DEVELOPMENTAL DYNAMICS 193266276 (1992)

Cartilage Matrix Protein is a Component of the Collagen Fibril of Cartilage NEIL WINTERBOTTOM, M. MEHRDAD TONDRAVI, TERRILL L. HARRINGTON, F. GEORGE KLIER, BARBARA M. VERTEL, AND PAUL F. GOETINCK La Jolla Cancer Research Foundation, La Jolla, California 92037 (N.W., M.M.T.,F.G.K., P.F.G.);Department of Cell Biology and Anatomy, Chicago Medical School, 3333 Green Bay Road, North Chicago, Illinois 60064 (T.L.H.,B.M.V.)

INTRODUCTION Cartilage contains a relatively low number of chondrocytes interspersed in a substantial extracellular matrix. The molecules which comprise the extracellular matrix, and the interactions between them, determine the structural and functional properties of cartilaginous tissues. Cartilage plays a variety of essential roles in the body. In the embryo, cartilage contributes to the growth of the embryo skeleton (Goetinck, 1985); with the onset of ossification, cartilage is restricted to the growth zone and the articular regions of the developing skeleton. In the mature animal, cartilaginous structures provide flexibility, as well as shock absorbing and articular functions (Hascall, 1977). Collagen fibrils provide the scaffolding of the cartilaginous matrix. These structures consist of at least three types of collagen. The major collagen is type 11. The cores of these fibrils contain type XI collagen and on the periphery there is covalently-attached type IX collagen (Mendler et al., 1989; Mayne, 1990). As collagen fibrils mature, they are stabilized by covalent intermolecular cross-links (Bornstein and Traub, 1979). Aggregates of the large cartilage proteoglycan, aggrecan, occupy most of the extracellular space between the collagen fibrils. As is the case for most of the molecules of the extracellular matrix, the protein core of aggrecan is a modular structure consisting of a series of domains, each conferring some structural or functional character (Goetinck and Winterbottom, 1991). The polyanionic glycosaminoglycan side chains of each aggrecan monomer are covalent modifications of the core protein and restrain large volumes of water within the matrix, thus providing the shock-absorbing properties of the adult tissue and contributing to the growth of the embryonic skeleton. The proteoglycan monomers form large aggregates by interacting with hyaluronic acid, These aggregates are stabilized by link protein which binds to both the hyaluronic acid and aggrecan. Several other proteins and small proteoglycans are also present in the cartilaginous matrix. Not all are well characterized, though it is readily recognized that the pattern of intermolecular interactions which stabilize the components of the matrix confer the strength and resilience characteristic of these tissues. Cartilage matrix protein (CMP) is a 54 kDa protein which occurs in cartilage as a disulfide bonded multi0 1992 WILEY-LISS, INC.

mer. Although CMP has been reported to be present only in cartilage, it is not necessarily present in all cartilages. For example, it could not be detected in articular and intervertebral cartilages (Paulsson and Heinegbrd, 1981, 1982). The amino acid sequence of this molecule has been deduced from the corresponding cDNA (Argraves et al., 1987) and genomic DNA (Kiss et al., 1989), and shows a central region with considerable homology to an EGF-precursor motif. On either side of this EGF-like region is a 190-amino acid motif (the CMP-motif) with homology to regions of several proteins, including von Willebrand factor (Sadler et al., 1986; Shelton-Inloes et al., 1986; Titani et al., 1986), complement factors B (Mole et al., 1984) and C2 (Bentley, 19861, type VI collagen (Bonaldo and Colombatti, 1989; Koller et al., 1989; Bonaldo et al., 1990), and the alpha chains of the integrins Mac-1 (Pytela, 1988; Corbi et al., 19881, p150,95 (Corbi et al., 1987), LFA-1 (Corbi et al., 1987), and VLA-2 (Takada and Hemler, 1989). Based on the observation that CMP copurifies with collagen and the demonstration that other molecules containing the CMP-motif also bind to collagen, we examined the interaction of CMP and collagen using in vitro binding studies and immunolocalization. In this report we show that CMP is a component of the cartilage collagen fibril. RESULTS Characterization of Antibodies The rabbit polyclonal anti-CMP and anti-Phe380Val424sera and the monoclonal antibody (mAb) 111-D5 recognize CMP in western blots (Fig. 1).The antigen recognized by these antibodies may be either tissuederived or the product of bacterial expression. Bacteria transformed with cDNA encoding the complete sequence of CMP produce a protein of Mr 54kDa; this, and the expression products of cDNA corresponding to Received October 23, 1991; accepted January 6, 1992. Paul F. Goetinck’s and M. Mehrdad Tondravi’s present address is Cutaneous Biology Research Center, Massachusetts General Hospital-East, Bldg. 149, 13th Street, Charlestown, MA 02129. Address reprint requestslcorrespondence to Paul F. Goetinck. F. George Klier’s present address is The Research Institute of Scripps Clinic, 10666 North Torrey Pines Road, La Jolla, CA 92037. Neil Winterbottom’s address is Metra Biosystems, 3181 Porter Drive, Palo Alto, CA 94304.

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Fig. 1. Characterization of antibodies against CMP. SDS-PAGE of CMP on gels of 7.5% acrylamide transferred electrophoretically to Immobilon-P. Samples of CMP in lanes 1, 4 and 7, and 2, 5, and 8 were electrophoresed under non-reducing and reducing conditions, respectively. Samples of CMP in lanes 3, 6, and 9 were reduced and alkylated before electrophoresis under non-reducing conditions. Detection of CMP was with anti-CMP mAb lll-D5 (lanes 1-3), rabbit anti-CMP (lanes 4-6) and affinity purified rabbit anti-Phe380-Va1424 antibodies (lanes 7-9).

partial lengths of CMP, are also recognized by the above antibodies (Fig. 21, clearly identifying the recognized antigen as CMP. CMP purified from cartilage, electrophoresed under nonreducing conditions, reveals two major bands of Mr 200 kDa and 250 kDa. Under reducing conditions, the same samples produce a single band of Mr 54 kDa. Samples which have been reduced and alkylated migrate as a band of Mr 60 kDa (Fig. l). mAb I-B4 recognizes collagen types I, 11, 111, IV, V, and VI, and Clq, a complement cascade protein which contains a collagenous triple helical domain (Fig. 3). Because of the diversity of the structures of these molecules, we assume that this antibody recognizes a common feature of the triple helical domain. It has also been demonstrated that this antibody recognizes the individual chains of collagens type I and type I1 in Western-blot analysis (data not shown). Isotype analysis determined that mAb I-B4 is an IgM (K) and that mAb III-D5 is an IgG2, (K). This assay and the initial screening of the antibodies were undertaken using ELISA; all the antibodies, therefore, recognize their epitopes in ELISA.

Binding Studies Demonstrate the Interaction of CMP and Type I1 Collagen An ELISA was developed which demonstrates the interaction of CMP with type I1 collagen (Fig. 4). This assay was also used to demonstrate the binding of reduced and alkylated CMP to type I1 collagen. CMP and reduced and alkylated CMP bound in a concentrationdependent manner, as detected by mAb 111-D5. The in-

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Fig. 2. Detection of bacterially expressed CMP. Detection was with anti-CMP mAb lll-D5 (lane 1) and affinity purified rabbit anti-Phe380-Va1424 antibodies (lane 2).

teraction between CMP and collagen could also be demonstrated when CMP was immobilized and the binding of soluble type I1 collagen was detected with mAb I-B4. The specificity of the interaction between CMP and collagen was demonstrated by inhibiting the binding of collagen to immobilized CMP with soluble CMP (Fig. 5). Reduced and alkylated CMP was equally effective in this inhibition (data not shown). Bovine serum albumin did not inhibit binding in this assay, whereas relatively high concentrations of link protein showed a minor inhibition which could be due to residual traces of CMP in the link protein preparation. However, link protein has previously been shown to bind to types I and I1 collagen (Chandrasekhar et al., 1983) and, a t high levels, this binding may interfere with the binding of CMP to collagen.

Rotary Shadowing Studies Reveal the Molecular Interaction of CMP and Type I1 Collagen In order to characterize the molecular sites of interaction of CMP and collagen, electron microscopic studies, using rotary shadowing of the components, were initiated. Isolated CMP molecules are globular proteins that appear to comprise two or three spherical domains connected by bent rods (Fig. 6, upper panels). Variations that are sometimes observed may be due t o different angles of view. Isolated type I1 collagen molecules show a typical rod-like structure of 280 nm in

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Collagen Type Fig. 3. Recognition of collagens by mAb 184. Various types of collagens and C1q were immobilized at decreasing concentration (1,800,570, 180, 57, 18, and 6 ng/well) from left to right in each collagen panel. Binding of mAb 1-84 was quantified using a peroxidase conjugated secondary antibody. Values represent the means of 4 replicates, error bars show the standard errors about the means.

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Inhibitor (nglml) Fig. 5. Competitive inhibition of the binding of type I1 collagen to CMP. CMP was immobilized from a solution of 1.2 pglml. Binding of type II collagen (95 pglml), mixed with varying concentrations of inhibitor, to the CMP was detected using anticollagen mAb 184 and peroxidase conjugated secondary antibody. The proteins tested for inhibitory activity were: CMP (filled circle), link protein (open triangle), and bovine serum albumin (open square). Values represent the means of 4 replicates, error bars show the standard deviation about the means.

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Immunolocalization Studies Demonstrate the Co-Localizationof CMP and Type I1 Collagen in w the Cartilage Extracellular Matrix 0.6 i z T The co-localization of CMP with type I1 collagen and aggrecan was examined in 5-day-old cultures of 0 chicken sternal chondrocytes using double immunoflua 0.4 orescent staining reactions. Chondrocytes were grown in the presence of ascorbic acid to facilitate the deposition of extracellular type I1 collagen fibrils. In Figure 0.2 7 the extracellular localization of the monoclonal antibody 111-D5 against CMP is compared with th at of a polyclonal antiserum to type I1 collagen. A dense filamentous extracellular matrix pattern is observed for 0.C Reduced & Alkylated CMP Native CMP both CMP (Fig. 7A) and type I1 collagen (Fig. 7B). In Figure 8 the extracellular localization of CMP is conFig. 4. Binding of CMP to type I1 collagen. Type II collagen was im- trasted with that of aggrecan. The filamentous distrimobilized (at 1.2 pg/well). CMP or reduced and alkylated CMP was allowed to bind from solutions of decreasing concentration (20, 5, 2, l , and bution of extracellular CMP (Fig. 8A) does not co-localize with the amorphous pattern of aggrecan (Fig. 0.3 pg/ml) from left to right in each collagen panel. Binding of CMP was quantified using anti-CMP mAb WD5 and a peroxidase conjugated sec8B). Immunofluorescent staining of chondrocyte culondary antibody. Values represent the average of duplicates, error bars tures carrying the genetic defect nanomelia, which is show the range. characterized by the absence of aggrecan i n the cartilage extracellular matrix, demonstrates a similar colength (Fig. 6, lower left). In mixtures of CMP and type localization of CMP with type I1 collagen (data not I1 collagen, CMP is localized at both ends of the colla- shown). gen molecule, resulting in the formation of concatemAs shown in Figure 8, there is no apparent co-localers (Fig. 6, lower right). In addition to simple concate- ization of CMP and aggrecan in cultures of normal nation, networks of collagen molecules are also chondrocytes. However, material remaining after treatment with testicular hyaluronidase reacts with observed, with CMP-globules at the branch-points. 0

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Fig. 6. Electron micrographs of rotary shadowed preparations of CMP, collagen, and CMP-collagen mixtures. Upper panels: Individual molecules of CMP. Lower left panel: Type I1collagen molecules. Lower right panel: CMP-type I1 collagen mixture. Note the concatemerization of collagen molecules in the mixture of CMP and collagen. Bar, 0.2 pm.

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Fig. 7. Extracellularco-localization of CMP and type I1 collagen. After 3 days in the presence of ascorbic acid supplements to promote matrix deposition and assembly, 5-day-old cultures of chicken sternal chondrocytes were fixed in 75% ETOH, treated for immunofluorescentstaining, and incubated with antibodies in the following order: mouse monoclonal

lll-D-5 anti-CMP; TRITC-coupled goat IgG anti-mouse IgG; rabbit antitype I1 collagen antisera; and FITC-coupled goat IgG anti-rabbit IgG. The same field of double labeled cells is shown stained for both CMP (A) and type I1 collagen (B). lmmunofluorescentstaining of both molecules colocalizes in a filamentous pattern. Bar, 2 +m.

antibodies to the aggrecan core protein and co-localizes with the filamentous CMP/type I1 collagen pattern (Fig. 9). To examine the ultrastructural associations of CMP with the cartilage ECM, chondrocytes in culture were examined after immunoperoxidase reactions (Fig. 10). A periodic distribution of CMP along the network of collagen fibrils was observed at both low (Fig. 10A) and high (Fig. 10B) magnification. The ultrastructural immunostaining pattern for type I1 collagen (Fig. 1OC) is similarly periodic. Quantitative analysis revealed a periodic repeat of 59.3 nm (standard error = ? 0.6 nm) for CMP and 60.3nm (standard error = k 0.6 nm) for type I1 collagen. This observed repeat is consistent with the 60-65 nm periodicity known to be characteristic of type I1 collagen. The normal serum control appears non-reactive (Fig. 10D).

sults of the present study show that CMP is also a component of this fibril. The solid phase assay shows that CMP binds to collagen in a specific and concentration-dependent manner. Electron-microscopyfollowing rotary shadowing shows that CMP binds to the ends of individual triple helical collagen molecules. This binding can apparently mediate concatenation of collagen molecules and a more complex networking, within which CMP forms the branch points of the network. Immunofluorescence experiments demonstrate the colocalization of CMP with type I1 collagen in the extracellular matrix of cultured chondrocytes and suggest a weaker secondary association of aggrecan with the collagen-CMP complexes. The specificity of binding of CMP is further demonstrated by immunoelectron-rnicroscopy of the extracellular matrix of cultured chondrocytes. The specificity of this binding is supported by the matching periodicity of CMP binding to collagen fibrils and the periodicity of the collagen fibril banding pattern. Extracted from tissue, CMP exists as a disulfide bonded trimer of 148 kDa, as measured by sedimentation equilibrium centrifugation. Estimates of the molecular weight of this protein by SDS-PAGE yield a

DISCUSSION The collagen fibril of cartilage is emerging as a complex heterogenous structure. This fibril, consisting largely of type I1 collagen, has been shown to have a core of type XI collagen and to have type IX collagen molecules covalently attached to its exterior. The re-

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Fig. 8. Differing patterns of extracellular localization of CMP and aggrecan. Chondrocytes treated as described in Figure 7 were incubated with antibodies in the following order: mouse monoclonal Ill-D-5 antiCMP; TRITC-coupledgoat IgG anti-mouse IgG; rabbit anti-aggrecan core protein IgG; and FITC-coupled goat IgG anti-rabbit IgG. The same field of double labeled cells is shown stained for both CMP (A) and aggrecan (B). The extracellular filamentous pattern exhibited by CMP does not co-localize with the amorphous pattern exhibited for aggrecan. Bar, 2 p n

higher value (Paulsson and Heineglrd, 1981; the present study). It is assumed that the cysteine residues within the central EGF-like motif of each CMP polypeptide are involved in disulfide bonds identical to those found in this motif within true EGF and that the disulfide-mediated oligomerization of CMP therefore involves some of the other cysteines. The solid phase assay demonstrates that reduced and alkylated CMP can bind to collagen and inhibit the binding of native CMP to collagen in a manner identical to that of free native CMP. The disulfide bonds of CMP, therefore, appear not to be necessary for the binding of this molecule to collagen. These bonds are, however, involved in the multimerization of CMP and this in turn may be required for the in vivo function of this molecule. Although a single CMP polypeptide is capable of binding to collagen in vitro, it may be necessary that a CMP multimer be available in vivo, either to bridge two collagen molecules or to provide sufficient multivalency with regard to the binding site of collagen. The collagen binding of another molecule, von Willebrand factor, which contains motifs homologous to the internal repeat of CMP, has been shown (Pareti et al.,

1987). Assuming that the CMP motifs found in other molecules are also involved in collagen binding, the absence of cysteine residues in these motifs would suggest that disulfide bonding is not critical in collagen binding. This conclusion is consistent with our data, which suggest that the disulfide bonded structure of CMP is not essential for its binding to collagen. As rotary shadowing electron-microscopy shows the binding of CMP to the end of collagen molecules, and as immuno-stained transmission electron-micrographs show the characteristic 60-64 nm collagen banding periodicity for the binding of CMP to collagen, we suggest that CMP binds to the exterior of the collagen fibril at the gap-zone between two collagen molecules by binding to each of those collagen molecules. The extent to which CMP is distributed within the collagen fibril is not known. Our model (Fig. 111, therefore, shows the interaction of CMP only with the surface of the collagen fibril. Whereas this model and the collagen binding abilities of CMP emphasize a role for the molecule in the bridging of collagen fibrils to other collagen fibrils, it is also possible that CMP plays additional roles in the matrix of cartilage.

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Fig. 9. Extracellular co-localization of CMP and residual aggrecan after testicular hyaluronidase digestion. Chondrocytes were cultured and fixed as described in Figure 7. Prior to fixation, cells were treated with testicular hyaluronidase for 15 minutes at 37°C to remove most of the extracellular aggracan. Chondrocytes were incubated with antibodies in

the same manner as described for Figure 8 . The removal of large quantities of extracellular proteoglycan revealed residual material that is immunoreactive with antibodies to the aggrecan core protein (B) and that colocalizes with extracellular CMP (A) in a filamentous pattern. Bar, 2 wm.

EXPERIMENTAL PROCEDURES Purification of CMP Finely chopped xyphoid cartilage of 4-6 week-old white Leghorn chickens was stirred, overnight a t 5"C, in 15 volumes of extraction buffer: 4 M guanidine hydrochloride/50 mM sodium acetate, pH 5.8, containing protease inhibitors (5 pM benzamidine HC1, 0.5 pM PMSF, 5 pM N-ethylmaleimide, 10 pM EDTA, 10 pM 6-aminocaproic acid). This extract was then fractionated using cesium chloride density gradient centrifugation under associative conditions (Hascall and Sajdera, 1969). The pellicle of insoluble material which forms at the top of the gradient was collected and stirred overnight in extraction buffer. Following clarification of this extract by centrifugation (25,OOOg, 30 min), it was concentrated 7-fold using Centriprep-10 ultrafiltration units (Amicon, Danvers, MA). This concentrated extract was chromatographed on a column of Sephadex G-200 eluted with extraction buffer. The void volume-pool from this chromatography was dialyzed against water and redissolved in 4 M guanidine hydrochloride for use in ELISA. Alternatively, the G-200 void volume-pool was concentrated by ultrafiltration using Centriprep concentrators and applied to a column of Sepharose CL4 B eluted with extraction buffer.

4 to 6 weeks old Aliquots of each fraction were TCAprecipitated and screened by SDS-PAGE to allow pooling of pure CMP for electron microscopy. Protein concentrations were determined using the Biorad Protein Assay (BioRad Laboratories, Richmond, CA) with bovine serum albumin as a standard.

Bacterial CMP Expression The isolation of partial cDNA clones of CMP has been previously described, and the entire CMP mRNA sequence was determined from these cDNA clones and from sequencing of the exonic fragments of a genomic clone (Argraves et al., 1987; Kiss et al., 1989). The longest cDNA clone pCMP4 (1,304 nucleotides long) spanned a n Eco RI site 53 nucleotides 3' to the translation termination codon and extended in the 5' direction to contain 1,251 out of 1,482 nucleotides of the coding sequence. A full-length CMP cDNA was constructed by using a n oligonucleotide primer overlapping a unique Sst I site near the 5' end of pCMP4 clone to synthesize a first strand cDNA extending from the Sst I site to the 5' end of the mRNA. A second oligonucleotide which overlaps the AUG initiation codon of CMP and generates a restriction site to facilitate cloning of the CMP cDNA in the same reading frame as the

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Fig. 10. lmmunoelectron microscopic localization of CMP and type II collagen. Electron microscopy utilizing immunoperoxidase reactions with chondrocytes grown as described for Figure 7 is described in Materials and Methods. The periodic distribution of CMP along collagen fibrils is shown (A, B). The periodicity of CMP is similar to that of type II collagen (C), as indicated by small arrows in A and C. Panel D demonstrates a normal serum control. Bar A, C, and D, 0.5 km; for 6,0.2 km.

P-galactosidase gene of pUC119 was then used to amplify this fragment by the polymerase chain reaction (PCR). This PCR product was cloned as a Kpn I-Sst I fragment into the same sites of pUC119 and sequenced

to insure that no mutations were inadvertently introduced during the reverse transcriptase or the PCR reactions. This clone is referred to as pCMP5’. Subsequently, the Sst I-Eco RI fragment of pCMP4 was gel

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Fig. 11. Model of collagen fibril of cartilage. A three-dimensional representation of the collagen fibril of cartilage showing the core of type XI collagen surrounded by the principal component of the fibril, type It collagen. Type IX collagen is shown cross-linked to type II collagen at the surface of the fibril, and CMP is shown in the gap regions, between the ends of adjacent type II collagen molecules at the surface of the fibril.

Electrophoresis SDS-PAGE (Laemmli, 1970) was carried out on gels of various acrylamide concentrations, as indicated in the figure legends, blotted (Towbin et al., 1979) to Immobilon-P (Millipore Corp., Bedford, MA), and stained using the above antibodies followed by alkaline phosphatase conjugated goat anti-mouse IgG (H + L) or goat anti-rabbit IgG (H + L) (Biorad Laboratories) as appropriate, and using 5-bromo-4-chloro-3-indolylphosphate p-toluidine salthitroblue tetrazolium chloride (Gibco BRL, Gaithersburg, MD) as chromogenic substrate.

Binding Assay The interaction of CMP with collagen was studied using ELISA. Immobilization of CMP to microtitration plates (EIA, Linbro; Flow Laboratories, McLean, VA) was carried out in 0.05 M sodium carbonate buffer, pH 9.6 (60 pl/well), overnight a t room temperature. Collagens (Type I, Collaborative Research Inc., Bedford, purified and cloned in the Sst I-Eco RI site of pCMP5' MA; Type I1 Nitta Gelatin Inc., Osaka, Japan; Types to generate the full-length CMP cDNA clone, pCMP111, IV, and V, Calbiochem Corp., La Jolla, CA; Type F1. For expression of full length mature CMP protein, VI, Telios Pharmaceuticals, San Diego, CA; Clq, Centhe fragment of pCMP-F1 from the mature end of the ter for Blood Research, Boston, MA) were immobilized protein (Alanine 24) to the end of the CMP cDNA inin the same buffer by allowing the coating solution to sert was amplified by PCR. The 5' PCR oligomer indry a t 37"C, overnight. All subsequent additions were cluded an ATG initiation codon incorporated as part of for 1 hr in PBS containing 0.05%Tween-20 (PBS-T). an Nco I site; the 3' PCR oligomer also introduced an After each addition, plates were washed with PBS-T. Nco I site at the 3' end of the PCR fragment. This The binding of collagen to plastic or to immobilized fragment was purified, cut with Nco I and cloned into CMP was detected with mAb I-B4. The binding of CMP the Nco I site of the E. coli expression vector PET-lld to plastic or to immobilized collagen was detected with (Novagen). The clone PET-CMP 1containing the insert mAb 111-D5. The detecting monoclonal antibodies were in the correct orientation was selected and sequenced followed in each case by peroxidase-conjugated rabbit to insure that inadvertent mutations were not introanti-mouse IgG (H + L) and then peroxidase-substrate duced during the cloning procedure. The E. coli strain (BioRad Laboratories). Absorbance a t 405 nm was deBL21(D3)pLysS(Novagen) was transformed with PETtermined and recorded using a microtitration-plate CMP 1 and at appropriate times CMP expression was reader (Titertek Multiskan Plus, Flow Labs Inc., induced by IPTG. McLean, VA). Inhibition of collagen-CMP binding was undertaken Antibodies by mixing various concentrations of inhibitor with colPolyclonal antisera were generated in rabbits lagen and allowing the mixture to stand for one hour against CMP and a synthetic peptide corresponding to prior to adding to CMP coated plates. Detection of of CMP (the numbers identify- bound collagen was as above. residues Phe380-Va1424 ing the amino acids refer to the residue numbers in the primary translation product) conjugated to keyhole Cell Culture limpet haemocyanin, as described by Goetinck et al. Chondrocytes were obtained from the sterna of 15 (1987). Monoclonal antibodies were raised against a days old White Leghorn chicken embryos as described crude guanidine hydrochloride extract of sternal carti- by Cahn et al. (1967). For immunofluorescence studies, lage from 4 to 6 week-old chickens. ELISA screening cells were cultured in monolayer on gelatinized, carbon showed that mAb 111-D5 recognized CMP and I-B4 rec- coated-coverslips at a density of 6 x lo5 cells per 60 mm tissue culture dish in 3 ml Ham's F-12 medium ognized type I1 collagen. For use in electron microscopy, 111-D5 ascites fluid containing 10% fetal bovine serum and 1%antibioticand rabbit anti-Phe380-Va1424 serum were purified by antimycotic mix. Cultures were fed fresh medium conaffinity chromatography on a column of CMP- taining 50 mM ascorbic acid on days 3 and 4 and fed Sepharose. Polyclonal antisera to chicken cartilage again 2 hr before fixation in 75% ethanol. For immutype I1 collagen and aggrecan used in immunofluores- noelectron microscopy, cells were cultured on gelaticence studies were as described previously (Vertel and nized 35 mm tissue culture dishes a t equivalent densiDorfman, 1979; Upholt et al., 1979). ties in 1.5 ml of the same medium. Cell cultures were

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(quartz crystal) film thickness monitor (FTM4) (Peters, 1979).The resulting films were backed by carbon evaporation, floated on a clean water surface, and picked up Immunofluorescent Staining on 300 mesh grids for electron microscopy. Samples At 5 days of culture, chondrocytes were washed sev- were viewed and photographed in an Hitachi H-600 eral times with Hank’s Balanced Salt Solution (HBSS), STEM. Nanomelic embryos were obtained from fertile eggs fixed with 75% ethanol and prepared for immunofluorescence staining as described in Vertel et al. (1985).In resulting from mailings between parents heterozygous some experiments, glycosaminoglycans were removed for the nanomelic mutation. These eggs were obtained from extracellular aggrecan prior to fixation by a brief from the Department of Animal Genetics, the Univerdigestion with testicular hyaluronidase (Vertel et al., sity of Connecticut, through the courtesy of Dr. Louis J. Pierro. 1985). Cells were incubated for 20 min at room temperature ACKNOWLEDGMENTS with primary antibodies and FITC or TRITC-coupled secondary antibodies as indicated in the figure legends. This work was supported by grants ND22016 and Cells were washed extensively with HBSS between an- DK28433 from the National Institutes of Health. M. tibody incubations. After further washes with HBSS, Mehrdad Tondravi is an Arthritis Foundation Postdocthe coverslips were mounted in phosphate bufferlglyc- toral Fellow. erol (1:9, v/v). Samples were observed and photographed using a Leitz Ortholux microscope with phase REFERENCES and epifluorescence optics. Fields were selected from Argraves, W.S., Debk, F., Sparks, K.J., Kiss, I., and Goetinck, P.F. double-stained specimens and photographed sequen(1987) Structural features of cartilage matrix protein deduced from cDNA. Proc. Natl. Acad. Sci. U.S.A. 84:464-468. tially for FITC and TRITC staining.

fed fresh medium with ascorbate as described above prior to fixation with 2.5% gluteraldehyde on day 5.

Bentley, D.R. (1986) Primary structure of human complement component C2. Biochem. J. 239:339-345. Immunoelectron microscopy Bonaldo, P. and Colombatti, A. (1989) The carboxyl terminus of the After 5 days of culture as described above, chondrochicken a3 chain of collagen VI is a unique mosaic structure with cytes were fixed in 2.5% gluteraldehyde for 30 min. glycoprotein Ib-like fibronectin type 111, and Kunitz modules. J. Biol. Chem. 264:20235-20239. Immunoperoxidase studies with mouse monoclonal IIID5 anti-CMP and rabbit anti-type I1 collagen were con- Bonaldo, P., Russo, V., Bucciotti, F., Doliana, R., and Colombatti, A. (1990) Structural and functional features of the a3 chain indicate a ducted according to the procedure of Brown and Farbridge role for chicken collagen VI in connective tissues. Biochemquhar (1984), with some modifications (Vertel et al., istry 29:1245-1254. 1989). Samples were counterstained for 5 min with Bornstein, P. and Traub, W. (1979) The chemistry and biology of collagen. In: “The Proteins,” Neurath, H. and Hill, R.L. (eds.). New Reynolds lead citrate and observed and photographed York Academic Press 4:411-632. using a Zeiss 10 electron microscope. Brown, W.J. and Farquhar, M.G. (1984) The mannose-6-phosphate Analysis of the periodicity of CMP and type I1 collareceptor for lysosomal enzymes is concentrated in cis Golgi cistergen immunostaining was performed using a computernae. Cell 36:295-307. assisted image analysis system and Microcomp soft- Cahn, R.D., Coon, H.G., and Cahn, M.B. (1967) Cell culture and cloning techniques. In: “Methods in Developmental Biology.” Wilt, F.H. ware. Actual readings were taken directly from and Wessells, N.K. (eds.) New York Thomas Y. Crowell Co., pp photographic prints of electron micrograph negatives 493-530. magnified 4.65 x . Seven samples were measured from Chandrasekhar, S., Kleinman, H.K. and Hassell, J.R. (1983) Interaceach enlargement. Measurements of over 200 samples tion of link protein with collagen. J . Biol. Chem. 258:6226-6231. were used in each group. Absolute calibrations were Corbi, A.L., Miller, L.J., OConnor, K., Larson, R.S., and Springer, T.A. (1987) cDNA cloning and complete primary structure of the a based on the use of a carbon replica standard. All elecsubunit of leukocyte adhesion glycoprotein, p150, 95. EMBO J . 6: tron micrographs and enlargements were photo4023-4028. graphed, developed, and printed at the same magnifi- Corbi, A.L., Kishimoto, T.K., Miller, L.J., and Springer, T.A. (1988) The human leukocyte adhesion glycoprotein Mac-1 (complement cations during the same photographic sessions.

Rotary Shadowing Preparations of purified CMP or collagen were dialyzed against 0.1M ammonium acetate (HPLC grade, Sigma) pH 7.4, mixed with glycerol (40%)to a concentration of 25-50 Fg/ml and nebulized onto freshly cleaved mica substrates (Engvall et al., 1986). Each substrate was attached to a rotary stage in an Edwards E 306 vacuum evaporator (pumped to lop6Torr) a t an angle of 5-10” to the twin electron beam source approximately 10 cm from the sample. Replicas were generated by evaporation of pure tungsten deposited to a thickness of 19-30 A, as measured by a water cooled

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Cartilage matrix protein is a component of the collagen fibril of cartilage.

DEVELOPMENTAL DYNAMICS 193266276 (1992) Cartilage Matrix Protein is a Component of the Collagen Fibril of Cartilage NEIL WINTERBOTTOM, M. MEHRDAD TON...
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