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INTRODUCTION |
Articular cartilage matrix can be regarded as a fiber-reinforced
composite material (1), where aggrecan complexes are entangled within a
network of collagen fibrils. The aggrecan complexes, constituting about
90% of the proteoglycan content (2), endow the matrix with high
osmotic pressure, compressive stiffness, and resilience, whereas
collagen is essential for the tensile strength of the tissue (3).
Different mechanical properties of the composite depend on these major
constituents and how they are assembled and stabilized by
intermolecular interactions. The capacity of cartilage to withstand
mechanical stress depends upon its structural integrity and, hence,
numerous interactions between the matrix components. Indeed, the
molecules are so tightly associated that most of the tissue
constituents require denaturing solvents or proteases for extraction.
This has hampered studies of molecular function. To gain insight into
the physiology of articular cartilage, it is necessary to identify and
characterize interactions between the matrix constituents, particularly
those involving the collagen.
In the present study we focused on a domain of the aggrecan molecule
containing the majority of the keratan sulfate
(KS)1 chain substituents and
a protein segment with a hexapeptide repeat sequence (4). The second
globular domain (G2) is localized adjacent to this KS-rich region, on
the N-terminal side. The first globular domain (G1), which represents
the hyaluronan-binding region (HABr), is localized next to G2 as the
very N-terminal portion of aggrecan. On the C-terminal side of the
KS-rich region, there is a large chondroitin sulfate (CS)-rich region
that accounts for more than 80% of the molecular mass. The very
C-terminal end contains a number of distinct domains including one with
homology to C-type lectins.
Proteoglycan aggregates are made up of several aggrecan molecules that
are bound via their G1 domains to hyaluronan. The binding is stabilized
by a third component, the link protein, that binds simultaneously to
aggrecan and hyaluronan (see Heinegård and Sommarin, Ref. 5). In the
aggregate, the G2 and KS-rich portions are interspaced between the
central hyaluronan strand and the region with the densely packed CS
chains. The structure of the KS-rich region suggests a relatively
extended and rigid conformation. This is supported by electron
microscopy of spread proteoglycan aggregates after rotary shadowing,
showing that the CS-rich region is clearly separated from the central
filament of hyaluronan (6). It appears that the KS-rich region acts as
a spacer within the proteoglycan aggregate. Interestingly, the
dimensions of this region are similar to the diameter of the collagen fibril.
The primary structure of the KS-rich region polypeptide encompasses a
number of consecutive proline-rich hexapeptide repeats, where the
bovine form contains 23 of these repeats (4). The proline-rich repeat
may form a polyproline coil.2
Such a structure has the potential for interacting with the collagen fibril, which in this case could find sufficient space to run through
the center of the proteoglycan aggregate and by interactions reinforce
the network of assembled aggrecan molecules. This hypothesis was put to
test in the present work.
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EXPERIMENTAL PROCEDURES |
Proteoglycan Fragments--
Fragments of bovine nasal cartilage
aggrecan were prepared according to previously described procedures
(7). The trypsin-resistant fragment KS.t represents the KS-rich region
extended to and including parts of the G2 domain (8). A more
extensively trimmed fragment, KS.t.c, which was obtained by additional
digestion with chymotrypsin, represents exclusively the KS-rich region
(7). The tryptic fragment HABr represents the hyaluronan-binding
region, whereas the preparation CS-pep contains several peptides
derived from the CS-rich region. Intact aggrecan core protein devoid of
CS was prepared by digestion with chondroitinase ABC (9).
Purification of Antibodies--
The antibodies against the
KS-rich portion of aggrecan were prepared from a polyclonal antiserum
raised against the entire bovine aggrecan molecule (10). Two affinity
columns containing CNBr-activated Sepharose 4B (Amersham Pharmacia
Biotech), to which isolated aggrecan fragments had been coupled, were
used for the purification. The antiserum was first passed through a
column containing the fragment HABr coupled at 2 mg/ml of gel. The
flow-through fraction of the antiserum, thus devoid of unspecifically
binding antibodies or antibodies recognizing epitopes within the HABr, was subsequently passed through a column containing gel with coupled KS.t. Bound antibodies were eluted with 3 M potassium
isocyanate followed by dialysis against 0.15 M NaCl, 5 mM NaPi, pH 7.4.
Enzyme-linked Immunosorbent Assay--
Specificity of purified
antibodies was examined by using an inhibition-type enzyme-linked
immunosorbent assay (10). Samples of antibody solution were
preincubated with different isolated aggrecan fragments. The incubation
mixtures were then transferred to microtiter plates coated with core
protein of bovine aggrecan. Antibodies bound to this protein coat were
quantified by incubation with a swine anti-rabbit (IgG) alkaline
phosphatase conjugate (Orion Chemicals), followed by paranitrophenyl
phosphate as a substrate for bound enzyme.
A microplate solid-phase assay was used to measure binding of
proteoglycan fragments to immobilized collagen molecules. The assay
procedure was similar to that described previously (11), with some
modifications as detailed in the following. Microplate wells were
coated overnight with pepsin-extracted collagen I at 10 µg/ml or
collagen II at 50 µg/ml in 0.14 M NaCl, 30 mM
NaPi, pH 7.3 and after that coated for 1 h with
-casein at 100 µg/ml to block unspecific interactions. The wells
were washed with buffer containing 0.05% Tween 20 and then incubated
with ligand solution. Antibodies used for detection of bound ligand
were either the affinity purified anti-KS.t antibodies or the
monoclonal 5D4 that specifically recognizes KS (12). These primary
antibodies were detected with a secondary antibody, the former with a
swine anti-rabbit (IgG) alkaline phosphatase conjugate and the latter
with a goat anti-mouse (IgG) (Orion Chemicals), and the substrate
paranitrophenyl phosphate. Absorbance was measured at 405 nm, and the
data were processed as described (11).
Before some of the binding experiments, KS was digested using either
endo-
-galactosidase (Boehringer Mannheim) or keratanase II
(Seikagaku Corp.). Samples of KS.t and KS.t.c were digested with
approximately 5 milliunits of enzyme/mg of substrate in 0.14 M NaCl, 30 mM NaPi, pH 7.3, for
2 h at 37 °C. Efficiency of digestion was confirmed by
SDS-polyacrylamide gel electrophoresis, showing higher migration rates
of fragments after enzyme treatment.
In one set of experiments, the binding of KS.t after papain cleavage
was examined. An aliquot of a solution containing the proteoglycan
fragment was combined with papain (200 µg/mg of proteoglycan) in 10 mM EDTA, 2 mM dithioerythritol, 0.14 M NaCl, 30 mM NaPi, pH 7.3, and
digested at 40 °C for 24 h. The enzyme was then inactivated by
addition of iodoacetamide to a concentration of 20 mM and
heating in a boiling water bath for 5 min. A control sample was
prepared from another aliquot of the KS.t solution and some papain
solution, treated identically as the digested sample except that the
enzyme and the proteoglycan fragment were kept separated until the
inactivation procedure had been completed.
Electron Microscopy--
The distribution of the aggrecan
KS-rich region in bovine articular cartilage was examined in low
temperature-embedded tarsometatarsal (fetlock) joints. Slices
comprising the entire cartilage with adjoining subchondral bone, were
cut from central areas of the proximal medial metatarsal condyle and
processed as described elsewhere (13). In short, fixation was performed
in a mixture of 0.3% glutaraldehyde and 0.3% paraformaldehyde and
then dehydrated by stepwise increasing concentrations of methanol at
temperatures progressively lowered to 228 K. Embedding was performed in
Lowicryl K11M (Chemische Werke Lowi GmbH, Waldkraiburg, Germany).
Ultrathin (40-50 nm) sections were placed on Formvar-coated nickel
grids and preincubated with 10% bovine serum albumin for 2 h,
followed by incubation with primary antibody in phosphate-buffered
saline containing 0.1% bovine serum albumin. The uncalcified cartilage was stratified into three zones. The zones and the compartments around
the cells were those defined previously (14, 15).
Sampling from each of two blocks per animal (n = 5) and
measurements were performed as described elsewhere (14, 15).
Immunoreactivity was visualized with protein-A conjugated to 10-nm gold
probes (Amersham Pharmacia Biotech, UK) in sections contrasted with
uranyl acetate and lead citrate.
For assessing the association between collagen fibrils and
labeling (n = 3), matrix was divided into two
compartments. Fibrillar labeling was defined as gold particles
projected over collagen fibrils. Interfibrillar labeling was defined as
all that outside the border of the fibril. The number of gold probes
was counted, and the collagen volume density (v/v) in the pericellular
and territorial compartments of zone II was measured by point counting (16). Immunoreactivity was then correlated to collagen v/v of the
pericellular and territorial compartments and a ratio between fibrillar
and interfibrillar labeling was calculated for each compartment.
To study the distribution of the aggrecan KS-rich region along the
collagen fibrils, bovine tarsometatarsal joint cartilage (n = 3) was prepared for cryosectioning as described
elsewhere (15). Ultrathin cryosections (100 nm) were incubated with
primary antibodies and immunoreactivity was detected with
protein-A-coated 10 nm gold probes. From random high power (× 250,000)
electron micrographs, the distribution of gold probes along the fibrils was measured (15).
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RESULTS |
Specificity of Antibodies--
Binding of affinity purified
antibodies to the aggrecan core protein was measured by enzyme-linked
immunosorbent assay. To determine the localization of epitopes within
the aggrecan structure, inhibition of binding by different aggrecan
fragments was examined (Fig. 1). Neither
isolated HABr nor peptides representing the CS-rich region inhibited
the binding of antibodies. Two different preparations of fragments
containing the KS-rich region strongly inhibited binding. The fragments
prepared using trypsin in combination with chymotrypsin inhibited the
antibody binding to the same extent as the fragments prepared using
trypsin only. Because the fragments remaining after chymotrypsin
digestion essentially represent the KS-rich region proper,
i.e. the hexapeptide repeat structure with the KS chains,
the antibodies used here mainly recognize epitopes within this
region.

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Fig. 1.
Specificity of antibodies toward the keratan
sulfate-rich region of aggrecan. Affinity purified antibodies were
incubated in microplate wells coated with aggrecan core protein.
Inhibition of antibody binding was tested by preincubation of the
antibodies with enzymatically generated cleavage products of aggrecan,
i.e. peptides representing the chondroitin sulfate-rich
region (CS pep.), the hyaluronan-binding region
(HABr), the entire core protein (Core), a
fragment containing the keratan sulfate-rich region and parts of the
second globular domain (KS.t), and a somewhat smaller
fragment corresponding essentially to the keratan sulfate-rich portion
only (KS.t.c). Bound antibodies were detected by a swine
anti-rabbit (IgG) alkaline phosphatase conjugate and paranitrophenyl
phosphate as a substrate for bound enzyme.
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Binding of Aggrecan KS-rich Fragments to Collagens in
Vitro--
As discussed above, the KS-rich region has a structure
indicative of a potential for binding to collagen. To examine this possibility, we studied binding of the isolated domain to collagen in vitro. A microplate solid-phase binding assay was used.
The fragment KS.t, which contains the KS-rich region and the
polypeptide up to and in some fragments actually including parts of the
second globular domain, showed saturable binding to collagen I as well as collagen II (Fig. 2, top).
The amounts of KS.t bound at saturation were larger for wells with
collagen I than for wells with collagen II, probably reflecting
differences in the amounts of immobilized collagen. In a Scatchard-type
plot, the data displayed linear correlations, indicating that the
binding sites were homogeneous (Fig. 2, bottom). The
affinity of the KS.t fragment was the same for collagen I and collagen
II, with an apparent dissociation constant of 1.1 µM.
This represents a fairly weak interaction. For comparison, the
affinities shown by the small proteoglycans decorin and fibromodulin in
the same type of assay were about 100 times higher (11).

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Fig. 2.
Binding of an aggrecan fragment
(KS.t) containing the keratan sulfate-rich region and
parts of the second globular domain, to isolated collagens in
vitro. Microplate wells were coated with collagen I
( ), collagen II ( ), or -casein only ( ), followed by
incubation with samples of KS.t at concentrations from 0 to 256 µg/ml. Bound KS.t was determined by enzyme-linked immunosorbent assay
using affinity purified antibodies as in Fig. 1. Conversion of the
substrate to colored product was measured as increase of absorbance at
405 nm (top). Specific absorbance changes ( A)
were calculated by subtraction of the absorbance values for control
wells coated with -casein. These data were used for a Scatchard-type
plot (bottom), assuming a molecular mass of the KS.t
fragment of 160 kDa (7). The dissociation constants, calculated from
the slopes of the fitted lines, were 1.1 µM both with
collagen I and collagen II.
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Binding of the fragment KS.t.c, apparently representing the repeat
polypeptide with the KS-chains only (7), to collagen I was not
different from that of KS.t (Fig. 3).
Hence, the peptide portions adjacent to the repeat defining the true
KS-rich region did not contribute significantly to the collagen
interactions. Moreover, the fragments still had capacity to bind
to collagen after enzymatic hydrolysis of the KS (Fig. 3) using either
keratanase II or endo-
-galactosidase. However, the binding capacity
was dramatically reduced if the KS.t core protein was extensively cleaved by digestion with papain (Fig.
4). These observations support the view
that the interaction depends on the core protein.

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Fig. 3.
Binding of aggrecan fragments to isolated
collagen I in vitro. Microplate wells, coated
with collagen I, were incubated with samples of aggrecan fragments KS.t
or KS.t.c at either 5 µg/ml or 50 µg/ml. In some cases, KS was
digested with keratanase II (k'ase). Some wells served as
negative controls and were incubated with buffer only. Affinity
purified antibodies were used for detection as in Fig. 1.
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Fig. 4.
Binding to collagen I in vitro
of aggrecan fragments derived from the KS-rich region.
Microplate wells, coated with collagen I ( , ), or -casein
only( , ), were incubated with samples of KS.t fragments that had
been pre-treated with active papain ( , ). Alternatively, the KS.t
samples contained papain that already was inactivated when added to the
proteoglycan fragments ( , ). Bound fragments were detected by
enzyme-linked immunosorbent assay with KS-specific monoclonal
antibodies and a goat anti-mouse (IgG) alkaline phosphatase conjugate.
Conversion of the substrate paranitrophenyl phosphate to colored
product was measured as increase of absorbance at 405 nm.
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To further characterize the collagen interaction of the KS.t
fragment, the sensitivity to increased ionic strength was examined. Binding was not affected by small differences in salt concentration within the near-physiological range, but it was gradually decreased at
NaCl concentrations higher than 0.15 M (Fig.
5). This suggests that charged groups are
important for the interaction. The primary structure of the KS.t core
protein consists of repeated hexapeptide units, typically with the
sequence Glu-Xaa-Pro-Phe-Pro-Ser (4). Glutamic acid residues occupy the
first position in 22 of the 23 units and, in addition, the second
position in 11 of the units. Therefore, it is likely that the
negative charges of the glutamates have a role in the interaction.
Still, the observation that significant binding occurred at the
relatively high NaCl-concentrations of 0.5-0.6 M (Fig. 5)
suggests that other forces in addition to pure electrostatic attraction
are involved in this protein-protein interaction.

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Fig. 5.
Binding of the aggrecan KS.t fragment to
collagen I in vitro at different concentrations of
NaCl. Microplate wells were coated with collagen I ( ) or
-casein only ( ) and were then washed with 30 mM
phosphate buffer containing 1 M NaCl and with buffer
containing 0.1 M NaCl. Next, the wells were incubated with
samples of KS.t at 50 µg/ml in phosphate buffer containing NaCl at
concentrations ranging from 0.1 to 0.6 M. Unbound KS.t was
removed by washing the wells four times with solvent of unchanged
composition, followed by washing of all wells with buffer containing
0.1 M NaCl. Bound KS.t was detected by enzyme-linked
immunosorbent assay, which was done similarly as in Fig. 4 except that
a solvent buffer containing 0.1 M NaCl was used.
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Ultrastructural Immunolocalization of the Aggrecan KS-rich
Region--
Electron microscopy of bovine articular cartilage after
immunogold-labeling with the antibodies, showed that reactivity was lowest in the superficial part and about two times higher in the middle
and deep parts (Table I). Labeling of the
interterritorial matrix was slightly lower than that for the matrix
closer to the cells. Because it is likely that all aggrecan molecules
retained in the matrix will contain the KS-rich region, regardless of
partial fragmentation, the distribution of the KS.t immunoreactivity
can be taken as a measure of aggrecan distribution. The results
corroborate and extend those obtained by other techniques (17-19).
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Table I
Immunoreactivity of aggrecan keratan-sulfate-rich region in uncalcified
bovine articular cartilage
Zone I is the superficial part; zone II is the upper part of the radial
zone; and zone III is the lowermost part of the radial zone.
Resin-embedded sections; mean (S.D.) (n = 5); *,
denotes significant difference between zone I and II; #,
denotes significant difference between zone I and III.
p < 0.05.
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Immunoreactivity for the aggrecan KS-rich region was preferentially
localized within the proximity of collagen fibrils. Thus, fibrillar
labeling was about 2-4 times higher than interfibrillar labeling in
the pericellular and territorial compartments (Table II). Only probes projected on a collagen
fibril or tangent to the border of the fibril were counted as
fibril-associated (Fig. 6). This should
represent an underestimation of the true fibrillar labeling because the
dimensions of the immunogold complex allow a proportion of the gold
particles attached to targets on the collagen fibril to be projected
outside the fibril. The expected diameter of the complex is about 23 nm
(20), and consequently a major proportion of the probes within this
distance from the fibril surface may reflect the fibril-associated
KS-rich region. However, we refrained from regarding any probes not
touching the fibrils as associated because this would have imposed
difficulties in defining the interfibrillar matrix.
Unfortunately, it was still not possible to distinguish between
fibrillar and interfibrillar labeling on micrographs showing
interterritorial matrix. This was because of the high volume density of
fibrils and concomitant overprojection of gold particles.
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Table II
Correlation of aggrecan keratan sulfate-rich region to collagen fibrils
in the pericellular and territorial compartments of zone II in bovine
articular cartilage
Resin-embedded sections; mean (S.D.) (n = 3).
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Fig. 6.
Electron microscopic immunolocalization of
the aggrecan keratan sulfate-rich region in bovine articular
cartilage. The micrograph was taken from the pericellular area
within the superficial zone of the articular cartilage, and it shows
collagen fibrils together with colloidal gold probes for
immunoreactivity. Probes projected over the fibrils or tangent to the
border of the fibrils (fibrillar labeling) were counted as
collagen-associated (open arrows), whereas probes projected
outside the border (interfibrillar matrix) were counted as
nonassociated (solid arrows). Resin-embedded section,
magnification × 95,000.
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The distribution of gold probes along the individual fibril was
assessed with cryosections at high power magnification. Cryosections were preferred because they give a higher intensity of labeling than
that obtained with sections of resin-embedded tissue. In this case,
immunolabeling with antibodies against the KS-rich region displayed a
non-random distribution along the fibril axis. Labeling showed a marked
preference to the gap region within the D-period (Fig.
7). A more precise identification of the
localization within the gap region is not feasible with the present
technique because of limitations imposed by the size of the immunogold
complex, which represents about one-third of the length of the D-period (20).

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Fig. 7.
Histogram showing the distribution of
aggrecan keratan sulfate-rich region immunoreactivity in relation to a
collagen fibril in bovine articular cartilage. Measurements were
performed on high-power micrographs from cryosectioned cartilage. To
delineate the distribution, the first thin band of one D-period was
identified, and the distance to each probe was measured. Note the
non-random distribution within the D-period, with preference to the gap
region (dark field). n = 123.
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DISCUSSION |
Previous histochemical studies have indicated a close spatial
relationship between proteoglycans and collagen in soft tissues, e.g. in tendon and annulus fibrosus (21) and in articular
cartilage (22). Immunohistochemical examination has shown that the
small dermatan sulfate proteoglycan decorin is associated with fibrils in tendon (23) and dermis (24), apparently because of interactions between the core protein and collagen. In cartilage, both decorin and
collagen IX occur as fibril-associated proteoglycans (25, 26), albeit
that most of the collagen IX molecules in mammalian cartilage do not
appear to have an attached glycosaminoglycan chain (27). Furthermore,
we have observed that the keratan sulfate proteoglycan fibromodulin is
distributed along collagen fibrils in articular cartilage (15), also
apparently bound via its core protein (11, 28). These small
proteoglycans are bound to the fibrils by strong interactions, in the
case of collagen IX stabilized by covalent bonds. Therefore, these
molecules can be considered as fibril constituents. In contrast, the
large cartilage proteoglycan aggrecan occurs in the tissue in a well
characterized supramolecular form that is distinct from the fibrils.
This aggregate consists of up to 100 aggrecan monomers and an equal
number of link proteins, associated with a single strand of hyaluronan.
It is likely that there are interactions between collagen fibrils and
the proteoglycan aggregates, important for the mechanical properties of
cartilage, but the mechanisms are not known. Based on work with
in vitro systems, it has been proposed that the CS-rich
region of aggrecan is essential for interactions (29, 30). The
existence of an interaction of some kind within the tissue, is
supported by observations made by immunoperoxidase electron microscopy
indicating a periodic arrangement of the proteoglycan along cartilage
fibrils (31).
The results presented here provide strong evidence that a proteoglycan
aggregate interacts with a collagen fibril located within the
aggregate, near the central hyaluronan strand. It is reasonable to
assume that the KS-rich region represents an extended structure
surrounded by free space. This molecular architecture may allow
diffusion of some matrix constituents, e.g. procollagen, and
may participate in specific interactions. Hypothetically, a collagen
fibril may be positioned as a backbone within the aggrecan complex and
connect several aggrecan KS-rich regions (Fig.
8). Even though each KS-rich region forms
a fairly weak interaction, the multiplicity existing in the aggregate
would result in a very tight complex with the collagen fibril. Such an
arrangement would considerably influence the structural integrity of
the proteoglycan aggregate. It could also have an important role in
providing the microenvironment for the formation of fibrils in
cartilage.

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Fig. 8.
Schematic illustration of a hypothetical
interaction between a cartilage collagen fibril and aggrecan monomers
via the keratan sulfate-rich region. Because of multiple
interactions with the proteoglycan aggregate, the fibril may serve as a
backbone to the aggrecan complex.
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