(Received for publication, September 8, 1994; and in revised form, November 9, 1994)
From the
The small proteoglycan decorin is known to interact with type I
collagen fibrils, thereby influencing the kinetics of fibril formation
and the distance between adjacent collagen fibrils. The structurally
related proteoglycan biglycan has been proposed not to bind to
fibrillar collagens. However, when osteosarcoma cells were cultured on
reconstituted type I collagen fibrils, both decorin and biglycan were
retained by the matrix. Immunogold labeling at the electron microscopic
level showed that both proteoglycans were distributed along collagen
fibrils not only in osteosarcoma cell-populated collagen lattices but
also in human skin. Reconstituted type I collagen fibrils were able to
bind in vitro native and N-glycan-free biglycan as
well as recombinant biglycan core protein. From Scatchard plots
dissociation, constants were obtained that were higher for glycanated
biglycan (8.7 10
mol/liter) than for
glycanated decorin (7
10
mol/liter and 3
10
mol/liter, respectively). A similar
number of binding sites for either proteoglycan was calculated.
Recombinant biglycan and decorin were characterized by lower
dissociation constants compared with the glycanated forms. Glycanated
as well as recombinant decorin competed with glycanated biglycan for
collagen binding, suggesting that identical or adjacent binding sites
on the fibril are used by both proteoglycans. These data suggest that,
because of its trivalency, biglycan could have a special organizing
function on the assembly of the extracellular matrix.
In the extracellular matrix, large aggregating proteoglycans as well as small, non-aggregating proteoglycans are found. Members of the former family, for example aggrecan and versican, provide the tissues with resistance to compressive forces. Members of the latter family, for example decorin and biglycan, are multifunctional proteoglycans that interact with other matrix macromolecules and regulate their functions (see (1, 2, 3) for reviews). Decorin and biglycan both are chondroitin/dermatan sulfate proteins. Decorin carries one (and biglycan most often two) glycosaminoglycan chains. Their core proteins are characterized by N-terminally located glycosaminoglycan attachment sites followed by a cysteine-rich region, several leucine-rich repeats, and a C-terminally located conserved disulfide loop(4, 5, 6) . Homologous core proteins are found in the keratan sulfate proteins lumican (7) and fibromodulin(8) .
The specific association of decorin with fibrillar collagen has been shown by immunostaining at the electron microscopic level (9, 10, 11) and by in vitro binding studies(12, 13) . While the dermatan sulfate chain plays only a minor role in this interaction(14) , the importance of the core protein for binding has convincingly been demonstrated(12, 13) , and a binding domain has been assigned to its C-terminal part(15, 16) . At the electron microscopic level, decorin appeared regularly arrayed at or near the d- and e-bands in the gap zone of type I collagen fibrils(9, 17, 18) . Lumican and fibromodulin are also interacting with fibrillar collagens, but their binding sites are different from the one occupied by decorin(14, 19) . As a consequence of the binding of decorin to the surface of collagen fibrils, the lateral assembly of triple helical collagen molecules is delayed(12, 20) , and the final diameter of the collagen fibrils becomes thinner(21) .
The specificity of the interaction between decorin and type I collagen has recently been questioned(22) . In a solid phase binding assay, decorin interacted efficiently only with type VI and not with type I collagen. Immunoelectron microscopy of several tissues(23, 24) suggests the participation of type VI collagen mainly in distinct microfibrillar and ``beaded filament'' structures deposited between collagen fibrils. Nevertheless, it cannot be excluded that type VI collagen acts as linking moiety between type I collagen and decorin. Tight association of decorin with type VI collagen was found in rabbit cornea(25) .
In situ hybridization and immunolocalization studies indicated the presence of biglycan in non-mesenchymal tissues where decorin had not been found(26) . However, biglycan was also detected in connective tissues, e.g. in dermis, bone, cartilage, and blood vessels(5, 26, 27) . It exhibited a predominantly pericellular location and was considered to play a role in growth control(28) . Despite the homology of the core protein of biglycan with the collagen-binding family members decorin, fibromodulin, and lumican, its co-localization with dermal collagen fibrils could not be detected by immunocytochemistry at the electronmicroscopic level(10) . Likewise, biglycan did not show a specific effect on collagen fibril formation in vitro(29) . In this paper, however, evidence for an interaction between biglycan and fibrillar collagen will be provided, thus adding this proteoglycan to the list of connective tissue-organizing macromolecules.
Type I collagen was prepared from calf skin by pepsin digestion. Further purification for the removal of types III and V collagen was performed by sequential NaCl precipitation at acid and neutral pH, respectively(31) . Jacalin-agarose was from Sigma, and collagenase was from Advance Biofactures (Lynbrook, NY).
For the preparation of radioactively labeled biglycan,
MG-63 cells were similarly incubated with S-labeled
metabolic precursors. Labeling was also performed in the presence of
tunicamycin (0.2 µg/ml) after preincubating the cultures for 16 h
with the same inhibitor concentration. Secreted proteoglycans were
obtained by chromatography on DEAE-Trisacryl M as
described(32) , except that 6 M urea was included in
the starting buffer. The ion exchange column (0.8
6 cm) was
then washed with 10 ml of 20 mM Tris/HCl buffer, pH 7.4,
containing 0.3 M NaCl, 6 M urea, and protease
inhibitors followed by 10 ml of the same solution without urea. Small
proteoglycans were desorbed with 1 M NaCl in urea-free buffer.
For the removal of proteoglycan-100, which is a major contaminant in
these preparations (33) , the eluate of the DEAE column was
directly applied to a 0.8
6-cm column of jacalin-agarose and
equilibrated and eluted with 20 mM Tris/HCl buffer, pH 7.5,
containing 1.0 M NaCl. Biglycan and decorin are not retained
by the lectin. Decorin was removed by immune reaction with a
monospecific antiserum against deglycosylated human decorin, using
immune globulins that had been immobilized on protein A-Sepharose.
Employing 9 mg of protein A-Sepharose for the treatment of the
secretions from a 75-cm
culture flask yielded preparations
where at the most 2% of the radioactivity was represented by decorin.
For the expression of recombinant decorin core protein, a BstUI-EcoRI fragment was prepared, which carried the information for amino acids 15-329 of the mature protein. It was cloned into the BamHI-EcoRI restriction site of pRSET A. All further steps were precisely as described above.
For the preparation of
radioactively labeled proteins, M9 minimal medium was used, which was
substituted with amino acids (except methionine and cysteine) and with
thiamine. MgSO was replaced by MgCl
. Induced
cultures (A
about 0.5) were then incubated for 3
h in the presence of 0.37 MBq/ml of [
S]sulfate,
and the same amount of radiosulfate was added after the first 1.5 h of
incubation. The purification procedures were as described above.
For immunocytochemistry at the electron
microscopic level, collagen lattices were pre-fixed and incubated under
sterile conditions with affinity-purified antibodies against decorin
(17 µg of protein/ml of PBS) and biglycan (20 µg of
protein/ml), respectively, or with monoclonal anti-type VI collagen
antibodies (30 µg/ml) followed by incubation with either
gold-labeled protein A (12 nm) or gold-labeled goat anti-mouse IgG (6
nm) as described(11) . Lattice samples were then washed, fixed
with Karnovsky's reagent, post-fixed with OsO, and
embedded in Epon 812(41) . Ultrathin sections were double
stained with uranyl acetate and lead citrate. For immunocytochemistry
of skin, a biopsy was taken from the upper arm of a 7-year-old boy and
immediately fixed in 4% formaldehyde, 0.25% glutaraldehyde in PBS.
Ultrathin sections were processed and embedded in Lowicryl K4M as
described(38) . Immunocytochemical labeling was performed by
floating formavar-coated nickel grids, section side down, on 20-µl
droplets of 10 mM NH
Cl, affinity-purified
antibody, diluted 1:50 in PBS and protein A-gold. The specificity of
the immunolabeling was tested by omitting the primary antibody and by
using non-immune serum or ``unspecific'' IgG instead. Further
processing was as described above.
Figure 1: Retention of small proteoglycans in a collagen lattice. 300,000 MG-63 osteosarcoma cells and skin fibroblasts (MG-63 Fibr.), respectively, were cultured in type I collagen lattices for 4 days. The lattices were then digested with collagenase, the cells were removed, and proteins were precipitated with chloroform/methanol. Identical portions from each lattice were digested with chondroitin ABC lyase and subsequently subjected to SDS-polyacrylamide gel electrophoresis. After electro-transfer, individual lanes were incubated with antisera against biglycan (BGN, 250-fold dilution) or decorin (DCN, 500-fold dilution).
The retention of biglycan was not due to its higher molecular weight compared with decorin because PG-100, which is similar in size to biglycan and which does not bind to collagen(33) , was predominantly released into the medium, and only minor quantities were retained in the lattice (Fig. 2). Further experiments demonstrated that the collagenous matrix was required for the retention of biglycan. MG-63 cells were maintained on coverslips on which distinct spots had been coated with reconstituted collagen fibrils. Immunostaining for biglycan, decorin, and fibromodulin indicated that these proteoglycans were associated with the fibrillar matrix and with the cells themselves. PG-100 was observed exclusively in association with the cells (Fig. 3).
Figure 2:
Comparison of the distribution of small
proteoglycans synthesized by osteosarcoma cells cultured in a collagen
lattice. 400,000 MG-63 cells were maintained in collagen lattices in
the continuous presence of [S]sulfate (2.8
MBq/plate). At the times indicated, the medium was replaced by unused
medium containing the same concentration of radiosulfate. The sum of
individual small proteoglycans in the media found over the incubation
time is given. Proteoglycans in lattices were quantified after 80 h of
incubation.
, biglycan;
, decorin;
,
PG-100.
Figure 3: Immunostaining of MG-63 cells plated on top of reconstituted collagen type I fibrils with monospecific antisera for small proteoglycans. In vitro reconstituted type I fibrils were bound to some areas of coverslips, and MG-63 cells were grown for 24 h on these slips. Fixed cells were stained with Coomassie Blue (A) to demonstrate the presence and absence of coated proteins. Immunostainings were performed with the preimmune serum for biglycan (B, 500-fold dilution) and with antisera for biglycan (C, 500-fold dilution), decorin (D, 500-fold dilution), PG-100 (E, 100-fold dilution), and fibromodulin (F, 500-fold dilution), respectively. Arrowheads indicate the border between collagen-covered and collagen-free areas. Bar, 50 µm.
Retention of biglycan within a collagenous network does not necessarily imply that it was associated with collagen fibrils. Immunoelectron microscopy was therefore used to study the localization of small proteoglycans along collagen fibrils. It is shown in Fig. 4that after 2 days of culture, the cells had produced sufficient quantities of biglycan and decorin to show their association with fibrils. Because of the reported high affinity binding of decorin to type VI collagen, we also tested for the presence of this collagen type. Though the most prominent staining for type VI collagen was present on non-fibrillar extracellular material (not shown), we also found type VI-specific staining associated with collagen fibrils (Fig. 4C). Thus, the formation of a sandwich between types I and VI collagen and the small proteoglycans cannot be excluded, but it may also be possible that type VI collagen is bound by fibril-associated proteoglycans.
Figure 4: Immunoelectron microscopical localization of biglycan (A), decorin (B), and type VI collagen (C) in a collagen lattice culture of MG-63 osteosarcoma cells. 300,000 MG-63 cells were maintained in a collagen lattice for 2 days prior to immunocytochemistry. Protein A-gold of 6 nm was used to demonstrate the presence of type VI collagen. Bars, 0.2 µm
Figure 5: Immunogold localization of small proteoglycan core proteins along collagen fibrils in human dermis. Affinity-purified antisera against biglycan (A, C) and decorin (B) or ``unspecific'' rabbit IgG (D) were used. In C, the antiserum against biglycan (10 µl) was preincubated with about 1 µg of decorin for 48 h at 4 °C. Bars, 0.2 µm
Figure 6:
Scatchard plots of the interaction of
[S]sulfate-labeled native biglycan (A) and
native decorin (B), respectively, to reconstituted type I collagen
fibrils.
The interaction between type I
collagen and biglycan was independent of the presence of N-linked oligosaccharides of the core protein.
[S]Methionine-labeled biglycan was prepared from
the secretions of MG-63 cells treated with tunicamycin. It is evident
from Fig. 7that N-glycan-free biglycan bound at least
as well as the fully glycanated form of the proteoglycan.
Figure 7:
In vitro interaction of
[S]methionine-labeled biglycan with type I
collagen. [
S]Methionine-labeled biglycan was
prepared from MG-63 cells, which had been maintained in the presence or
absence of tunicamycin (TM). 20,000 cpm of purified
proteoglycan were incubated with reconstituted collagen fibrils for 24
h at 37 °C. Unbound macromolecules were precipitated with
chloroform/methanol prior to chondroitin ABC lyase digestion. Bound
proteoglycans were similarly digested after three washing steps with
PBS. Tracks1, collagen-bound material; tracks2, parallel incubations without collagen (subsequent
immune precipitation with decorin antiserum to show the almost complete
absence of decorin); tracks3, unbound
material.
In an
independent approach to study small proteoglycan binding,
[S]methionine/cysteine-labeled fusion proteins
of biglycan and decorin were expressed in Escherichia coli.
Renatured proteins were used in binding assays. It is shown in Fig. 8that both proteins were able to interact with type I
collagen fibrils. The Scatchard plots obtained indicated that the
carbohydrate-free biglycan exhibited with 5
10
mol/liter (3
10
mol/liter in a
separate experiment), a much smaller K
value than
the native proteoglycan (Fig. 9). The K
for
decorin was with 3
10
mol/liter similar to
the high affinity value obtained for glycanated decorin.
Figure 8:
Binding of
[S]methionine/cysteine-labeled fusion protein of
biglycan (BGN) and decorin (DCN), respectively, to
reconstituted type I collagen fibrils. 10,000 cpm of either protein
were allowed to bind to collagen fibrils. Bound material (B)
or 10,000 cpm each of the original proteins (C) were
electrophoresed on a 3-12% SDS/polyacrylamide gel prior to
fluorography. The migration distance of reference proteins is indicated
on the leftmargin.
Figure 9: Scatchard plots of the interaction of recombinant biglycan (A) and recombinant decorin (B), respectively, to reconstituted type I collagen fibrils. One of three independent experiments is shown.
The availability of recombinant proteins made it possible to investigate whether or not identical binding sites on collagen were occupied by both core proteins. Recombinant biglycan as well as recombinant and native decorin were able to inhibit the binding of native biglycan to type I collagen (Table 1). This conclusion was corroborated in two further experiments using different preparations of recombinant core proteins.
This communication provides evidence for an interaction of biglycan with reconstituted collagen fibrils by showing that biglycan associates with the fibrils in cell-populated collagen lattices and by the demonstration that native as well as recombinant biglycan bind to purified type I collagen fibrils. These in vitro observations are strengthened by the finding of a co-localization of biglycan and fibrillar collagen in human dermis. Furthermore, in a recent study, collagen fibrils of human articular cartilage were found to be decorated with biglycan as well as with decorin(41) , thus supporting the potential relevance of the biglycan-collagen interaction under in vivo conditions.
Previous studies had shown that chondroitin/dermatan sulfate proteins occupied d- or e-bands in the gap zone of collagen fibrils, whereas a- or c-bands were the locations of keratan sulfate proteins(19) . Fibromodulin is located at a different site than decorin(14) . The competition experiments performed in this study allow the conclusion that decorin and biglycan either use the same site along the fibril or that steric hindrance occurs when one of two adjacent binding sites is occupied.
With regard to decorin-collagen interaction, Scott (17, 42) has proposed a model where the glycosaminoglycan chains of fibril-bound decorin form antiparallel duplexes with each other, thereby regulating the distance between adjacent fibrils. An analogous binding of biglycan and collagen adds a further dimension to this model since the two glycosaminoglycan chains of biglycan could form bridges to two separate collagen fibrils. It could easily be envisaged that biglycan as a trivalent ligand contributes even more substantially than the bivalent decorin to the organization of a collagenous matrix.
A further aspect of the
potential biological importance of the interaction between type I
collagen and biglycan can be deduced from the findings that osteoblasts
and MG-63 cells respond to treatment with transforming growth factor
with an induction of type I collagen and biglycan
biosynthesis(43, 44) . Biglycan and decorin were both
found to bind and potentially to inactivate this cytokine(2) .
An increase of transforming growth factor
, e.g. after
bone injury, could, therefore, initiate a self-regulatory circuit of
extracellular matrix production and growth factor activity. This
circuit would depend on the interaction of biglycan and type I collagen
since decorin becomes down-regulated upon transforming growth factor
treatment (44) .
In light of the present results, it
seems surprising that until very recently previous attempts failed to
demonstrate the association of biglycan with collagen. A negative
immunostaining result(11) , however, could be explained by the
assumption that the reactive epitope(s) of the biglycan core protein
became masked by the interaction with matrix molecules. In a very
recent study(45) , it was found indeed that on a light
microscopic level, biglycan staining co-localized with intense collagen
type I and III staining in atherosclerotic lesions. The failure of
observing biglycan-collagen interactions under in vitro conditions (29) is more difficult to explain. It seems
reasonable to assume that even subtle changes in the tertiary structure
of the core protein and/or differences in the composition of the
glycosaminoglycan chains and the asparagine-bound oligosaccharides may
influence the binding properties. Our purification procedure for intact
biglycan avoided the use of guanidine hydrochloride, a strong
denaturing agent. On the other hand, recombinant core proteins could
successfully be renatured, as indicated, for example, also by the
strong competition of recombinant decorin for endocytosis of glycanated
decorin by fibroblasts. ()
Though biglycan was detected on
the surface of fibrils in cell-populated collagen lattices, the
apparent K value for biglycan was 2 orders of
magnitude higher than the respective value for decorin, the data being
calculated on the basis of in vitro studies with reconstituted
type I collagen fibrils. As the core proteins of both proteoglycans
have a basic isoelectric point(4, 5) , it seems
possible that in case of a biglycanated core protein there is a greater
chance than in case of a monoglycanated one that the collagen binding
site becomes inaccessible, due to interactions with acidic
glycosaminoglycan chains. The data could indicate that the stability of
biglycan collagen complexes is also lower in vivo than the
stability of decorin collagen complexes. However, Scatchard plots with
reconstituted fibrils should be interpreted cautiously since in
tissues, mixed fibrils of tightly regulated diameter are
present(46, 47) , which may behave differently with
respect to proteoglycan binding.
An unexpected result was the
finding of the very low dissociation constant for the complex between
recombinant biglycan and collagen fibrils. In interpreting this K value, the observation should be considered that
biglycan appears to be capable of self-association(48) , which
also seems to be relevant under physiological ionic
conditions(30) . If self-association occurs preferentially on
surfaces, the quantity of fibril-bound biglycan would have been
selectively overestimated. This aspect of the study deserves further
attention.
In summary, our data provide evidence for an interaction between biglycan and fibrillar collagen, which can be found in vitro and in vivo. This conclusion is not at variance with previous proposals that biglycan may play a role in the regulation of cell behavior (26) but adds a further aspect to the potential functions of the proteoglycan with respect to matrix assembly and growth factor binding.