding, we performed solid phase binding assays with the
affinity-purified radioiodinated full-length aggrecan. The full-length
proteoglycan indeed shows the same binding characteristics as the
aggrecan rCLD. Aggrecan binds tenascin-R (Fig.
4A) and fibulin-2 (Fig.
4C) in a calcium-dependent manner, whereas no binding was observed to fibulin-1C (Fig. 4B). Full-length
versican isolated from cell cultures was radiolabeled and
affinity-purified using the same protocol as for aggrecan (not shown).
In solid phase assays, versican bound tenascin-R (Fig. 4D),
fibulin-1 (Fig. 4E), and fibulin-2 (Fig. 4F),
which was dependent on calcium and comparable to the versican rCLD.

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Fig. 4.
Binding of purified native full-length
aggrecan and versican to tenascin-R and fibulins in solid phase
assays. Radiolabeled aggrecan and versican were tested for binding
to the known lectin domain ligands tenascin-R fibronectin type III
repeats 3-5 (A and D), fibulin-1C (B
and E), and fibulin-2 (C and F).
Circles show results upon incubation in the presence of
CaCl2 and triangles in the presence of
EDTA.
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The Proteoglycan rCLD Interacts with the Calcium Binding EGF
Repeats of Fibulin-2--
The interaction sites for the proteoglycan
rCLDs on fibulin-2 were mapped using a collection of different
fragments of the molecule (Fig.
5A) recombinantly expressed in
293-EBNA cells. As displayed in Table II,
the central domain II (fragment II) is sufficient for proteoglycan rCLD
binding with similar affinities as for the full-length fibulin
molecule. Binding, albeit weak, was also observed to some subfragments
of domain II (fragments EG + III, (EG)2 + III and IIb-e)
but not to fragment IId-g. This suggests the presence of two
cooperative binding sites for the proteoglycan rCLDs on the fibulin-2
molecule, as outlined in Fig. 5B. The previously mapped
binding sites on fibulin-1C (18) are shown for comparison.

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Fig. 5.
A, schematic outline of the different
recombinant mouse fibulin-2 proteins used for mapping the binding site
for aggrecan and versican C-type lectin domains. Full-length fibulin-2,
different combinations of domains (fragments N + I and II + III),
isolated domains (fragments N, I, and II), and subdomain fragments (Na,
EG + III, (EG)2 + III, IIb-e, and Iid-g) were used. The
domain structure of dimeric fibulin-2 as seen in rotary shadowing is
outlined on top. B, summary of BIAcore interaction mapping
of aggrecan and versican rCLD binding sites on fibulin-1 (18) and
fibulin-2. Binding regions are indicated by filled domain
symbols.
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Table II
Mapping of the binding site on fibulin-2 for the proteoglycan C-type
lectin-like domains by surface plasmon resonance affinity measurements
The units used are as follows: ka,
M 1 s 1 × 10 3;
kd, s 1 × 106;
KD, nM.
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Electron Microscopy--
We used negative staining electron
microscopy to confirm the results of the mapping experiments.
Fibulin-1C was found to be dumbbell-shaped monomers (Fig.
6A) as previously reported
using rotary shadowing microscopy (45). Fibulin-2 (Fig. 6B)
was found predominantly as X-shaped dimers (compare schematic in
Fig. 5A, top). A morphometric analysis was based on a large
set of molecular complexes (Fig. 6, G-N). In agreement with
the different numbers of EGF-like repeats present in the two fibulins,
the center-to-center distances between the globular domains in
fibulin-1C and fibulin-2 were found to be 15 and 20 nm, respectively
(Fig. 6, G and H). In the latter case the two
globular domains I and III could be separately identified due to the
additional N-domain. Gold-labeled aggrecan rCLD (Fig. 6, C
and D) and versican rCLD (Fig. 6, E and F) bound to the central stretch of calcium binding EGF-like
repeats in domain II of both fibulin-1C (Fig. 6, C and
E) and fibulin-2 (Fig. 6, D and F).
When we measured the distance from the bound proteoglycan lectin domain
to the closest globular domain of fibulin-1C, we found that the
aggrecan binding distribution was restricted to a narrow region of
domain II close to that globular domain (Fig. 6I). The
versican binding was more widely distributed along domain II (Fig.
6M). These interaction sites correspond well
with the BIAcore binding site mapping (Fig. 5B). Aggrecan
rCLD binding to fibulin-2 showed a bimodal distribution on domain II
with some binding close to the globular domain I (Fig. 6J)
and more extensive binding closer to domain III, also in correspondence
with biochemical data (Fig. 5B). Versican rCLD showed a more
equal binding distribution for the same two regions along domain II
(Fig. 6N), comparable to mapping data. More than 70% of the
aggrecan and versican rCLDs were bound, whereas brevican and neurocan
rCLDs showed no significant binding (below 5% of total rCLD) to both
fibulins. Interactions of unlabeled native full-length aggrecan with
fibulin-1 (Fig. 6K) and fibulin-2 (Fig. 6L) were
shown to be in accordance with those of the lectin domain alone.
Interestingly, both negative staining (Fig.
7) and rotary shadowing (not shown)
electron microscopy data of full-length aggrecan and fibulins-1 and -2 demonstrate the ability of dimers of both fibulins to act as
cross-linkers between different hyaluronan-aggrecan complexes. The
binding was to the aggrecan G3 domain positively identified by
antibodies against the C-type lectin. There was no indication of an
association of the fibulins with the filament representing hyaluronan
with bound link protein and hyaluronan-binding region.

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Fig. 6.
Visualization of aggrecan and versican rCLD
interactions with fibulin-1 and fibulin-2 by negative staining electron
microscopy. Fibulins were visualized alone or after incubation
with gold-labeled alkaline phosphatase-tagged proteoglycan rCLDs.
A, fibulin-1C. The arrow points to a fibulin-1
dimer. B, fibulin-2. C, aggrecan rCLD binding to
fibulin-1C. D, aggrecan rCLD binding to fibulin-2.
E, versican rCLD binding to fibulin-1C. F,
versican rCLD binding to fibulin-2. Arrowheads point to
gold-labeled rCLDs. Note binding of the rCLDs to the central
inter-globular EGF-like repeat stretch of both fibulins. The measured
center-to-center distances between the globular domains of fibulin-1C
and fibulin-2 are shown in G and H, respectively.
I-N show the relative frequency distribution of the
distance from the closest globular domain of fibulins for bound
proteoglycan rCLD and full-length proteoglycan core proteins. Binding
of aggrecan rCLD and full-length aggrecan is restricted to a narrow
region of fibulin-1C domain II close to a globular domain (I or III not
distinguishable) (I and K, respectively).
Versican rCLD (M) binding is more widely distributed on
fibulin-1C. Aggrecan rCLD binding to fibulin-2 shows a bimodal
distribution on domain II with some binding close to the globular
domain I and more extensive binding closer to domain III
(J). Full-length aggrecan is binding fibulin-2 in the same
pattern as isolated lectin domain (L). Versican rCLD show a
more equal binding distribution for the same two regions along domain
II (N). The distances were measured on >100 molecules for
each panel.
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Fig. 7.
Fibulin dimers act as cross-linkers.
Full-length aggrecan forming networks with fibulin-1 (A) and
fibulin-2 (B) as visualized using negative staining electron
microscopy. The black dots in the panels are colloidal
thiocyanate gold attached at the globular domains of the fibulin
molecules. Both panels are sections of larger networks. The aggrecan
interactions with fibulin-1 and -2 are shown in detail in C
and D, respectively. The aggrecan G3 domains are denoted by
arrowheads. In E and F the G3 domain
(arrowheads) is identified by the use of anti-aggrecan
lectin domain antibodies (arrows). Scale bar = 80 nm in A and B and 20 nm in
C-F.
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Immunohistochemistry--
Immunohistochemical stainings to
identify the different proteins in various tissues show an overlapping
pattern of aggrecan and fibulin-1 and/or fibulin-2 epitopes in growth
plate and adult articular and adult ear cartilage (Fig.
8). Aggrecan and the two fibulins all
show an intense staining in embryonic day 15.5 growth plate cartilage
templates, whereas in the two adult tissues, fibulin-1 is predominantly
found in articular cartilage and fibulin-2 in ear cartilage. It is
noteworthy that much of the staining is found in the cartilage
pericellular/territorial regions, with little staining of the
interterritorial matrix (see articular cartilage).

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Fig. 8.
Immunohistochemical staining for fibulin-1,
fibulin-2, and aggrecan. Fetal femur growth plate embryonic day
15.5, adult articular cartilage, and adult ear cartilage were
paraffin-embedded and cut in 4-µm thick sections. These were stained
with antibodies against fibulin-1 (left panels), fibulin-2
(center panels), or aggrecan (right panels).
Scale bar = 100 µ m.
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DISCUSSION |
In this work we demonstrate that fibulin-2 is a high affinity
ligand for the C-type lectin domains of the proteoglycans aggrecan, brevican, and versican. The affinities for versican and aggrecan rCLDs
are in the sub-nanomolar to nanomolar range, as measured by surface
plasmon resonance technology. No binding to the neurocan rCLD could be
detected. Furthermore, we show that fibulin-2 competes for the same
binding site as tenascin-R on the aggrecan and versican rCLDs and at
least on the versican rCLD also with the site for fibulin-1 binding. In
addition, we confirm that full-length proteoglycans can interact with
the rCLD ligands by affinity chromatography on tenascin-R and in solid
phase assays bind to tenascin-R and fibulin-2. We do not yet have an
explanation why no interaction is seen between fibulin-1 and aggrecan
rCLD or full-length aggrecan in the solid phase assay. Adsorption may,
however, lead to conformational changes in the fibulin-1 molecule
affecting binding site functionality. In previous work, we did find a
strong calcium-dependent interaction between the aggrecan
rCLD and fibulin-1 in solution through copurification and through
surface plasmon resonance assays (18). However, in contrast to the
versican rCLD interaction, fibulin-1 does not compete for aggrecan rCLD
binding to tenascin-R or fibulin-2 in solid phase assays. This
indicates separate binding sites for tenascin-R/fibulin-2 and fibulin-1
on the aggrecan lectin domain, but this awaits more detailed analysis.
In this respect it is interesting that the versican lectin domain
showed good binding. This may indicate differences in the binding sites
of the lectins. In agreement with BIAcore data, in solid phase assays
neurocan rCLD linked to AP showed no binding to the fibulins nor did AP alone.
Mapping of the interaction site on the fibulin-2 molecule revealed that
the proteoglycan rCLDs bind to two nonoverlapping sites on calcium
binding EGF-like repeats in the central rod-like domain of the
molecule. We observed a large drop in KD for the
interaction with lectican rCLDs when using shorter fibulin-2 fragments
containing only one binding site. This probably reflects cooperative
binding of the two sites in the larger fragment to the multiple rCLD
molecules immobilized on the BIAcore flow cell surface. Measurements of
the distance between the gold-labeled rCLDs and the globular domains of
the fibulins in negative staining electron microscopy showed distinct
narrow binding of aggrecan rCLD and full-length core protein to the
parts of domain II in fibulin-2 corresponding to the binding sites
observed in the BIAcore mapping experiments. In the fibulin-1 case we
also found a narrow binding site for the aggrecan rCLD and core
protein, which presumably represents the carboxyl-terminal site of
interaction disclosed in the BIAcore experiments. The versican rCLD
showed a wider range of binding within domain II of fibulin-1 and two
distinct populations of binding sites for fibulin-2. This is in good
agreement with the BIAcore mapping of the fibulin binding sites. By
using negative staining electron microscopy, we were also able to
observe directly fibulin-mediated cross-linking of hyaluronan-aggrecan
complexes. The fibulin-2 dimers were under these experimental
conditions predominantly found in the X-shaped form (30). We failed to observe cross-linking mediated by the rod- or Y-shaped fibulin-2 dimers, suggesting that the interactions between domains N and II may
render the lectican-binding sites inaccessible. Fibulin-1 was mostly
found in monomeric form although some dimers were observed. Since only dimers could act as aggrecan cross-linkers, fibulin-1 may be
less efficient in organizing hyaluronan-lectican complexes.
Immunohistochemical staining show that both fibulins are codistributed
with proteoglycan in different tissues, suggesting that the examined
interactions are of physiological relevance. Striking examples are the
strong expression of versican and both fibulins in the endocardial
cushion tissue during heart development (26, 31, 32, 34, 46) and the
prominent expression of aggrecan and both fibulins in newly formed
cartilage and bone. Indeed, treatment of endocardial cushion tissue
sections with hyaluronidase to degrade hyaluronic acid led to removal
of both versican and fibulins, whereas fibronectin staining remains
unaffected (26). Furthermore, disruption of the versican gene results
in heart malformation and early embryonic death (24, 25). This indicates that the correct formation of a hyaluronan-versican-fibulin network may be important for heart development.
Both fibulins are present in precartilaginous mesenchymal condensations
and developing cartilage (18, 32, 33). It may be that fibulin-1 and -2 binding by the aggrecan C-type lectin domain plays a significant role
during the development of cartilage and bone, perhaps in the
organization process of the early matrix. Indeed, in the embryonic day
15.5 femur growth plate aggrecan and both fibulins are expressed in an
overlapping pattern. However, versican may also in this context be
another relevant binding partner for the two fibulins, since versican
expression precedes the one of aggrecan in cartilage development (47,
48).
In adult tissue, fibulin-1 staining in articular cartilage overlaps
with that of aggrecan, whereas no fibulin-2 is detected. In view of the
more rapid turnover of aggrecan in the territorial domain of articular
cartilage, it is interesting to note that the staining for aggrecan, as
well as fibulin-1, is more pronounced in this region. On the other
hand, in ear cartilage, staining for fibulin-2 is more intense, and
colocalization with aggrecan is seen. It should be noted, however, that
we only find fibulin-1 staining in some of many examined joints.
In this context it is interesting to note that an increasing fraction
of aggrecan molecules in the extracellular matrix is truncated with age
leading to the loss of G3 domain (9). This suggests that the lectin
domain interactions are important during development or in assembling
newly synthesized aggrecan into the matrix.
The proteoglycan rCLDs bind to fibulin-1 and -2 through EGF-like
repeats but bind to a fibronectin type III repeat in tenascin-R (15)
and tenascin-C (16). Interestingly, the lectican binding is in all
these cases directed to extended rod-like stretches of multimeric
extracellular matrix molecules. Although the biological functions of
these interactions remain to be investigated, the notion that all four
ligands form multimeric complexes in the extracellular matrix suggests
that these proteins act to cross-link the proteoglycan-hyaluronan
complexes in the tissue.