Proc. Nati. Acad. Sci. USAVol. 83, pp. 5126-5130, July 1986Cell Biology

In vitro regulation of cartilage matrix assembly by a Mr 54,000collagen-binding protein

(type H collagen fibril assembly/proteoglycans)

SRINIVASAN CHANDRASEKHAR*, GORDON W. LAURIE, FRANCES B. CANNON, GEORGE R. MARTIN,AND HYNDA K. KLEINMANLaboratory 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 asthin, short, randomly oriented fibrils. In vitro, however, typeII collagen forms fibrils of large diameter, indicating thatadditional factors may be involved in the regulation of collagenfibril formation. We have examined extracts of a cartilage-producing tumor for the presence of collagen-binding proteins.In addition to fibronectin and link protein, a Mr 54,000 proteinwas found to bind to collagen fibrils as well as to native anddenatured type II collagen. Immunological studies using anti-body against the protein indicate that it is a cartilage matrixprotein, not present in bone or in several other tissues. In vitrostudies show that the Mt 54,000 protein in combination withcartilage proteoglycan decreases the rate of type II fibrilformation and causes the fibrils to be of small diameter (24 ±8 nm). These studies indicate that complexes between collagenand proteoglycans mediated by this protein may regulate theassembly of cartilage matrix.

Cartilage has a distinctive histology, containing chondro-cytes surrounded by a rather homogeneous matrix. Thematrix is composed of small collagen fibrils (1-3) widelyseparated by large aggregate structures of proteoglycan,hyaluronic acid, and link protein (4-10). This arrangement ofcollagen fibrils and space-filling proteoglycan aggregatesgenerates a cushioning matrix that resists compression. It isnot known whether or how collagen fibrils and proteoglycanaggregates are linked in the matrix.Most (>90%) of the collagen in cartilage matrix is type II

collagen, although certain other collagens such as types IXand X and the la, 2a, and 3a collagens are also present(11-13). Type II collagen is secreted from chondrocytes as asoluble precursor, known as type II procollagen, that israpidly converted to type II collagen by specific proteases.Under physiological conditions, collagen is insoluble andassembles into fibrils (14). Type I collagen has the capacityto self-assemble into fibrils in vitro with the same packing ofmolecules in the fibrils as that observed in vivo (15-24). Thisis also true with type II collagen, but the fibrils obtained arevery large in diameter, in contrast to the small fibrilsobserved in hyaline cartilage (25, 26). These observationssuggest that information for fibril formation is inherent in themolecule and/or that other factors exist that regulate the sizeof the collagen fibrils in cartilage.

Observations on type I collagen fibril formation indicatethat several noncollagenous glycoproteins and proteoglycansinfluence fibril assembly (27-33). The large chondroitinsulfate proteoglycan ofcartilage and its isolated glycosamino-glycan side chains cause some alterations in the kinetics oftype I fibril formation but do not reduce the diameter of thefibrils (33). Further, the diameter ofthe type II collagen fibrils

is not noticeably changed in the cartilage of a chicken mutantthat lacks proteoglycan (34). Such observations indicate thatproteoglycans may not be the most important regulatorymolecule in determining fibril size.

In this study, we have identified a collagen-binding proteinof Mr 54,000 in extracts of a well-characterized chondrosar-coma that produces cartilage macromolecules. We call thisMr 54,000 protein CBP. Immunological studies indicate thatCBP is also a constituent of normal cartilage but not of othertissues. CBP binds to type II collagen and, in combinationwith cartilage proteoglycan, limits the growth of the type IIcollagen fibrils. This protein may regulate fibril size incartilage.

MATERIALS AND METHODSIsolation and Purification of CBP. The Swarm chondro-

sarcoma (35) was used as the source of cartilagenous pro-teins. The tumor was grown in rats and harvested about 4weeks after inoculation. All subsequent procedures wereperformed at 40C unless otherwise indicated. Freshly har-vested tissue (200 g/liter) was homogenized in a solutioncontaining 0.05 M Tris'HCl (pH 7.2), 20% NaCl, 0.01 MEDTA, 0.1 M 6-aminohexanoic acid, and 5 mM benzami-dine HCl to remove serum contaminants and was centrifugedat 25,000 x g for 30 min. The residue was reextracted with 500ml of 2 M urea/0.05 M Tris-HCl, pH 7.2, containing theprotease inhibitors used above. This latter extract was usedas the source of collagen-binding proteins.

Portions of the urea extract were chromatographed on acolumn (25 x 5 cm) of DEAE-cellulose equilibrated with thesame solvent. The unbound fraction, containing CBP, wasdialyzed against 2M urea/0.,15 M NaCl/5 mM phosphate, pH6.8, and was chromatographed on a column (25 x 5 cm) ofhydroxyapatite (Ultrogel; LKB) equilibrated in the samebuffer. Bound materials were eluted with a linear gradient to0.3 M phosphate in the same buffer.Type II Collagen. Type II collagen was isolated from the

chondrosarcoma grown in lathyritic rats (35). This materialcontained a small amount (usually <2%) of type I collagen,which has been shown to originate from the capsule. In somestudies, native or denatured type II collagen was coupled toCNBr-activated Sepharose (Pharmacia).

Collagen Fibrillogenesis. Type II collagen (1 mg/ml in 0.5-Macetic acid) was dialyzed against 0.01 M phosphate buffer(pH 6.8) at 40C overnight to induce fibrils to form. Fibril-logenesis was also initiated by incubation at 370C, after asolution of type II collagen (in 0.5 M acetic acid) was mixedat 40C with a concentrated buffer solution such that the finalconcentration of collagen was 0.6 mg/ml in 0.135 MNaCl/0.03 M sodium phosphate/0.02 M N-tris(hydroxy-

*To whom correspondence should be addressed at present address:Lilly Research Laboratory, Building 98, Room 4331, Division ofImmunology and Connective Tissue Research, 307 East McCartyStreet, Indianapolis, IN 46285.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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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-tively, by measuring changes in the optical density of thesample in a Gilford spectrophotometer at 314 nm (15, 25, 30).

Aliquots taken from these samples after incubation for 6 hrat 350C were diluted 1:30 with distilled water; 10 A.l of thediluted samples were placed on a Formvar-covered grid,stained with 1% uranyl acetate for 5 min, and examined in aJEOL 100C electron microscope.For quantitation of fibril diameter, electron microscopic

negatives of incubations of type II collagen alone, type IIcollagen plus chondroitin sulfate proteoglycan, type IIcollagen plus CBP, or type II collagen plus chondroitinsulfate proteoglycan and CBP were projected onto a ZIDAS(Zeiss) digitizing tablet at a final magnification of 200,000. Onthe tablet, a grid was drawn with lines separated by 4 cm.Fibrils that fell under the intersection of two lines were thenmeasured to determine diameter.

Preparation of Antibodies to CBP. The 2 M urea extract ofchondrosarcoma was dialyzed against phosphate-bufferedsaline (PBS, 0.02 M sodium phosphate, pH 7.2/0.15 M NaCl)and 'centrifuged to remove insoluble material. The superna-tant fluid was mixed with type 1Lcollagenfibrils (prepared asdescribed above) and incubated at 370C for 1 hr. Theprecipitate was collected by centrifugation at 25,000 x g for15 min, washed with PBS, solubilized in 1% NaDodSO4, andsubjected to electrophoresis. Subsequently, the portion of gelcontaining CBP (100 Izg in 1.5 ml) was mixed with completeFreund's adjuvant and injected into rabbits. At least threebooster injections of the same material in incomplete adju-vant were administered at 2-week intervals.We used immunoblotting techniques to detect the antibody

in the sera of immunized rabbits, to establish the specificityof the antibody, and to determine the distribution of theprotein in various tissues (36). Extracts (4.0 M guanidine-HCl/0.05 M Tris HCl, pH 7.2) of the Swarm chondrosarcomawere used as a source of antigen. After dialysis, the proteinsin the extract were separated by electrophoresis in aNaDodSO4/7.5% polyacrylamide gel and were transferredelectrophoretically to nitrocellulose paper ("blots"). Theblots were exposed to various dilutions of antisera, andbound antibody was detected using a goat anti-rabbit IgGcoupled to peroxidase with 4-chloro-1-naphthol as substrate(36). The distribution of CBP was determined in a similarfashion in normal serum and in 4M guanidine HCl extracts ofvarious tissues. Frozen sections of tissues were also stainedby indirect immunofluorescence according to published pro-cedures (37), using the anti-CBP sera at a 1:10 dilution.

RESULTSCBP in Cartilage Extracts. In preliminary experiments,

attempts were made to identify the collagen-binding proteinsin extracts of a cartilage-producing tumor. In these studies,the residue of cartilage matrix obtained by homogenizingtissue in 20% NaCl to remove serum and the cellularcomponents was reextracted with a mild chaotropic solvent(2M urea). After centrifugation and dialysis against PBS, thesupernatant fraction was incubated with fibrils of type IIcollagen at 370C for 15 min. Collagen fibrils plus boundproteins were then collected by centrifugation, washed inPBS, dissolved in NaDodSO4/PAGE sample buffer, andelectrophoresed (Fig. 1). In addition to the componentspresent in the original collagen preparations, fibrils incubatedwith the cartilage extract bound a protein of Mr 54,000 (Fig.1, lane 5).This collagen-binding protein was also identified by affinity

chromatography using immobilized type II collagen. In thesestudies, the urea extract of the tissue was dialyzed againstPBS and passed over a column of native type II collagen

200- -

116- -93- at

68- 1 -mm

'U

s-l

-54

42- eU

1 2 3 4 5

FIG. 1. Binding of CBP to collagen fibrils. The 2 M urea extractwas dialyzed against PBS and centrifuged, and the supernatantfraction (0.5 mg of protein in 0.5 ml) was incubated with or withoutcollagen fibrils (1.0mg in 0.5 ml) at 350C for 15 min. After incubation,the solutions were centrifuged and the pellets were examined byNaDodSO4/7.5% PAGE in the presence of a reducing agent. Lane 1:molecular weight standards (M, x 10-3 at left). Lane 2: 2 M ureaextract. Lane 3: the material precipitated when the 2 M urea extractwas incubated alone. -Lane 4: the material precipitated when type IIfibrils were incubated alone. Lane 5: the material precipitated whentype II fibrils and 2 M urea extract were incubated together. Positionof CBP (M, 54,000) is indicated at right.

coupled to Sepharose. Bound material was eluted with 6 Murea and was found to consist of three major components(Fig. 2, lane 3). These were (i) the Mr 54,000 protein observedabove; (ii) a Mr 220,000 protein, tentatively identified asfibronectin on the basis of its size and its ability to bindcollagen; and (iii) a M, 42,000 protein, tentatively identifiedas link protein on the basis of its size and affinity for collagen(38). Similar results were observed when type II collagen wasdenatured before coupling to the Sepharose, but the proteindoes not bind to Sepharose-conjugated bovine serum albumin(data not shown). These studies indicate that there are threespecies of proteins in these extracts that bind to collagen,including the M, 54,000 protein, which we call CBP.

Isolation of CBP. The urea extract was chromatographedon a DEAE-cellulose column in 2 M urea/0.15 M NaCl/0.05M Tris HCl, pH 7.2. CBP did not bind to the column and wasrechromatographed on a column of hydroxyapatite, using agradient of phosphate buffer from 0.005-0.3 M as eluent.CBP was eluted at 0.1 M phosphate (Fig. 3a). The migrationposition of the protein in the NaDodSO4/polyacrylamide gelwas identical before and after reduction (Fig. 3b), indicatingthat the protein does not contain interchain disulfide bonds.

Tissue Distribution of CBP. The ability of CBP to bindcollagen fibrils was utilized to isolate the protein for antibody

200-

116 _93 -

68 -54-

42 --

FIG. 2. Type 11 couagen-sepnaroseaffinity chromatography. The 2 M ureaextract (10 ml) was dialyzed against

I1 * >PBS and chromatographed on a nativetype II collagen-Sepharose column.

* Bound material was eluted with 6 Murea and examined by NaDodSO4/PAGE in the presence of a reducing

_ agent. Lanes: 1, molecular weight stan-dards; 2, 2 M urea extract; 3, bound

1 2 3 4 fraction; 4, unbound fraction.

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a

0

Nc

b

54-'W"CL

Fraction

FIG. 3. (a) Isolation of CBP. The 2 M urea extract (100 ml) waschromatographed on a DEAE-cellulose column, and the unboundfraction was further chromatographed on a hydroxyapatite column(see Materials and Methods). Bound material was eluted by agradient of phosphate buffer (0.005-0.3 M) at pH 6.8. (Inset)Fractions were pooled and examined by NaDodSO4/PAGE in thepresence ofa reducing agent. Proteins were visualized by Coomassieblue. Lanes: 1, molecular weight standards; 2, 2 M urea extract; 3,peak I; 4, peak II; 5, peak III; 6, peak IV. (b) CBP before (lane 2) andafter (lane 1) reduction. CBP was isolated as described in a and wasexamined by NaDodSO4/PAGE.

preparation. Collagen fibrils were equilibrated with the ex-tract, isolated by centrifugation, and electrophoresed, andrabbits were given multiple injections with segments of gelscontaining the CBP band over a period of several weeks untilantibody was detected by immunoblotting.The antibody was found to react with a single peptide in the

extract, with mobility identical to the antigen (Fig. 4). Asimilar approach was used to survey for the presence of theprotein in serum and in extracts of normal cartilage, liver,kidney, brain, and muscle. These studies showed a positivereaction with extracts of chondrosarcoma and cartilage butnot with other tissues (Table 1). Thus, the antibody is specificfor CBP, and CBP appears to be concentrated in cartilage.Finally, immunofluorescence localization demonstrated thatCBP is present in the matrix of cartilage (Fig. 5) and notpresent in other tissues (data not shown).

Regulation of Collagen Fibril Formation by CBP. Since CBPbinds to collagen, we tested it, alone and with proteoglycan,for its effects on the fibrillogenesis of type fl collagen. CBPalone (Fig. 6) increased the rate of fibril formation (t12 = 3.34

FIG. 4. Specificity of antiserum to CBP. An-

tiserum (1:10 dilution) was used in a "transblot"

procedure against an unfractionated extract of

chondrosarcoma. The protein was identified byusing peroxidase-conjugated second antibody and

4-chloro-1-naphthol substrate. Lanes: 1, molecu-

lar weight markers; 2, chondrosarco'ma extract

stained with amido black; 3, chondrosarcoma

1 2 3 extract stained with anti-CBP.

Table 1. Tissue distribution of CBP

Tissue CBP

Cartilage (sternal) + + +Chondrosarcoma +++BoneSkinTendon +LiverKidneyBrainSerum

Various rat tissues were extracted with 4 M guanidine hydrochlo-ride for 12 hr. The extracts were dialyzed, lyophilized, and subjectedto electrophoresis followed by transfer to nitrocellulose. The proteinwas identified by using anti-CBP at a 1:10 dilution.

hr) in comparison to type II collagen (tl2 = 2.83 hr) and incombination with proteoglycan further increased the rate (tin= 4.54 hr). The proteoglycan alone had very little effect.However, major differences were observed when the mor-phology and diameters of the fibrils were compared (Figs. 7and 8). More than 85% of type II fibrils (control) were ofwider diameter (>100 nm). In contrast, 99% of type II fibrilsformed in the presence of CBP plus proteoglycan werethinner in diameter (<40 nm). The fibrils formed in thepresence of either proteoglycan or CBP were somewhatlarger. These results suggest that CBP binds to type IIcollagen and in combination with chondroitin sulfateproteoglycan limits the growth of collagen fibrils.

DISCUSSIONIn recent years, a number of collagen binding proteins havebeen identified, including fibronectin, chondronectin,laminin, and link protein. Fibronectin, chondronectin, andlaminin bind to one or more collagen types and also to cellsurfaces and thus link cells to matrix (39). These collagen-binding proteins may also regulate the organization of theextracellular matrices. For example, laminin causes type IVcollagen to precipitate (40), and fibronectin binds to type Icollagen and alters its rate of fibrillogenesis (31). Antibodiesto the collagen-binding domain on fibronectin disrupt thenormal arrangement in which collagen fibrils are deposited incell culture (32). In addition, the link protein, along withvarious proteoglycans, has been found to bind to fibrils oftype I collagen, to change the kinetics offibril formation, andto change the size of the fibril formed (33, 38). Such effectsare not unexpected, given that these proteins bind tightly tocollagen.The size of collagen fibrils in vivo is rather constant,

particularly in fibrous tissues (21, 22, 24). However, the sizeof fibrils formed in vitro is nonuniform. It is likely thatadditional factors normally regulate their formation. Forexample, using procollagen, it has been noted that fibrils ofdifferent size are generated, depending upon the order inwhich the procollagen precursor-specific peptides arecleaved (41).The hyaline cartilage matrix is highly organized. Its

collagen fibrils are then arranged in a nonparallel fashion inwhich proteoglycan aggregates occupy spaces betweenfibrils. The factors regulating this arrangement of macromo-lecular structures are not known. Since type II collagen canform large fibrils in vitro, careful regulation, presumably byother macromolecular constituents of the cartilage matrix,must be involved.

Cartilage contains a variety of proteins of unknown func-tion (42). We have sought to identify putative regulators ofcollagen fibril assembly by isolating collagen-binding pro-teins from the matrix of the Swarm chondrosarcoma, whichis an abundant source of newly deposited cartilage proteins

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FIG. 5. Indirect immunofluorescence of cartilage and the chondrosarcoma after reaction with anti-CBP. (a) Phase-contrast micrograph ofunstained rib cartilage. (b) Rib cartilage stained with anti-CBP. (c) Chondrosarcoma stained with preimmune serum. (d) Chondrosarcoma stainedwith anti-CBP.

(35, 43). Although the cells in this chondrosarcoma aretransformed, they produce apparently normal cartilage ma-trix macromolecules and deposit them in a regular fashion.Matrix components were solubilized in a chaotropic solvent,and their affinity for type II collagen was tested in physio-logical solvents. CBP was the major component found to bindto type II collagen fibers, although proteins tentativelyidentified as fibronectin and link protein also showed somebinding. Immunization of rabbits with CBP produced anantibody that recognized CBP in extracts of the chondrosar-coma and of normal cartilage but not in extracts of various

11 + CBP

C,, II~~~~~~1+PG0

0.5-_O I + PG +CBP0D

Time, hr

FIG. 6. Effect ofCBP and proteoglycan on type II collagen fibrilformation. Type II collagen (0.6 mg) was mixed with CBP (0.05 mg)and/or chondroitin sulfate proteoglycan (0.1 mg) and incubated at0C for 10 min in a final volume of 1.0 ml. The solutions were quicklybrought to 350C and monitored for changes in optical density at 314nm, and the turbidity curves were recorded. The maximal opticaldensity recorded was for type II collagen alone (OD = 2.2). The otherOD values are expressed relative to those obtained for type IIcollagen alone, which is considered as 1. The tln for fibril formationfor type II collagen alone is 3.34 hr; for type II collagen plusproteoglycan (PG), 3.63 hr; for type II collagen plus CBP, 2.83 hr;and for type II collagen plus CBP and PG, 4.43 hr.

FIG. 7. Visualization of the effect of CBP and proteoglycan ontype II collagen fibril formation. Samples were withdrawn aftera 6-hrincubation at 35°C and examined in the electron microscope. (a)Type II collagen alone. Most of the fibrils are large and cross-striated. (b) Type II collagen plus CBP and proteoglycan. All of thefibrils are narrow throughout the field; the fibrils show a slightcross-striation. Some branching is seen. (Bar = 100 am.)

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0

50C.

U-

0Type II Type 11 Type 11 Type 11

+ CBP + PG + PG + CBP

FIG. 8. Diameter oftype II collagen fibrils formed in the presenceor absence of CBP and proteoglycan (PG). Fibril diameters weremeasured after a 6-hr incubation at 350C. Open bars represent fibrils<40 nm in diameter; solid bars represent fibrils 100-250 nm indiameter. Number of fibrils scored was as follows: for type IIcollagen alone, 84; for type H collagen plus CBP, 114; for type IIcollagen plus PG, 108; for type II collagen plus PG and CBP, 105.

other tissues or in serum. Taken together, these resultssuggest that CBP is a cartilage-specific protein.A possible function for this protein in the regulation of

collagen fibrillogenesis was sought based on its interactionwith type II collagen. In confirmation of past studies (26), wefound that purified type II collagen reconstitutes into large,well-ordered fibrils in vitro. Addition of either CBP orchondroitin sulfate proteoglycan gave rise to a mixture oflarge plus small fibrils. However, addition of CBP andproteoglycan together with type II collagen resulted in fibrilsof uniformly small diameter (24 ± 8 nm). These resultssuggest that CBP in combination with the cartilage-specificcollagen and proteoglycan generates small fibrils character-istic of hyaline cartilage. Thus, it is likely that multipleinteractions are involved in the formation of the cartilagematrix. Various data suggest that the formation of otherextracellular matrices also requires several interacting com-ponents. For example, ternary complexes of this type havebeen noted before with type I collagen, fibronectin, andheparan sulfate (44). In addition, type IV collagen, laminin,and heparan sulfate proteoglycan likely interact (40) to formnetworks of 3- to 8-nm-wide cords which are concentrated inthe lamina densa part of basement membrane (45). Thus, ageneral feature determining matrix structures may be thatcollagens interact with other matrix components. In the caseof cartilage, mutants lacking chondroitin sulfate proteoglycanhave a matrix containing thin collagen fibrils, suggesting thatproteoglycan is not essential (34). On the other hand, CBPmay well bind to proteoglycan as well as to collagen and couldlink these apparently independent aggregates into one com-mon structure.

We thank Gertrude Goping and William Johnson for technicalassistance and Irma Burke for preparing the manuscript. This workwas supported by grants from the intramural division of the NationalInstitute of Dental Research and the Kroc Foundation.

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