In Vitro Regulation of Cartilage Matrix Assembly by a Mr 54,000 Collagen-Binding Protein
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Proc. Nati. Acad. Sci. USA Vol. 83, pp. 5126-5130, July 1986 Cell Biology In vitro regulation of cartilage matrix assembly by a Mr 54,000 collagen-binding protein (type H collagen fibril assembly/proteoglycans) SRINIVASAN CHANDRASEKHAR*, GORDON W. LAURIE, FRANCES B. CANNON, GEORGE R. MARTIN, AND HYNDA K. KLEINMAN Laboratory of Developmental Biology and Anomalies, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892 Communicated by Victor A. McKusick, March 18, 1986 ABSTRACT In cartilage, type II collagen is present as is not noticeably changed in the cartilage of a chicken mutant thin, short, randomly oriented fibrils. In vitro, however, type that lacks proteoglycan (34). Such observations indicate that II collagen forms fibrils of large diameter, indicating that proteoglycans may not be the most important regulatory additional factors may be involved in the regulation of collagen molecule in determining fibril size. fibril formation. We have examined extracts of a cartilage- In this study, we have identified a collagen-binding protein producing tumor for the presence ofcollagen-binding proteins. of Mr 54,000 in extracts of a well-characterized chondrosar- In addition to fibronectin and link protein, a Mr 54,000 protein coma that produces cartilage macromolecules. We call this was found to bind to collagen fibrils as well as to native and Mr 54,000 protein CBP. Immunological studies indicate that denatured type II collagen. Immunological studies using anti- CBP is also a constituent of normal cartilage but not of other body against the protein indicate that it is a cartilage matrix tissues. CBP binds to type II collagen and, in combination protein, not present in bone or in several other tissues. In vitro with cartilage proteoglycan, limits the growth of the type II studies show that the Mt 54,000 protein in combination with collagen fibrils. This protein may regulate fibril size in cartilage proteoglycan decreases the rate of type II fibril cartilage. formation and causes the fibrils to be of small diameter (24 ± 8 nm). These studies indicate that complexes between collagen MATERIALS AND METHODS and proteoglycans mediated by this protein may regulate the Isolation and Purification of CBP. The Swarm chondro- assembly of cartilage matrix. sarcoma (35) was used as the source of cartilagenous pro- teins. The tumor was grown in rats and harvested about 4 Cartilage has a distinctive histology, containing chondro- weeks after inoculation. All subsequent procedures were cytes surrounded by a rather homogeneous matrix. The performed at 40C unless otherwise indicated. Freshly har- matrix is composed of small collagen fibrils (1-3) widely vested tissue (200 g/liter) was homogenized in a solution separated by large aggregate structures of proteoglycan, containing 0.05 M Tris'HCl (pH 7.2), 20% NaCl, 0.01 M hyaluronic acid, and link protein (4-10). This arrangement of EDTA, 0.1 M 6-aminohexanoic acid, and 5 mM benzami- collagen fibrils and space-filling proteoglycan aggregates dine HCl to remove serum contaminants and was centrifuged generates a cushioning matrix that resists compression. It is at 25,000 x g for 30 min. The residue was reextracted with 500 not known whether or how collagen fibrils and proteoglycan ml of 2 M urea/0.05 M Tris-HCl, pH 7.2, containing the aggregates are linked in the matrix. protease inhibitors used above. This latter extract was used Most (>90%) of the collagen in cartilage matrix is type II as the source of collagen-binding proteins. collagen, although certain other collagens such as types IX Portions of the urea extract were chromatographed on a and X and the la, 2a, and 3a collagens are also present column (25 x 5 cm) of DEAE-cellulose equilibrated with the (11-13). Type II collagen is secreted from chondrocytes as a same solvent. The unbound fraction, containing CBP, was soluble precursor, known as type II procollagen, that is dialyzed against 2 M urea/0.,15 M NaCl/5 mM phosphate, pH rapidly converted to type II collagen by specific proteases. 6.8, and was chromatographed on a column (25 x 5 cm) of Under physiological conditions, collagen is insoluble and hydroxyapatite (Ultrogel; LKB) equilibrated in the same assembles into fibrils (14). Type I collagen has the capacity buffer. Bound materials were eluted with a linear gradient to to self-assemble into fibrils in vitro with the same packing of 0.3 M phosphate in the same buffer. molecules in the fibrils as that observed in vivo (15-24). This Type II Collagen. Type II collagen was isolated from the is also true with type II collagen, but the fibrils obtained are chondrosarcoma grown in lathyritic rats (35). This material very large in diameter, in contrast to the small fibrils contained a small amount (usually <2%) of type I collagen, observed in hyaline cartilage (25, 26). These observations which has been shown to originate from the capsule. In some suggest that information for fibril formation is inherent in the studies, native or denatured type II collagen was coupled to molecule and/or that other factors exist that regulate the size CNBr-activated Sepharose (Pharmacia). of Collagen Fibrillogenesis. Type II collagen (1 mg/ml in 0.5-M the collagen fibrils in cartilage. acetic acid) was dialyzed against 0.01 M phosphate buffer Observations on type I collagen fibril formation indicate (pH 6.8) at 40C overnight to induce fibrils to form. Fibril- that several noncollagenous glycoproteins and proteoglycans logenesis was also initiated by incubation at 370C, after a influence fibril assembly (27-33). The large chondroitin solution of type II collagen (in 0.5 M acetic acid) was mixed sulfate proteoglycan ofcartilage and its isolated glycosamino- at 40C with a concentrated buffer solution such that the final glycan side chains cause some alterations in the kinetics of concentration of collagen was 0.6 mg/ml in 0.135 M type I fibril formation but do not reduce the diameter of the NaCl/0.03 M sodium phosphate/0.02 M N-tris(hydroxy- fibrils (33). Further, the diameter ofthe type II collagen fibrils *To whom correspondence should be addressed at present address: The publication costs of this article were defrayed in part by page charge Lilly Research Laboratory, Building 98, Room 4331, Division of payment. This article must therefore be hereby marked "advertisement" Immunology and Connective Tissue Research, 307 East McCarty in accordance with 18 U.S.C. §1734 solely to indicate this fact. Street, Indianapolis, IN 46285. 5126 Downloaded by guest on September 29, 2021 Cell Biology: Chandrasekhar et al. Proc. NatL. Acad. Sci. USA 83 (1986) 5127 methyl)methyl-2-aminoethanesulfonic acid, pH 7.4. The ef- fects of CBP and cartilage proteoglycan on collagen fibril- logenesis were examined at 50 ,ug and 100 Ag/ml, respec- 200- - tively, by measuring changes in the optical density of the sample in a Gilford spectrophotometer at 314 nm (15, 25, 30). 116- s-l for 6 hr - Aliquots taken from these samples after incubation 93- at at 350C were diluted 1:30 with distilled water; 10 A.l of the diluted samples were placed on a Formvar-covered grid, stained with 1% uranyl acetate for 5 min, and examined in a 68 - 1 -mm JEOL 100C electron microscope. For quantitation of fibril diameter, electron microscopic -54 negatives of incubations of type II collagen alone, type II 'U collagen plus chondroitin sulfate proteoglycan, type II 42- eU collagen plus CBP, or type II collagen plus chondroitin sulfate proteoglycan and CBP were projected onto a ZIDAS 1 2 3 4 5 (Zeiss) digitizing tablet at a final magnification of200,000. On the tablet, a grid was drawn with lines separated by 4 cm. FIG. 1. Binding of CBP to collagen fibrils. The 2 M urea extract Fibrils that fell under the intersection of two lines were then was dialyzed against PBS and centrifuged, and the supernatant to determine diameter. fraction (0.5 mg of protein in 0.5 ml) was incubated with or without measured collagen fibrils (1.0 mg in 0.5 ml) at 350C for 15 min. After incubation, Preparation of Antibodies to CBP. The 2 M urea extract of the solutions were centrifuged and the pellets were examined by chondrosarcoma was dialyzed against phosphate-buffered NaDodSO4/7.5% PAGE in the presence ofa reducing agent. Lane 1: saline (PBS, 0.02 M sodium phosphate, pH 7.2/0.15 M NaCl) molecular weight standards (M, x 10-3 at left). Lane 2: 2 M urea and 'centrifuged to remove insoluble material. The superna- extract. Lane 3: the material precipitated when the 2 M urea extract tant fluid was mixed with type 1Lcollagenfibrils (prepared as was incubated alone. -Lane 4: the material precipitated when type II described above) and incubated at 370C for 1 hr. The fibrils were incubated alone. Lane 5: the material precipitated when precipitate was collected by centrifugation at 25,000 x g for type II fibrils and 2 M urea extract were incubated together. Position 15 min, washed with PBS, solubilized in 1% NaDodSO4, and of CBP (M, 54,000) is indicated at right. subjected to electrophoresis. Subsequently, the portion ofgel containing CBP (100 Izg in 1.5 ml) was mixed with complete coupled to Sepharose. Bound material was eluted with 6 M Freund's adjuvant and injected into rabbits. At least three urea and was found to consist of three major components booster injections of the same material in incomplete adju- (Fig. 2, lane 3). These were (i) the Mr 54,000 protein observed vant were administered at 2-week intervals. above; (ii) a Mr 220,000 protein, tentatively identified as We used immunoblotting techniques to detect the antibody fibronectin on the basis of its size and its ability to bind in the sera of immunized rabbits, to establish the specificity collagen; and (iii) a M, 42,000 protein, tentatively identified of the antibody, and to determine the distribution of the as link protein on the basis ofits size and affinity for collagen protein in various tissues (36).