©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interaction of Biglycan with Type I Collagen (*)

(Received for publication, September 8, 1994; and in revised form, November 9, 1994)

Elke Schönherr (1)(§) Petra Witsch-Prehm (1) Bärbel Harrach (2) Horst Robenek (2) Jürgen Rauterberg (2) Hans Kresse (1)

From the  (1)Institute of Physiological Chemistry and Pathobiochemistry and the (2)Institute of Arteriosclerosis Research, University of Münster, D-48129 Münster, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 times 10 mol/liter) than for glycanated decorin (7 times 10 mol/liter and 3 times 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.


INTRODUCTION

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.


EXPERIMENTAL PROCEDURES

Materials

The human osteosarcoma cell line MG-63 was obtained from the American Type Culture Collection. Rabbit antisera against biglycan, decorin, and proteoglycan-100 were those previously used(30) . An antiserum against human fibromodulin was kindly provided by Dr. Anna Plaas (Shriners Hospital, Tampa, FL). There was no cross-reactivity between the different antisera. A mouse monoclonal antibody was obtained after immunization with pepsin-solubilized bovine type VI collagen. For immunocytochemistry of biglycan and decorin, affinity-purified antisera were used.

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).

Preparation of Metabolically Labeled Small Proteoglycans

Decorin was prepared from the secretions of human skin fibroblasts after labeling with [S]sulfate for 72 h or with [S]methionine for 16 h(30) , respectively, as described(32) . Due to the low expression of biglycan in cultured skin fibroblasts, at the most 5% of the total radioactivity in the final preparation was represented by this proteoglycan.

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 times 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 times 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^2 culture flask yielded preparations where at the most 2% of the radioactivity was represented by decorin.

In Vitro Binding of Small Proteoglycans to Type I Collagen

Acid-soluble type I collagen was dissolved in 17 mM acetic acid (4.4 mg/ml), neutralized by adding PBS (^1)(140 mM NaCl in 18 mM sodium phosphate buffer, pH 7.4), and incubated for 30 min at 37 °C. The fibrils formed were collected, suspended in 300 µl of PBS/mg of collagen, and dispersed by ultrasonication. The incubation mixture contained (in a final volume of 600 µl) 0.17 mg of collagen and radioactively labeled ligand in PBS, 0.1% bovine serum albumin. Controls were incubated without collagen. After 5 h at 37 °C under constant mixing, the pellet was washed with PBS, and bound and free radioactive ligands were quantitated. Proteins remaining in the supernatant were precipitated by addition of 1 volume of chloroform and 3 volumes of methanol, digested with chondroitin ABC lyase(34) , and subjected to SDS-polyacrylamide gel electrophoresis.

Expression and Purification of Recombinant Core Proteins

Plasmid P16 containing a full-length human biglycan cDNA sequence (5) was a generous gift from Dr. L. Fisher (NIDR, National Institutes of Health). Clone D6 containing the coding region of human decorin core protein has been previously described(35) . From the biglycan cDNA, a HindIII fragment (36) was generated that contained the information for amino acids 4-343 of the secreted form of the core protein. The fragment was cloned into the BamHI restriction site of pRSET A (Invitrogen) and expressed as recommended by the manufacturer. Recombinant proteins were extracted with 6 M guanidine hydrochloride, 0.5 M NaCl, and 20 mM sodium phosphate, pH 7.8, and purified on a nickel-nitrilotriacetic acid-agarose spin column (Qiagen, Hilden, Germany) equilibrated with 8 M urea and 0.5 M NaCl in 20 mM sodium phosphate, pH 7.8 (buffer A). The column was washed with buffer A until the A was below 0.01. Further washings were with 8 M urea, 0.5 M NaCl, 0.05% Tween 80 (Sigma) in 20 mM sodium phosphate, pH 6.5 (buffer B). Elution was achieved by applying 8 M urea, 0.5 M NaCl, 0.05% Tween 80 in 20 mM sodium phosphate, pH 4.0. Appropriate fractions were diluted with buffer B to an A of about 0.03, made 0.05% with bovine serum albumin, and dialyzed for 48 h at 4 °C against 20 times the sample volume of 1 M urea, 2 mM glutathione (reduced), 0.005% Tween 80 in 50 mM Tris/HCl, pH 8.0. After dialysis overnight against 0.2 times PBS, insoluble proteins were removed by centrifugation, and 1.5-ml aliquots of the supernatant were lyophilized and reconstituted with 300 µl of H(2)O by brief sonication.

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(4) was replaced by MgCl(2). 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.

Cell Culture and Immunocytochemistry

Human skin fibroblasts and MG-63 osteosarcoma cells were maintained in monolayer culture and embedded in type I collagen lattices exactly as described before(11) . In some experiments, metabolic labeling with [S]sulfate (final radioactivity, 0.74 MBq/ml) started at the time of the preparation of cell-populated collagen lattices, and the media components were supplemented with the radioactive precursor accordingly. In other experiments, type I collagen fibrils (0.67 mg) were suspended in 200 µl of medium, and 5-µl aliquots were dotted on coverslips. 30 min later, MG-63 cells were transferred to the coverslips and maintained in culture for 1 day. Immunostaining for biglycan and decorin was performed as described(27) . Staining for PG-100 and fibromodulin was performed analogously.

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(4), 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(4)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.

Analysis of Collagen Lattice-associated Proteoglycans

For biochemical analysis of proteoglycans retained in the collagen lattice, the lattices were incubated with 50 bovine tendon collagen units of collagenase in 100 µl of 25 mM Tris/HCl, 0.15 M NaCl, 10 mM CaCl(2) containing additionally 10 mM CaCl(2) for 15 min at 45 °C. Cells were collected by centrifugation, the supernatant was removed, and the cells were washed with 100 µl of enzyme-free buffer. Both supernatants were combined and subjected to immune precipitations. Neither biglycan nor decorin core proteins were degraded by the enzyme.

Other Methods

SDS-polyacrylamide gel electrophoresis and Western blotting (39) were performed as described (30) . For the quantification of recombinant proteins, standards (bovine serum albumin, 1-30 µg) and samples were electrophoresed on a 12.5% polyacrylamide gel and transferred to nitrocellulose membranes. The membranes were stained with 0.1% (w/v) amido black 10B in methanol/acetic acid/H(2)O (9:2:9 by volume) and destained in methanol/acetic acid/H(2)O (45:1:4, by volume). Stained bands were cut out and eluted with 300 µl of 25 mM NaOH, 0.04 mM EDTA in 50% aqueous ethanol before the absorbance at 630 nm was determined.


RESULTS

Retention of Biglycan in Collagen Lattices

Under our tissue culture conditions, normal skin fibroblasts produce only very small quantities of biglycan. To study the interaction between biglycan and collagen, MG-63 osteosarcoma cells were used, which usually synthesize more biglycan than decorin. These cells, like fibroblasts, are able to retract collagen gels albeit at somewhat slower velocity. After 4 days of culture in collagen lattices, biglycan was the predominant small proteoglycan retained in gels populated by MG-63 cells, whereas decorin was retained in fibroblast cultures (Fig. 1).


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; circle, 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



Presence of Collagen Biglycan Complexes in Tissue

Immunostaining experiments using cell-populated collagen lattices suffer from the inherent drawback that the antigens under investigation have to be synthesized within the short experimental period. Immunoelectron microscopy of human dermis was therefore used to study the localization of biglycan and decorin under the condition of a metabolic equilibrium. Fig. 5indicates that both biglycan and decorin could be found in association with fibrillar collagen. Excessive quantities of decorin did not quench the positive staining for biglycan (Fig. 5C).


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



In VitroInteraction between Biglycan and Type I Collagen

To investigate the specificity of [S]sulfate-labeled biglycan for the binding to collagen fibrils, highly purified type I collagen was used for fibril reconstitution. In a Scatchard plot, a straight line was obtained (Fig. 6A). From the known specific radioactivity (17 times 10^4 cpm/µmol uronic acid) and the previously determined composition of biglycan (160 uronic acid residues/molecule) (33) , a K(d) value of 8.7 times 10 mol/liter was calculated. This value is very close to the K(d) value of 3 times 10 mol/liter reported for the interaction of decorin from bovine tendon with type I collagen(40) . Using 300 kDa as relative molecular mass of collagen, the number of biglycan binding sites per collagen molecule was estimated to be 0.046. This value, too, is similar to 0.054 binding sites per molecule previously calculated for decorin(40) . However, decorin obtained from fibroblast secretions appeared to interact with reconstituted type I collagen fibrils with greater affinity than the proteoglycan from tendon (Fig. 6B). For a high affinity site, a K(d) value of about 7 times 10 mol/liter was calculated. The interaction with a second site, which accommodates the majority of ligand molecules, could be characterized by a K(d) value of 3 times 10 mol/liter. Using higher doses of decorin (up to 7 nmol per assay) additional binding could be detected (data not shown), and a K(d) value of 2 times 10 mol/liter and a total of 0.043 binding sites per collagen molecule were calculated. Shortage of material prevented a search for low affinity biglycan binding sites.


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 times 10 mol/liter (3 times 10 mol/liter in a separate experiment), a much smaller K(d) value than the native proteoglycan (Fig. 9). The K(d) for decorin was with 3 times 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.




DISCUSSION

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 beta 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 beta, 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 beta 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. (^2)

Though biglycan was detected on the surface of fibrils in cell-populated collagen lattices, the apparent K(d) 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(d) 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.


FOOTNOTES

*
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 310, Projects B2 and B3, and SFB 223, Project A4) and by BMFT Grant 01VM8805/7. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Institute of Physiological Chemistry and Pathobiochemistry, Waldeyerstrasse 15, D-48149 Münster, Federal Republic of Germany. Tel.: 251-835584; Fax: 251-835596.

(^1)
The abbreviations used are: PBS, phosphate-buffered saline; PG-100, proteoglycan-100.

(^2)
H. Hausser, E. Schönherr, and H. Kresse, unpublished result.


ACKNOWLEDGEMENTS

The skillful technical assistance of M. Bahl, P. Blumberg, and M. Opalka is gratefully acknowledged.


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