Complexes of Matrilin-1 and Biglycan or Decorin Connect Collagen VI Microfibrils to Both Collagen II and Aggrecan*

Native supramolecular assemblies containing collagen VI microfibrils and associated extracellular matrix proteins were isolated from Swarm rat chondrosarcoma tissue. Their composition and spatial organization were characterized by electron microscopy and immunological detection of molecular constituents. The small leucine-rich repeat (LRR) proteoglycans biglycan and decorin were bound to the N-terminal region of collagen VI. Chondroadherin, another member of the LRR family, was identified both at the N and C termini of collagen VI. Matrilin-1, -3, and -4 were found in complexes with biglycan or decorin at the N terminus. The interactions between collagen VI, biglycan, decorin, and matrilin-1 were studied in detail and revealed a biglycan/matrilin-1 or decorin/matrilin-1 complex acting as a linkage between collagen VI microfibrils and aggrecan or alternatively collagen II. The complexes between matrilin-1 and biglycan or decorin were also reconstituted in vitro. Colocalization of collagen VI and the different ligands in the pericellular matrix of cultured chondrosarcoma cells supported the physiological relevance of the observed interactions in matrix assembly.

chain. Collagen VI molecules associate laterally in an antiparallel fashion into dimers that are stabilized by disulfide bridges (3,4,7). The dimers aggregate further into tetramers that are secreted into the extracellular matrix (7), where they join end to end into microfibrils. These subsequently form characteristic thin beaded filaments that are found in a variety of tissues (3,8,9). The formation of microfibrils was recently shown to depend on the N5 vWFA-like domain of ␣3(VI) (10).
In addition to the collagens, the large hyaluronan-binding proteoglycan, aggrecan is a major constituent of the cartilage extracellular matrix. The aggrecan core protein has a molecular weight of ϳ220 kDa (11) and is heavily substituted with about 100 chondroitin sulfate chains, 30 keratan sulfate chains, and 60 N-and O-linked oligosaccharides (12)(13)(14)(15)(16). Aggrecan interacts with hyaluronan and other matrix molecules (reviewed in Ref. 17).
The LRR (leucine-rich repeat) protein family consists of 12 known extracellular members and the molecules appear to play major roles in modulating the functional properties of the collagen networks (17,30). Nine of the members are characterized by a core protein with 10 to 11 leucine-rich repeats, surrounded by disulfide-linked loops. Most LRR proteins have an N-terminal extension with specific characteristics. These range from clustered tyrosine sulfate residues, long stretches of aspartate residues, clustered basic amino acids as well as substitution with oligosaccharides or glycosaminoglycan chains. In many cases binding to triple helical collagens has been demonstrated. Decorin, a proteoglycan member of this family, interacts with fibrillar collagens in vitro (31) and interferes with collagen fibrillogenesis (32,33). It has also been shown to interact with collagen VI (34) at the same site as biglycan (35), another LRR-proteoglycan closely related to decorin. Biglycan is substituted with two glycosaminoglycan chains, often dermatan sulfate, in its N-terminal domain, while decorin carries a single chain (36 -40). Biglycan does not appear to be involved in the assembly of the fibrillar collagens. However, it was recently shown to bind to the vicinity of the collagen VI N-terminal by its core protein in a glycosaminoglycan-independent manner (35). Furthermore biglycan was demonstrated to induce and catalyze the formation of hexagonal-type collagen VI networks in vitro, a function that depends on its glycosaminoglycan substitution (41).
The tissue-specific structures and functions of the extracellular matrix involve complex, and as yet poorly understood, interactions between individual extracellular proteins. The formation of these specific architectural elements is critical for normal tissue development and function. The present study represents a detailed examination of networks of molecules isolated under mild conditions from the Swarm rat chondrosarcoma. We use molecular electron microscopy in combination with immunogold techniques to show that small LRR-proteoglycans together with matrilins form a linkage between collagen VI microfibrils and other macromolecular components in the cartilage extracellular matrix.

EXPERIMENTAL PROCEDURES
Preparation of Chondrons-Chondrons were isolated from rat chondrosarcoma tissue by a modification of a previously published method (42). In short, freshly isolated tumor tissue was dispersed in Nutrient Mixture F-12 (HAM's F 12) (Invitrogen) by passing it through a nylon filter (pore size 50 m 2 ). The dispersed particles were washed with HAM's F12 on a 25 m 2 pore sized nylon filter. Retained material was resuspended in HAM's F12 and prepared for scanning electron microscopy.
Scanning Electron Microscopy-For scanning electron microscopy (SEM), 50 l of the final suspension was gently drawn onto a wet Millipore filter, pore size 0.22 m. The whole filter was fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate, 0.1 M sucrose, pH 7.4, for two hours at 4°C. Alternatively, filters were fixed in 2% paraformaldehyde, 0.2% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.4, for 1 h at 4°C, pre-incubated with 0.5% BSA in the same buffer for 1 h at room temperature and subsequently incubated with an antiserum raised against bovine collagen VI for 1 h at room temperature, washed with 0.1 M sodium cacodylate, pH 7.4, and incubated with protein A-gold as described (43).
Fixed filter samples were dehydrated for 10 min at each step of an ascending ethanol series and critical point dried in a Balzers critical point dryer. They were mounted on aluminum stubs, gold/palladium coated and examined in a Jeol J-330 scanning electron microscope. Alternatively, immunolabeled filters were coated with a 20 nm carbon layer under rotation prior to SEM. Gold label was detected by recording back scattered electron images at an acceleration voltage of 30 kV and a working distance of 10 mm.
Double Immunolabeling of Chondrosarcoma Cells-The Swarm rat chondrosarcoma cell line (44) was a kind gift from Dr. J. Kimura (Henry Ford Hospital, Detroit). The immortalized cells were cultured in HAM's F12 supplemented with 10% fetal calf serum, 50 units/ml penicillin, 50 units/ml streptomycin, and 50 g/ml L-ascorbic acid.
For immunolabeling the cells were fixed for 20 min with 3.6% formaldehyde in phosphate-buffered saline. Nonspecific antibody binding was blocked by incubation for 30 min with 1% (w/v) BSA in TBS. Immunolabeling was done by consecutive treatment of the slides for 1 h with a mixture of the two appropriate primary antibodies and a mixture of the two secondary antibodies. As secondary antibodies a Cy™3conjugated affinity-purified donkey anti-chicken IgG (Jackson Immuno) was used in combination with either a Cy™2-conjugated affinity-purified goat anti-rabbit IgG (Jackson Immuno) or a Cy™2-conjugated affinity-purified goat anti-mouse IgG (Jackson Immuno). All antibodies were diluted in 1% (w/v) BSA in TBS.
Purification of Protein Complexes from the Swarm Rat Chondrosarcoma-Tissue was washed with five volumes of ice cold 0.05 M Tris-HCl, 0.15 M sodium chloride pH 7.4 (TBS) containing, 5 mM calcium chloride and extracted twice for 1 h and once overnight at ϩ4°C in TBS containing 10 mM EDTA (45). All buffers contained protease inhibitors (46). The tissue residues were removed by centrifugation at 10000 ϫ g for 20 min and the supernatant was centrifuged at 100 000 ϫ g for 8 h at ϩ4°C (Beckman 70.2 Ti). The bottom fraction, enriched in high molecular weight components, was dissolved in TBS at ϩ4°C for 48 h. Insoluble material was pelleted by centrifugation at 10 000 ϫ g for 30 min. Protein complexes in the supernatant were purified further by rate zonal centrifugation in a 12 ml glycerol gradient (10 -50%) for 3 h at 200 000 ϫ g in a Beckman SW41 Ti rotor. The tube content was divided into twelve equal fractions and their molecular constituents were analyzed by electron microscopy after negative staining.
Preparation of Gold-labeled Proteins and Antibodies-Colloidal gold particles of 4 nm Ϯ 15% were prepared by reduction of HAuCl 4 by thiocyanate (47) and conjugated after titration to polyclonal antibodies (against human proN(II)-peptide and murine matrilin-2), polyclonal, affinity-purified antibodies (against bovine biglycan, decorin, chondroadherin, matrilin-1, and against murine matrilin-3 and matrilin-4) and monoclonal antibodies (against human aggrecan). In some experiments, colloidal gold particles with a larger (12 Ϯ 20%) diameter were prepared by reduction of HAuCl 4 using sodium citrate (48) and conjugated after titration to the above-described antibodies against matrilin-1. The middle fraction from the rate zonal centrifugation was diluted into TBS and incubated with different dilutions of gold-labeled antibodies. As a control, samples were incubated with pre-immune serum under the same conditions. Samples were adsorbed to a 400-mesh carbon coated copper grid, which was rendered hydrophilic by glowdischarge at low pressure in air. The grid was immediately blotted, washed with two drops of water, and stained with 0.75% uranyl formate for 15 s. Samples were observed in a Jeol 1200 EX transmission electron microscope operated at 60 kV accelerating voltage and ϫ75,000 magnification. Evaluation of the data from electron micrographs was done as described previously (49).
Antibody specificity was determined by SDS-polyacrylamide gel electrophoresis and immunoblotting. Samples of TBS/EDTA extracts from chondrosarcoma tissue were digested with chondroitinase ABC prior to electrophoresis by incubation with 50 microunits/l chondroitinase ABC (Seikagaku). Samples were allowed to react at 37°C for 1-2 h, subjected to ethanol precipitation and resolved in sample buffer with 5% ␤-mercaptoethanol. SDS-polyacrylamide gel electrophoresis was performed as described by Laemmli (50). For immunoblots, the proteins were electroblotted to nitrocellulose membranes and incubated with a dilution of the appropriate antibody. Bound antibodies were detected by using peroxidase-conjugated swine anti-mouse IgG, 3-aminophtalhydrazide (1.25 mM), p-coumaric acid (225 M), and 0.1% H 2 O 2 .
Collagen VI Microfibrils-Collagen VI microfibrils without bound protein complexes were prepared from bovine cornea (52). In short, bovine corneas were cut into pieces and homogenized in Tris/saline buffer, pH 7.4, containing 5 mM calcium chloride and protease inhibitors. The homogenate was digested with collagenase type 1 (Worthington). Not dissolved material was pelleted by centrifugation at 48 000 ϫ g for 20 min. The supernatant was applied in 500-l aliquots onto a 25 ml Superose 6 column (Amersham Biosciences) equilibrated and eluted with homogenization buffer at 0.2 ml/min. Fractions of 0.5 ml from the void volume containing collagen VI microfibrils were collected and pooled.
Recombinant LRR-Proteoglycans/Proteins-Preparation of native recombinant LRR-proteoglycans/proteins in human kidney cells, human HeLa cells, and Chinese hamster ovary cells has been previously described (41).
Reconstitution of Complexes in Vitro-Colloidal gold particles were prepared through reduction with thiocyanate (smaller size) or citrate (larger size) as described above. Colloidal gold particles of smaller and larger size were titrated and conjugated to biglycan, decorin, chondroadherin, and matrilin-1, respectively. Gold-labeled proteins were incubated with purified collagen VI microfibrils from cornea, incubated for 15 min in ϩ4°C and then subjected to negative staining and electron microscopy as described above.

Structural Analysis of the Pericellular Matrix of Chondrons and Cultured
Chondrocytes-Chondrons, representing single or clustered chondrocytes surrounded by territorial matrix, were purified in an intact form from Swarm rat chondrosarcoma tissue using a modification of a method previously reported for mature cartilage (42). The high water content and the softness of the tissue allowed isolation of the chondrons by passing tissue samples through nylon filters. Chondrons isolated by this method exhibited the same overall appearance as those isolated from cartilage by Poole et al. (42) (Fig. 1a). SEM analysis showed that the chondron capsule contains different fibrillar networks where thin microfibrils contrast against thicker fibers (Fig. 1b). The thinner fibrils where identified as collagen VI by immunolabeling and SEM (Fig. 1c). Cultured Swarm rat chondrosarcoma cells deposit a pericellular matrix containing collagen VI microfibrils similar to those found in vivo (Fig. 1, d-f). Immunofluorescence microscopy of the chondrosarcoma cells showed co-localization of collagen VI microfibrils with both matrilin-1 and matrilin-3 (Fig. 2). Since the two matrilins showed extensive co-localization with each other and may well occur in hybrid molecules and hybrid filaments (21,26), only matrilin-3 was studied in further double immunofluorescence experiments (Fig. 2). Biglycan and decorin showed localizations partially overlapping with matrilin-3 in pericellular filaments. In addition, matrilin-3 was partially co-distributed with both aggrecan and collagen II.
To assure that all antibodies used were truly specific, these were tested in immunoblots against extracts of chondrosarcoma proteins and proteoglycans that had been resolved by SDS-PAGE performed under reducing conditions. All antibodies reacted specifically with bands with a mobility characteristic for the corresponding antigen (not shown).
Analysis of Isolated Collagen VI Microfibrils by Electron Microscopy-Collagen VI microfibrils were obtained in an intact form by extraction of rat chondrosarcoma tissue with EDTA-containing physiological saline, followed by a differential centrifugation procedure. The fibrils were examined by electron microscopy after negative staining. Collagen VI was visualized as beaded filaments where adjacent tetramers were linked together in the characteristical end-to-end manner (Fig.   FIG. 1. Scanning electron microscopy  3, a and i). These filaments formed complexes with a number of molecules giving their N termini a bulky appearance (Fig. 3a, arrow) compared with the collagen VI microfilaments without bound ligands (Fig. 3h, arrow). The latter filaments were prepared by collagenase digestion of bovine cornea (52). Two different kinds of particles were frequently observed at the N termini of collagen VI derived from the chondrosarcoma (Fig.  3a, insets). The smaller of these consisted of two globular domains connected by a smaller, central domain. The larger was in most cases composed of three globular domains of equal size (Fig. 3a, insets), but also structures made up of four subunits were frequently seen. The molecular identity of the particles was determined using gold-labeled, affinity-purified antibodies. Using this approach, biglycan (Fig. 3b), and decorin (Fig.   3c) were found located to the N-terminal globular domains of collagen VI and represented the smaller kind of particle. The larger molecules, typically bound to the small LRR-proteoglycans, and more distant from the collagen VI filaments, were identified as matrilin-1 (Fig. 3d), matrilin-3 (Fig. 3e), and matrilin-4 (Fig. 3f). Matrilin-2 was never detected as a component of the collagen VI microfibril complexes (not shown). We also identified chondroadherin bound both to the N-and C-terminal globular domains of collagen VI, but we did not observe other proteins interacting with chondroadherin in these samples (Fig. 3g).

Reconstitution of Collagen VI Complexes in Vitro-
The observations made on native, chondrosarcoma-derived collagen VI complexes were verified in recombination experiments employing highly purified proteins and recombinant native proteoglycans (Fig. 4). Corneal collagen VI microfibrils, free of associated proteins, were mixed with LRR-proteoglycans/proteins in vitro. Recombinant native biglycan, decorin, and chondroadherin were labeled with colloidal gold prior to incubation. Biglycan and decorin bound exclusively to the N-terminal parts of collagen VI, while chondroadherin bound both to the Nterminal and C-terminal portions (Fig. 4, a-c). In addition, matrilin-1 labeled with larger gold particles was found to bind FIG. 3. Structure of intact collagen VI from the Swarm rat chondrosarcoma as seen by electron microscopy after negative staining. a, collagen VI microfibrils with different molecules bound close to the N-terminal parts of the collagen VI tetramers are visible (arrows). Representative particles exhibiting multidomain structures of different sizes are shown at higher magnification (insets). Using specific gold-labeled antibodies these molecules are identified as biglycan (b), decorin (c), matrilin-1 (d), matrilin-3 (e), matrilin-4 (f), and chondroadherin (g). Thus, complexes of matrilin -1, -3, or -4 and the LRR proteoglycans biglycan or decorin, binding close to the collagen VI N termini, are identified. h, collagen VI microfilaments, extracted with collagenase from cornea tissue lack bound ligands as visualized with negative staining. The N-terminal regions of collagen VI are indicated (arrows). The bars represent 100 nm (a) and 20 nm (b-g). i, schematic presentation of collagen VI microfibrils with the dimensions and globular domains of collagen VI indicated (arrows).

FIG. 4. Reconstitution of collagen VI microfibril complexes in vitro.
Gold-labeled biglycan (a), decorin (b), and chondroadherin (c) bind to collagenase extracted corneal collagen VI microfibrils close to the N terminus (biglycan and decorin) or both the N-and C-terminal parts (chondroadherin). Matrilin-1, labeled with gold particles of a larger size than the LRR-proteoglycans is connected to the collagen VI microfibrils via gold-labeled biglycan (d) or decorin (e). The bar represents 100 nm.
to the collagen VI via biglycan (Fig. 4d) or decorin (Fig. 4e), labeled with smaller colloidal gold, when these molecules were mixed prior to incubation with the corneal collagen VI microfibrils.
Analysis of the Interactions between Matrilin-1 and LRRproteoglycans-The results obtained by electron microscopy indicated that matrilin-1 is able to bind the LRR-proteoglycans biglycan and decorin. This was further supported in binding experiments using purified proteins and proteoglycans and detecting bound proteins by surface plasmon resonance in the BIAcore TM 2000 system. Matrilin-1 was immobilized to the CM-5 chip and biglycan, decorin or chondroadherin added in the fluid phase (Fig. 5). For biglycan and decorin a concentrationdependent binding to matrilin-1 was observed, while chondroadherin did not interact. The lack of binding of chondroadherin to matrilin-1 is in agreement with the results from electron microscopy, where this LRR-protein was never seen associated with matrilins (Fig. 3g). The binding of native biglycan and decorin to matrilin-1, but not of the structurally releated chondroadherin, demonstrates the specificity of the assay.
Identification of Extracellular Matrix Components Interact-ing with Collagen VI Microfibrils via the LRR-proteoglycan/ matrilin Complex-In electron microscopy, the LRR-proteoglycan/matrilin complexes were frequently seen to mediate contacts between the collagen VI filaments and other matrix macromolecules. These peripherally attached molecules were identified as procollagen II (Fig. 6a) and aggrecan (Fig. 6d) by incubation with gold-labeled affinity-purified antibodies prior to negative staining and electron microscopy. Also assemblies in the form of collagen II fibers were shown to bind to collagen VI via the matrilin-biglycan/decorin linkage (Fig 6c). Binding to the various constituents occurred via matrilin-1, which was identified with a gold-labeled antibody (Fig. 6, b, c, and e). FIG. 6. Identification of extracellular matrix components interacting with collagen VI microfibrils via the LRR-proteoglycan/matrilin complex. Components of collagen II (a-c) and aggrecan (d and e) networks were found connected to collagen VI microfibrils via the matrilin-1/LRR-proteoglycan complex. Gold-labeled antibodies (white arrows) were used to directly identify procollagen (a), matrilin-1 (b and e) and the aggrecan core protein (d) in purified high molecular complexes. Arrowheads in (b and e) point to matrilin-1 consisting of three globular domains. In (a and d) arrowheads point to similar particles consisting of three globular domains, resembling matrilins. c, double staining with gold of different sizes occasionally located complexes of biglycan (white arrow, small gold) and matrilin-1 (black arrow, large gold) between collagen VI microfibrils and striated collagen II fibrils. Note that the enlargement in c is different from (a, b, d, e). The bars represent 100 nm.

DISCUSSION
Supramolecular assembly of collagen VI molecules into fibrillar networks has been evaluated in detail previously (3,4,7), but how these networks are associated with other interstitial network components at the molecular level is still elusive. Although collagen VI has been shown to interact with fibrillar collagens in vitro (6,34) it has been suggested that other molecules may be involved in the in vivo interaction (53).
In the present study we provide evidence for molecular mechanisms by which collagen VI microfibrils can interact with other interstitial extracellular matrix networks. We took advantage of the Swarm rat chondrosarcoma, a comparatively soft cartilaginous tissue, which allows purification of cartilage extracellular matrix components under native conditions. Electron microscopy in combination with negative staining and colloidal gold technology was used to visualize the extracellular matrix structures at high resolution and to identify individual components both in native extracts and reconstituted multimolecular assemblies. For the recombination experiments bovine proteins were used. Identical results were obtained when bovine matrilin-1, CHAD, biglycan, and decorin were used in combination with rat collagen VI (not shown). Despite the species difference the results did in all aspects agree with and confirm the observations made in the Swarm rat chondrosarcoma system.
Compared with "naked" beaded microfibrils extracted by limited collagenase digestion (52) the collagen VI microfibrils, extracted under conditions that preserve native structures, exhibited a variety of globular multidomain proteins surrounding the globular beads of the tetramer junctional complex. Gold-labeled antibodies confirmed that the smaller particles, binding close to the microfibril, were different LRR proteins or protoglycans, whereas the larger particles found more distant from the microfibril were identified as different matrilins. Biglycan and decorin were found in the vicinity of the N terminus, whereas chondroadherin bound close to both the N and C termini. In vitro reconstituted complexes consisting of collagenase-digested microfibrils with added gold-labeled biglycan, decorin, or chondroadherin, looked similar. This is in accordance with previous results showing in vitro binding of biglycan and decorin close to the N-terminal region of collagen VI tetramers (35), and chondroadherin to both N-and C-terminal globular moieties (41).
Native glycosylated biglycan and decorin, but not chondroadherin, interacted with matrilins in both native and reconstituted microfillar assemblies. The interactions were not dependent on glycosaminoglycan chains as neither pretreatment of native biglycan and decorin with chondroitinase ABC, nor the presence of purified chondroitin or dermatan sulfate chains inhibited the assembly (results not shown). Complexes between matrilin-1 and biglycan/decorin were examined in further detail as models for this interaction, where different complexes between LRR-proteoglycans and matrilins were found bound to native collagen VI microfibrils. The finding, that collagen VI is connected to the major constituents of extracellular matrix through the LRR-proteoglycans in complex with the matrilins, is in accordance with the fact that matrilin-1 interacts with both aggrecan and collagen II (27)(28)(29). By use of electron microscopy we visualized the individual molecules and their interrelations in native complexes extracted from tissue and demonstrate that the major structural entities, collagen II and aggrecan are joined to collagen VI via a complex of matrilins and LRR-proteoglycans. This observation offers new insights into the complex organization of the cartilage matrix and allows us to propose a model of the in vivo organization of the studied molecules (Fig. 7). We conclude that an important role of matrilin-1, biglycan and decorin is to serve as adapter proteins connecting macromolecular networks in the cartilage extracellular matrix. The members of the matrilin family are differentially expressed among and within cartilages. For example, the superficial zone of articular cartilage lacks matrilin-1 and -3 while matrilin-4 is present (54). It is likely that matrilin-4 takes over the adapter function in this tissue, but it may be that the levels of matrilin expression influence the kind of macromolecular assemblies formed in a particular tissue compartment. Other members of the matrilin and LRR-proteoglycan families may play similar roles in the tissues where they are expressed.
Our study visualizes collagen VI microfibrillar networks, which are connected to collagen II fibrils by biglycan/matrilin-1 complexes as linkers. These linkers also connect a number of procollagen molecules to the collagen VI scaffold. These may represent immobilized nucleation centers for collagen II fibril assembly. It is thus possible that a functional role of such collagen VI microfibril supramolecular assemblies is to act as scaffolds for the formation of the structurally critical fibrillar collagen networks. A further role of the collagen VI network may be to present fibrillogenesis modulators such as LRR proteins in proximity to the growing fibrils. In this way the formation of the collagen VI microfibrillar network in the early stages of tissue formation, or in repair processes such as wound and fracture healing, may play an important instructional role in tissue development, architecture, and homeostasis.