From the Connective Tissue Biology Laboratories,
School of Biosciences, Cardiff University, Museum Avenue, Cardiff
CF10 3US, Wales, United Kingdom and ¶ Collagen Research Group,
School of Veterinary Science, University of Bristol, Langford,
Bristol BS18 7DY, United Kingdom
Received for publication, September 26, 2000, and in revised form, November 9, 2000
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ABSTRACT |
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Using competitive binding experiments, it was
found that native type XI collagen binds heparin, heparan sulfate, and
dermatan sulfate. However, interactions were not evident with
hyaluronic acid, keratan sulfate, or chondroitin sulfate chains over
the concentration range studied. Chondrocyte-matrix interactions
were investigated using cell attachment to solid phase type XI
collagen. Pretreatment of chondrocytes with either heparin or
heparinase significantly reduced attachment to type XI collagen.
Incubation of denatured and cyanogen bromide-cleaved type XI collagen
with radiolabeled heparin identified sites of interaction on the
Type XI collagen is a component of the heterotypic collagen
fibrillar network found in cartilage that, along with proteoglycan, gives cartilage its unique structural and biomechanical properties. The
type XI collagen molecule consists of three genetically distinct polypeptide chains, namely By immuno-electron microscopy, it was found that the triple helical
domain of type XI collagen was buried within the heterotypic fibril
(4). However, it has been detected without the use of chaotropic agents
both pericellularly (5) and, also, more generally throughout the matrix
(4, 6), suggesting that some type XI collagen molecules in cartilage
are not buried and are therefore available for interaction.
Type XI collagen has been shown to be associated with the surface of
bovine articular chondrocytes in suspension culture (7). It is also
known that the triple helical domain binds to heparin-agarose with
greater affinity than other cartilage collagens, an activity that has
been exploited as a tool for its purification (8). Therefore, the
triple helix of type XI collagen may have the potential to bind
glycosaminoglycans present on the surface of chondrocytes.
Although type XI collagen is associated with type II collagen in
cartilaginous matrices, several studies show that chains of type XI and
type V collagen can coexist within tissues (9-11). Much work has been done on the glycosaminoglycan and cell binding
properties of type V collagen (for review, see Ref 15), and a region
within the The recognition of heparin and heparan sulfates as surface components
in various cell types (18, 19) suggests that type XI collagen may be
important in cell-matrix interactions. The glycosaminoglycans
associated with the cartilage matrix, however, are hyaluronic acid,
chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, and
keratan sulfate. In this study, we have used competitive binding
experiments to determine the relative affinities of the type XI
collagen molecule for various glycosaminoglycans. The contribution of
heparinase- and collagenase-labile interactions to chondrocyte binding
to type XI collagen has also been investigated. Furthermore, the use of
rotary shadowing techniques has enabled localization of heparin binding
sites on the triple helical molecule. These studies provide evidence
for a more complex range of interactions between the triple helical
domain of type XI collagen and glycosaminoglycans than was previously
thought. Such interactions may be partly responsible for the role of
type XI collagen in establishing and maintaining cartilage matrix integrity.
Purification of Type XI Collagen and Preparation of Cyanogen
Bromide Peptides--
Type XI collagen was isolated from porcine
articular cartilage by limited pepsin digestion. The cartilage was
extracted with 4 M guanidinium chloride in 0.05 M Tris-HCl buffer, pH 7.5, to remove proteoglycan. Pepsin
(1 mg/100 mg of tissue) was dissolved in 0.5 M acetic acid
and added to the insoluble material. After digestion at 4 °C
overnight, insoluble material was sedimented by centrifugation, and the
supernatant containing the pepsin-extractable material was precipitated
sequentially with 0.7, 0.9, 1.2, and 2.0 M NaCl. The
precipitates were collected by centrifugation. The 1.2 M
precipitable fraction was redissolved in acid and reprecipitated. This
was repeated until the fraction appeared to be homogeneous and free of
other collagen types, as determined by SDS-polyacrylamide gel
electrophoresis (PAGE)1
(20).
Cyanogen bromide (CNBr) peptides of type XI collagen were prepared by
incubating 5 mg of purified pepsinized type XI collagen with 10 mM dithiothreitol in phosphate-buffered saline (PBS) for 30 min at room temperature and subsequently with 5 mg of CNBr in 70%
(v/v) formic acid at 30 °C for 4 h. After digestion, the reaction mixture was diluted 10-fold with water, dialyzed extensively against 10 mM acetic acid, and lyophilized.
Heparin Binding Studies--
To quantify binding of heparin to
type XI collagen, native pepsinized type XI collagen diluted in 5 mM acetic acid was separated into aliquots and applied to
nitrocellulose discs 4 mm in diameter. The coated discs were air-dried
and incubated for 2 h in 96-well plates (Falcon) in 100 µl of
PBS-Tween containing radiolabeled (N-[35S]sulfonate) heparin solution (15.8 mCi/g; 2 mCi/ml; 4-6-kDa molecular mass) (Amersham Pharmacia Biotech)
at a final activity of 0.1-2.0 µCi/ml. After extensive washing in
PBS-Tween supplemented with NaCl to a final concentration of 0.25 M, the amount of radiolabeled heparin bound to the discs
was determined by scintillation counting.
To determine the specificity of the type XI collagen-glycosaminoglycan
interaction, competitive binding assays were performed. Type XI
collagen-coated nitrocellulose discs were incubated simultaneously with
radiolabeled heparin (1 µCi/ml; 64 µg/ml) and unlabeled heparin, heparan sulfate, de-N-sulfated heparin, chondroitin
4-sulfate, chondroitin 6-sulfate, keratan sulfate, dermatan sulfate,
and hyaluronic acid (0-1.05 mg/ml) in PBS. Unlabeled heparin
preparations (from porcine intestinal mucosa) and other
glycosaminoglycans were obtained from Sigma.
Preparation of Chondrocytes--
Articular chondrocytes were
isolated from porcine femoral head and condylar cartilage. The
cartilage was digested with 0.4% (w/v) Pronase (protease E, Sigma) in
Dulbecco's modified Eagle's medium containing 4 mM
glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml amphotericin B, and 5% (v/v) fetal calf serum at 37 °C.
Subsequently, the cartilage was digested with collagenase
(Clostridium histolyticum, Sigma) at 0.3% (w/v) in the same
medium. Cells were recovered and washed by centrifugation at 100 × g. The cells were maintained in suspension cultures
(5 × 105 cells/ml) in Dulbecco's modified Eagle's
medium containing 4 mM glutamine, 100 units/ml penicillin,
100 µg/ml streptomycin, 2.5 µg/ml amphotericin B, and 10%(v/v)
fetal calf serum for up to 48 h.
Cell Attachment and Blocking Assays--
Microtiter plate wells
were coated overnight with type XI collagen at 200 ng/well in 10 mM acetic acid at 4 °C. The plates were subsequently
blocked at room temperature using a 1 mg/ml solution of bovine serum
albumin in PBS. Control incubations were prepared by coating wells with
200 ng/well BSA. The plates were washed with PBS and used directly for
the attachment assays.
Cells were harvested from the suspension cultures and incubated for
2 h with a highly purified bacterial collagenase preparation that
exhibits no other proteinase activity (Form III, Advance Biofactures
Corp.) at 1.5 units/ml or with heparin (Sigma) at 10 units/ml. The
chondrocytes were collected by centrifugation at 100 × g for 10 min, washed twice in serum-free Dulbecco's
modified Eagle's medium by resuspension and centrifugation, and
dispensed onto the washed plates at a concentration of 5 × 104 cells/well. Cells were allowed to attach for 90 min at
37 °C in a humidified atmosphere containing 5% (v/v)
CO2. The plates were rinsed three times with PBS to remove
unattached cells, and the number of attached cells was determined by
measuring the N-acetylhexosaminidase activity (21). For
subsequent experiments, collagenase-pretreated cells were washed and
incubated on coated plates as above in serum-free Dulbecco's modified
Eagle's medium in the absence or presence of either heparinase
(heparinase III from Flavobacterium heparinum, Sigma) at 2.5 units/ml or heparin at 10 units/ml.
Identification of Binding Sites on Whole Chains and CNBr Peptides
of Type XI Collagen--
Whole and cyanogen bromide digests of
purified pepsinized type XI collagen were resolved using
SDS-polyacrylamide gel electrophoresis and electroblotted onto
polyvinylidene difluoride membrane (Immobilon P, Amersham Pharmacia
Biotech) (22). After incubation in PBS containing 0.05%(v/v) Tween 20, the membranes were incubated with 35S-radiolabeled heparin
at 2 µCi/ml for 2 h at room temperature. After washing with
PBS-Tween containing 0.25 M NaCl for 2 h, the blots
were air-dried and exposed to Hyperfilm-MP radiography film (Amersham
Pharmacia Biotech) for 5 days.
To isolate CNBr peptides containing heparin binding sites, CNBr digests
(5 mg) of type XI collagen in 10 mM sodium phosphate buffer, pH 7.4, containing 0.15 M NaCl were incubated
overnight at 4 °C with an aliquot of heparin-Sepharose (Amersham
Pharmacia Biotech) equilibrated in the same buffer. After extensive
washing of the heparin-Sepharose with 0.25 M NaCl in the
same buffer, the adsorbed peptide(s) was eluted using 0.5 M
NaCl, incubated with SDS-PAGE sample buffer at 60 °C for 30 min, and
resolved on a 10% (w/v) polyacrylamide gel.
Amino Acid Sequence Analysis--
Peptides separated by SDS-PAGE
on a 10% (w/v) polyacrylamide gel were electroblotted onto
Problott polyvinylidene membrane (Applied Biosystems) in 10 mM CAPS buffer, pH 11. NH2-terminal sequences
were determined by automated Edman degradation on a protein sequencing
system (Applied Biosystems) at the University of Manchester.
Identification of Binding Sites by Rotary Shadowing Electron
Microscopy of Type XI Collagen and Heparin-BSA Conjugate--
Type XI
collagen was solubilized in 10 mM acetic acid at a
concentration of 1 mg/ml. An aliquot of the collagen (25 µl) was added to 1.25 ml of 20 mM ammonium bicarbonate, pH 8, containing 25 µl of commercially available BSA-conjugated heparin
(Sigma). The mixtures were incubated at 4 °C for up to 24 h,
and subsequently, glycerol was added to the mixture to give a final
concentration of 30% (v/v). Samples were prepared by the sandwich
method. 25 µl of sample was spread between two 1.5-cm2
sheets of freshly cleaved mica (Agar Scientific Ltd., UK), which were
separated after 1 min and dried under vacuum in a metal-coating system
(Edwards 306). The samples were rotary-shadowed with platinum at an
angle of 9° and subsequently coated with pulse-evaporated carbon at
90°. The replicas were floated onto distilled water, picked up on
400-mesh copper grids, air-dried, and examined by electron microscopy
(Philips EM 400 or EM 208). Images were recorded at 50,000×
magnification and scanned at 600 dots/inch using an Epson GT-7000
scanner. Measurements of molecular length and respective sites of
heparin-BSA binding were acquired using a Windows 95 ProScan image
analysis program after calibration with reference to the 8.75 crystal
lattice-spacing of beef liver catalase.
Binding of Heparin to Native Type XI Collagen--
Heparin-agarose
has been employed successfully for the affinity purification of type XI
collagen, both before and after pepsinization. To ascertain the
specificity of this binding with respect to glycosaminoglycan species,
a nitrocellulose membrane solid phase assay was established in which
heparin-collagen binding could be optimized and quantified (Fig.
1). Optimal concentrations of the
collagen and of the radiolabel were established to be 10 µg of type
XI collagen/disc and 1 µCi/ml (64 µg/ml) radiolabeled heparin. The
heparin was maintained at ~64 µg/ml for subsequent assays.
The stoichiometry of the binding could not be calculated on a molar or
weight ratio by introducing unlabeled heparin preparations of a range
of molecular weight (average) into the assay (Fig. 2). The relationship between the
molecular weight of the glycosaminoglycan species and inhibition of
radiolabel bound was not linear. On a molar basis, this nonlinearity
was accentuated further with the concentration of the 3-kDa (average)
heparin preparation, required to reduce bound radiolabel by 50%, being
45-fold greater than the required concentration of the 17 kDa (average)
species. The average molecular mass of the radiolabeled heparin was 6 kDa. The pattern of charge densities along the heparin molecule appears to be a major factor in determining the affinity of binding, and this
is consistent with the lack of binding of de-N-sulfated
heparin.
These assays were not optimized for reliable measurement of binding
affinity, but the data obtained would suggest that the affinity is low
(not shown). Nevertheless, low affinity binding would still be
physiologically significant if the binding were complex, involving
multivalent interactions or several component molecules.
Inhibition of Heparin Binding to Native Type XI Collagen by Other
Glycosaminoglycans--
The specificity of the heparin binding site(s)
was investigated by introducing unlabeled glycosaminoglycan species
into the assay. Relative specificity of the glycosaminoglycans was
ascertained by their capacity to compete with the radiolabeled heparin
for type XI collagen binding. Over the concentration range studied, there appeared to be a hierarchy of binding affinity of the
glycosaminoglycans (Fig. 3). None of the
glycosaminoglycans could compete with radiolabeled heparin as
successfully as heparin itself. Both heparan sulfate and dermatan
sulfate significantly inhibited binding of radiolabel in a
concentration-dependent manner. Hyaluronic acid,
chondroitin 4-sulfate, chondroitin 6-sulfate, and keratan sulfate were
all ineffective over the concentration range studied (up to 1.05 mg/ml) (Table I).
Attachment of Chondrocytes to Native Type XI Collagen--
To
address the possibility that glycosaminoglycan type XI collagen
interactions may be of importance at the chondrocyte-matrix interface,
the binding of chondrocytes to native type XI collagen was investigated
using a cell binding assay. Fig.
4a summarizes the observed
effects of chondrocyte pretreatment with exogenous heparin and
bacterial collagenase on attachment to type XI collagen. The highly
purified bacterial collagenase preparation used only digests regions
containing the Gly-X-Y repeat of collagen,
without digesting the noncollagenous proteins or domains. Pretreatment of cells with purified bacterial collagenase to remove cell surface collagen reduced the binding. Heparin also significantly reduced cell
attachment to type XI collagen (p < 0.05) but also
increased nonspecific binding to BSA, suggesting that specific binding
was reduced to a greater extent. This indicates that heparin inhibits chondrocyte interaction with type XI collagen.
To ascertain whether the inhibitory effect of exogenous heparin was due
directly to a cell surface heparin-like molecule, chondrocytes
pretreated with collagenase were incubated with heparinase (Fig.
4b). Heparinase and heparin treatment both significantly (p < 0.05) reduced the specific cell attachment,
indicating that the type XI collagen-chondrocyte interaction observed
in vitro was due in part to a cell surface heparan
sulfate-like molecule.
Identification of Heparin Binding Sites in Denatured Type XI
Collagen by Blotting and NH2-terminal Sequencing--
Type
XI collagen binds to heparin-Sepharose with approximately the same
affinity, whether it has been digested with pepsin or not (data not
shown). Therefore, at least one binding site must be located within the
triple-helical region of the collagen molecule. The nature of this
binding was investigated further to establish whether a binding site(s)
could be found on individual chains resolved by SDS-PAGE, or were
composites of sequences contained within more than one chain.
Pepsinized type XI collagen was resolved into its component
The binding of heparin to electroblotted CNBr peptides of type XI
collagen indicated that the binding site was not susceptible to CNBr
cleavage (Fig. 5, lanes 3 and 4). NaCl
concentrations of greater than 0.35 M were required to
elute the radiolabel from the electroblotted type XI collagen chains or
peptides (data not shown), suggesting that the affinity of binding to
denatured collagen was similar to that of native type XI collagen.
CNBr peptides containing a heparin binding site were adsorbed onto a
heparin-Sepharose column at a NaCl concentration of 0.25 M
and, following extensive washing of the column with the same buffer,
were eluted using a NaCl gradient, a peak of eluant occurring at around
0.375 M NaCl (data not shown). This experiment was repeated using batch-wise elution of heparin-Sepharose to which type XI collagen
CNBr peptides had been bound (Fig. 6).
CNBr peptides of type XI collagen were applied to heparin-Sepharose at
0.15 M NaCl. After washing in 0.25 M NaCl, the
heparin-Sepharose was washed with an equal volume of 0.5 M
NaCl. The peptides contained in each fraction are shown in Fig. 6. The
eluted peptides were resolved on a 10% (w/v) polyacrylamide gel and
electrotransferred onto polyvinylidene difluoride (Problott). Several
faint bands are apparent in the 0.5 M NaCl fraction.
However, one main band was seen, as described earlier.
NH2-terminal sequencing revealed that the peptide was the
product of CNBr cleavage of Rotary Shadowing of Native Type XI Collagen Triple Helix and
Heparin-BSA Conjugate--
To establish whether heparin binds to other
sites along the triple helix, the binding of type XI collagen to a
heparin-BSA conjugate was observed using rotary shadowing (Fig.
7a); the heparin conjugate had
an approximate stoichiometry of five heparin molecules per BSA molecule
(information from supplier). Large aggregates were often observed,
presumably due to the presence of multiple heparin molecules on each
BSA molecule. However, in a sample of 115 isolated molecules (mean
length 306.7 ± 8.4 nm), 50 exhibited heparin-BSA binding. As
shown in the upper six panels of Fig. 7a, the
heparin-BSA appeared to bind predominantly to a site localized at
95.3 ± 11.7 nm from one end of the triple helix of the type XI
collagen molecules. In addition, a near-terminal site located ~281.7 ± 6.0 nm from one end of the molecule was observed (Fig. 7a, lower left and lower middle
panels). The frequency distribution of binding along the type XI
collagen triple helix is presented in Fig. 7b.
We have investigated the glycosaminoglycan binding properties of
the 300-nm triple helical domain of type XI collagen to evaluate further its potential to interact with proteoglycan species in cartilage, thereby extending previous studies on the type XI
collagen-glycosaminoglycan interaction (8, 23). Previously we have
found that type XI collagen interacts with other matrix molecules in
cartilage, necessitating the use of SDS to extract it from growth plate
and articular cartilage (6) after pepsin digestion.
From the current study, heparin binding to type XI collagen appears to
be independent of the triple helical conformation as it interacts with
both native and denatured preparations of the collagen. The
glycosaminoglycan specificity of the binding to the native triple
helical molecule is very similar to that reported for other heparin
binding molecules (24), with heparin and heparan sulfate showing the
greatest affinity for the type XI collagen. As confirmation that the
specificity observed was due to charge density alone, the
nonphysiological but highly sulfated glycosaminoglycan, dextran
sulfate, containing four sulfate groups per disaccharide and an average
molecular mass of 10 kDa (Sigma), was found to bind with greater
affinity than heparin itself (data not shown). Furthermore, in the
current study, de-N-sulfated heparin did not interact with
type XI collagen.
In our cell binding studies, we found that chondrocytes bind to type XI
collagen and that this binding was reduced when cells were incubated
with heparin or heparinase, suggesting that type XI collagen binds to a
cell surface heparan sulfate proteoglycan. Previous studies (7) showed
that chondrocytes in culture retained type XI collagen at the
cell-medium interface, whereas type II collagen could be released into
the medium. Also, immunolocalization studies showed that the triple
helix of type XI collagen and, presumably, the heparin binding site are
accessible in pericellular regions in cartilage (25, 5). It may be
speculated, therefore, from the specificity of the binding, that type
XI collagen triple helix binds to the small heparan sulfate or dermatan
sulfate containing proteoglycans that are present in cartilage,
particularly at the cell surface.
The binding of type XI collagen to cell surface proteoglycan may be
important in the organization and stabilization of the cartilage
matrix. Type XI collagen can regulate type II collagen fibril diameter
in vitro (26, 2), and as fibrillogenesis is initiated at the
cell surface, the anchoring of type XI collagen may in some way control
this process in vivo. Although alternative splicing of the
NH2-terminal domains of type XI collagen (27, 28) indicates
an important role for these domains in cartilage development, it is not
known whether these proteolytically susceptible domains (29, 30) are
retained in adult cartilage. Therefore, the interactions of
proteoglycans with the triple helical domain may be important for
maintaining tissue integrity, particularly in the pericellular environment.
We have identified two heparin binding sites on type XI collagen at
around 24 and 95 nm from the NH2-terminal of
pepsin-extracted collagen. The location of the 25-nm site is derived
from the rotary shadowing data that indicate the presence of a site at
around 280 nm from one end of the helix, which is located around 70 nm from the 95- or 100-nm site identified by sequence analysis and from
previous studies. The 95-nm site identified by rotary shadowing coincides with the sequence obtained from the heparin-binding CNBr
peptide of 1(XI) and
2(XI) chains. NH2-terminal sequence
data confirmed that the predominant heparin-binding peptide contained
the sequence GKPGPRGQRGPTGPRGSRGAR from the
1(XI) chain. Using
rotary shadowing electron microscopy of native type XI collagen
molecules and heparin-bovine serum albumin conjugate, an additional
binding site was identified at one end of the triple helical region of
the collagen molecule. This coincides with consensus heparin binding
motifs present at the amino-terminal ends of both the
1(XI) and the
2(XI) chains. The contribution of glycosaminoglycan-type XI collagen
interactions to cartilage matrix stabilization is discussed.
INTRODUCTION
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ABSTRACT
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1(XI),
2(XI), and overglycosylated
1(II) chains, and is typical of the fibrillar class of collagens having a 300-nm triple helical domain (1). Although type XI collagen is
a relatively minor collagen in cartilage, it is believed to be
important in the regulation of fibril diameter (2) and in maintaining
tissue integrity and cohesion. Mice homozygous for the autosomal
recessive chondrodysplasia (cho) mutation in the
col11a1 gene (3) do not synthesize
1(XI) chains and have larger cartilage collagen fibrils, less cartilage matrix cohesion, and
increased extractability of proteoglycans. However, neither the domains
of type XI collagen responsible for these activities nor the molecules
with which they interact are known.
1(XI) mRNA
has also been detected in tumors (12), placenta-derived cell lines
(13), and in a variety of noncartilaginous embryonic chick tissues
(14). As a result, type V and type XI collagen chains are believed to
participate in the formation of heterotypic molecules with
stoichiometries not previously assigned. They are therefore considered
as a single collagen type (15).
1(V) chain has been identified as the site through which
ionic interactions with glycosaminoglycan moieties occur (16). This
site contains several basic amino acid residues and is located ~100
nm from the amino-terminal end of the triple helical domain of the
molecule. Further studies (17) demonstrate that binding of heparin to
residues 905-921 in this
1(V) site is influenced by secondary
structure. The
1(XI) chain shows considerable homology with the
1(V) chain of type V collagen (12), and the sequence of amino acids
comprising the heparin binding site within
1(V) is also present
within
1(XI).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Concentration-dependent binding
of radiolabeled heparin to nitrocellulose discs coated with type XI
collagen. Binding of 35S-heparin to type XI collagen
was measured as described under "Experimental Procedures."
Discs were coated with 20 µg (open squares), 10 µg
(filled squares), 5 µg (open circles), 2.5 µg
(filled circles), and 1.25 µg (triangles) of
type XI collagen and subsequently incubated with radiolabeled heparin.
The values represent the amounts of radioactivity bound after extensive
washing of the discs.
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Fig. 2.
Inhibitory effect of unlabeled heparin
preparations on the binding of radiolabeled heparin to type XI
collagen. Discs were coated with 10 µg of type XI collagen. The
discs were incubated simultaneously with radiolabeled heparin (~64
µg/ml) and unlabeled heparin preparations of 6 kDa (open
squares), 3 kDa (filled triangles), and 17 kDa
(open triangles). Results of incubations with
de-N-sulfated heparin (filled squares) are also
shown. Values represent the amounts of radiolabeled heparin
bound.
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Fig. 3.
Inhibitory effect of unlabeled
glycosaminoglycans on the binding of radiolabeled heparin to type XI
collagen. Discs were coated with 10 µg of type XI collagen.
Radiolabeled heparin (~64 µg/ml) and glycosaminoglycans were
incubated with the coated discs simultaneously, as described under
"Experimental Procedures." The values represent the
radiolabeled heparin bound in the presence of competing
glycosaminoglycans: filled squares, dermatan sulfate;
filled circles, heparan sulfate; open squares,
chondroitin 4-sulfate; and open circles, unlabeled
heparin.
Inhibitory effect of glycosaminoglycans on binding of radiolabeled
heparin to type XI collagen immobilized on nitrocellulose
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Fig. 4.
Chondrocyte attachment to type XI
collagen. a, chondrocytes were harvested from
suspension cultures and incubated for 2 h with purified bacterial
collagenase or heparin. After washing, the cells were plated on
microtiter plate wells coated with type XI collagen (solid
bars) or BSA (hatched bars). b, chondrocytes
pretreated with collagenase were incubated in type XI collagen or
BSA-coated microtiter plate wells in the presence of heparin or
heparinase. The values represent the absorbance of the product of the
colorimetric N-acetylhexosaminidase assay used to quantify
numbers of cells attached to the wells. The values shown are means ± S.E. of mean (n = 6).
chains
on 7% (w/v) polyacrylamide gels by SDS-PAGE and transferred onto
polyvinylidene difluoride membrane. Autoradiography of radiolabeled
heparin bound to electroblotted type XI collagen revealed that both the
1 and the
2 chains of type XI collagen contained binding sites
(Fig. 5, lanes 1 and 2), suggesting that the primary structure of the individual
chains was sufficient for heparin binding. Heparin did not bind to the
1(II) chain.
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Fig. 5.
Binding of radiolabeled heparin to
electroblotted type XI collagen. Lane 1 shows
pepsinized type XI collagen resolved on a 7% polyacrylamide gel
stained with Coomassie blue. Simultaneously, another lane
loaded identically was electroblotted and incubated with radiolabeled
heparin as described under "Experimental Procedures."
Lane 2 shows the autoradiograph of electroblotted type XI
collagen after incubation. Similarly, CNBr peptides of type XI collagen
were resolved on a 10% polyacrylamide gel and either Coomassie
blue-stained (lane 3) or electroblotted and incubated with
radiolabeled heparin. The autoradiograph is shown in lane
4.
1(XI) at methionine 792 and commenced
with the sequence GLKGDRGEVGQ. From the human
1(XI) sequence (12) it
was deduced that this peptide would contain the sequence identified as
the heparin binding site within
1(V), GKPGPRGQRGPTGPRGSRGARGP (16),
confirming that this homologous region is a binding site in both type
XI and V collagen. An equivalent
2(XI) CNBr peptide bound to
heparin-Sepharose was not isolated.
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Fig. 6.
Amino-terminal sequencing of a
heparin-Sepharose-adsorbed type XI collagen cyanogen bromide
peptide. CNBr peptides of type II (lanes 1,
2, and 3) and type XI collagen (lanes
4, 5, and 6) were incubated with
heparin-Sepharose in 10 mM sodium phosphate buffer, pH 7.4, containing 0.15 M NaCl. Peptides that were eluted using
NaCl concentrations of 0.15 M (lanes 1 and
4), 0.25 M (lanes 2 and 5)
and 0.5 M (NaCl) were resolved by SDS-PAGE on a 10%
polyacrylamide gel. Peptides were resolved on a duplicate gel and
electrotransferred onto polyvinylidene difluoride membrane for
amino-terminal sequencing. The sequence GLKGDRGEVGQ was obtained for
the predominant type XI collagen peptide bound (indicated by an
asterisk).
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Fig. 7.
Rotary shadowing of heparin-BSA conjugate
bound to pepsinized type XI collagen. Type XI collagen was
incubated with heparin-BSA conjugate and visualized as described under
"Experimental Procedures." a, two predominant
binding sites were observed, at ~95 nm (upper six panels)
and at 280 nm (lower left and center panels).
Occasional molecules exhibited binding of heparin-BSA at both sites
simultaneously (lower right panel). Bar, 50 nm.
The distribution data for a representative number of labeled molecules
are summarized in the histogram (b).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1(XI) (Fig. 8a).
Both the
1(XI) and
2(XI) chains of type XI collagen contain, in
register, the motif of basic residues identified in the
1(V) chain
as the 100-nm heparin binding site. It is surprising therefore, that
the corresponding
2(XI) CNBr peptide was not identified by binding
of radiolabeled heparin. The sequence of amino acids in
2(XI) is not
identical to the KPGPRGQR sequence in
1(V) and
1(XI), defined as
the heparin binding motif (31). This difference would not, however,
explain the anomaly that the whole
2(XI) chain can bind heparin,
whereas the CNBr peptide containing the postulated site does not.
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Fig. 8.
Primary structure of the heparin binding
sites in type XI collagen. The amino acid sequences represent the
corresponding heparin binding sites observed by rotary shadowing
electron microscopy. Conserved basic residues are in
boldface. The available sequences for 1(V) (33),
1(XI)
(12), and
2(XI) (GenBankTM accession number CAA20240)
were aligned manually in the two regions of interest.
A recent study of type V collagen heparin binding has indicated that
the stoichiometries of type V collagen chains present may contribute to
binding sites of varying affinities (17) due to the presentation of
basic residues on one face of the polyproline II helix of each 1(V)
chain. Thus, the
1(V)3 homotrimer has a greater affinity
than the
1(V)2
2(V) heterotrimer. We have not been
able to isolate the
2(XI) peptide that corresponds to the 100-nm
site or that can bind heparin within the denatured
2(XI) chain.
Nevertheless, the
2(XI) chain may contribute to the 100-nm site in
the
1(XI)
2(XI)
1(II) heterotrimer, akin to the
1(V)2
2(V) molecule. Similar to the
1(V)2
2(V) heterotrimer, this site in type XI collagen
would have positive charges on the outside of the two contributing
-chains and would not form a continuous band around the triple
helical molecule. The 20-amino acid sequences depicted in Fig.
8a represent a distance of ~6 nm in the triple helix,
which would accommodate 7 disaccharide units. If the complete binding
site is required for optimal binding, this may explain why the 3-kDa
heparin (average 6 disaccharides) did not bind as well as the larger
heparin molecules. However, if the sequence (KPGPRGQR), defined as the
sequence accommodating a pentasaccharide (31), is the functional site,
the 3-kDa species should bind as well as the larger species.
The 25 nm binding site identified by rotary shadowing corresponds, in
both the 1(XI) and
2(XI) chains, to a region where there are a
number of basic residues (Fig. 8(b)). In fact, both chains contain the
postulated consensus heparin-binding motif XBBXBX (32) at this site,
where X represents any amino acid, while B represents a basic amino
acid. Such a site has been implicated in binding the triple helical
collagen tails of asymmetric acetylcholinesterase to heparin (33).
However, sequences R581 to R588 and R539 to R546 in the
1(XI) and
2(XI) chains respectively, also agree with the heparin binding motif
described earlier which accommodates a pentasaccharide (31). Thus, the
1(XI),
2(XI) and
1(V) chains have potential to bind heparin at
the NH2-terminal end by either of these motifs. While we
have identified this site in type XI collagen by rotary shadowing, this
site has not been reported for type V collagen. The corresponding type
XI collagen CNBr peptides of ~5 kDa were not detected in the assay in
which radiolabeled heparin was bound to CNBr peptides electroblotted
onto nitrocellulose. In both the
1(XI) and
2(XI) chains however,
the site is followed immediately by a methionine that would be cleaved
by CNBr. This may prevent the CNBr peptide assuming a structure
suitable for heparin binding. It has been suggested that there is a
requirement for conformational constraint within a polyproline II helix
on the heparin binding sequence of each chain; flanking sequences would
influence this secondary structure (17). The sequences may however also
need the constraints of being in a triple helical structure to function
as an efficient binding site.
It is unlikely that multivalent binding can occur between a single
glycosaminoglycan chain and both sites identified on one type XI
collagen molecule, either in our in vitro studies or
in vivo, due to spatial constraints. However, it is possible that both sites are accommodated by glycosaminoglycan chains present on
different cell surface heparan sulfate proteoglycans. This, along with
the formation of cross-links between type XI collagen monomers (34),
presents the possibility that type XI collagen-glycosaminoglycan interactions contribute to cell surface-matrix interactions of a range
of complexity and affinity. Indeed, the sum of a range of interactions
between type XI collagen and matrix or cell-associated proteoglycans
may be advantageous to matrix stabilization during periods of dynamic
remodeling. The interactions between the triple helical domain of type
XI collagen and glycosaminoglycan species may contribute therefore, to
the cohesiveness and integrity of articular cartilage matrix.
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ACKNOWLEDGEMENTS |
---|
We thank Linda Berry at the University of Manchester for assistance with the NH2-terminal sequencing and Sophie Gilbert for assistance with the graphics.
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FOOTNOTES |
---|
* This work was supported by Wellcome Trust Grant 048479/2/96 and by Arthritis Research Campaign Grant D0083.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 44-02920 875419; Fax: 44-02920 874594; E-mail: vaughan-thomas@cardiff.ac.uk.
Published, JBC Papers in Press, November 17, 2000, DOI 10.1074/jbc.M008764200
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ABBREVIATIONS |
---|
The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; BSA, bovine serum albumin; CAPS, 3-(cyclohexylamino)propanesulfonic acid.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Burgeson, R. E., and Hollister, D. (1979) Biochem. Biophys. Res. Commun. 87, 1124-1131[Medline] [Order article via Infotrieve] |
2. | Eikenberry, E. F., Mendler, M., Bürgin, R., Winterhalter, K. H., and Bruckner, P. (1992) in Articular Cartilage and Osteoarthritis (Kuettner, K. , Schleyerbach, R. , Peyron, J. , and Hascall, V., eds) , pp. 133-149, Raven Press, Ltd., New York |
3. | Li, Y., Lacerda, D. A., Warman, M. L., Beier, D. R., Yoshioka, H., Ninomiya, Y., Oxford, J. T., Morris, N. P., Andrikopoulos, K., Ramirez, F., Wardell, B. B., Lifferth, G. D., Teuscher, C., Woodward, S. R., Taylor, B. A., Seegmiller, R. E., and Olsen, B. R. (1995) Cell 80, 423-430[Medline] [Order article via Infotrieve] |
4. | Mendler, M., Eich-Bender, S. G., Vaughan, L., Winterhalter, K. H., and Bruckner, P. (1989) J. Cell Biol. 108, 191-197[Abstract] |
5. | Ricard-Blum, S., Hartmann, D. J., Herbage, D., Payen-Meyran, C., and Ville, G. (1982) FEBS Lett. 146, 343-347[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Wardale, R. J.,
and Duance, V. C.
(1993)
J. Cell Sci.
105,
975-984 |
7. | Smith, G. N., Jr., Hasty, K. A., and Brandt, K. D. (1989) Matrix 9, 186-192[Medline] [Order article via Infotrieve] |
8. | Smith, G. N., Jr., and Brandt, K. D. (1987) Collagen Relat. Res. 7, 315-321 |
9. | Niyibizi, C., and Eyre, D. R. (1989) FEBS Lett. 242, 314-318[CrossRef][Medline] [Order article via Infotrieve] |
10. | Kleman, J. P., Hartmann, D. J., Ramirez, F., and van der Rest, M. (1992) Eur. J. Biochem. 210, 329-335[Abstract] |
11. |
Mayne, R.,
Brewton, R. G.,
Mayne, P. M.,
and Baker, J. R.
(1993)
J. Biol. Chem.
268,
9381-9386 |
12. |
Yoshioka, H.,
and Ramirez, F.
(1990)
J. Biol. Chem.
265,
6423-6426 |
13. |
Bernard, M.,
Yoshioka, H.,
Rodriquez, E.,
van der Rest, M.,
Kimura, T.,
Ninomiya, Y.,
Olsen, B. R.,
and Ramirez, F.
(1988)
J. Biol. Chem.
263,
17159-17166 |
14. |
Nah, H. D.,
Barembaum, M.,
and Upholt, W. B.
(1992)
J. Biol. Chem.
267,
22581-22586 |
15. | Fichard, A., Kleman, J-P., and Ruggiero, F. (1994) Matrix Biol. 14, 515-531 |
16. | Yaoi, Y., Hashimoto, K., Koitabashi, H., Takahara, K., Ito, M., and Kato, L. (1990) Biochim. Biophys. Acta 1035, 139-145[Medline] [Order article via Infotrieve] |
17. |
Delacoux, F.,
Fichard, A.,
Geourjon, C.,
Garrone, R.,
and Ruggiero, F.
(1998)
J. Biol. Chem.
273,
15069-15076 |
18. |
Koda, J. E.,
Rapraeger, A.,
and Bernfield, M.
(1985)
J. Biol. Chem.
260,
8157-8162 |
19. | Bernfield, M., Kokenyesi, R., Kato, M., Hinkes, M. T., Spring, J., Gallo, R. L., and Lose, E. J. (1992) Annu. Rev. Cell Biol. 8, 365-393[CrossRef] |
20. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
21. | Landegren, U. (1984) J. Immunol. Methods 67, 379-388[CrossRef][Medline] [Order article via Infotrieve] |
22. | Towbin, H., Stachelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354[Abstract] |
23. |
Smith-Jr, G. N.,
Williams, J. M.,
and Brandt, K. D.
(1985)
J. Biol. Chem.
260,
10761-10767 |
24. | Ruoslahti, E., and Engvall, E. (1980) Biochim. Biophys. Acta 361, 350-358 |
25. | Duance, V. C., and Wotton, S. F. (1991) Biochem. Soc. Trans. 19 (suppl.), 376 |
26. | Vaughan-Thomas, A., and Duance, V. C. (1994) Int. J. Exp. Pathol. 75, (abstr.) 41-42 |
27. |
Zhidkova, N. I.,
Justice, S. K.,
and Mayne, R.
(1995)
J. Biol. Chem.
270,
9486-9493 |
28. |
Oxford, J. T.,
Doege, K. J.,
and Morris, N. P.
(1995)
J. Biol. Chem.
270,
9478-9485 |
29. | Maciewicz, R. A., Wotton, S. F., Etherington, D. J., and Duance, V. C. (1990) FEBS Lett. 269, 189-193[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Wu, J-J.,
Lark, M. W.,
Chun, L. E.,
and Eyre, D. R.
(1991)
J. Biol. Chem.
266,
5625-5628 |
31. |
Margalit, H.,
Fischer, N.,
and Ben-Sasson, S. A.
(1993)
J. Biol. Chem.
268,
19228-19231 |
32. | Cardin, A. D., and Weintraub, H. J. R. (1989) Arteriosclerosis 9, 21-32[Abstract] |
33. |
Deprez, P. N.,
and Inestrosa, N. C.
(1995)
J. Biol. Chem.
270,
11043-11046 |
34. |
Wu, J. J.,
and Eyre, D. R.
(1995)
J. Biol. Chem.
270,
18865-18870 |