no detectable formation of proteoglycan. Our results ... related glycoprotein that does not undergo the post- .... used to determine background disintegrations/min.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular’ Biology, Inc.
Vol. 263, No. 33, Issue of November 25, pp. 17749-17754,1988 Printed in U.S.A.
Nanomelic ChondrocytesSynthesize a Glycoprotein Related to Chondroitin Sulfate Proteoglycan Core Protein* (Received for publication, June 9, 1988)
Colleen M. O’Donnell, Karen Kaczman-Daniel, Paul F. GoetinckS, and Barbara M. VertelQll From the §Department of Cell Biology and Anatomy, University of Health ScienceslThe Chicago Medical School,North Chicago, Zllinois 60064, the Department of Biology, Syracuse University, Syracuse, New York 13210, and $La Jolla Cancer Research Foundation, Cancer Research Center, La Jolla, California 92037
Chicken embryos homozygous for the autosomal recessive gene nanomelia exhibit cartilage defects, synthesize low levels of cartilage chondroitin sulfate proteoglycan (CSPG), and are missing the CSPG core protein (Argraves, W. s., McKeown-Longo, P. J., and Goetinck, P. F. (1981) FEBS Lett. 131, 265). In our studies of nanomelic chondrocytes in culture, we detected neither sulfate-labeled CSPG noritsM, 370,000 core protein. However, in immunoprecipitation reactions usingboth polyclonal and monoclonal antibodies directed against the cartilage CSPG core protein, we identified a protein of M, 300,000 that contains an epitope found in the hyaluronic acid-binding region of the normal core protein. This protein was alsodetected among products synthesized by chondrocytes obtained from phenotypically normal embryos resulting from matings between parents heterozygous for nanomelia. Sensitivity to endoglycosidase H indicated that the product is a glycoprotein with attached mannose-rich oligosaccharides. Pulse-chase studies revealed the disappearance of the glycoprotein after 6 h of chase, but no detectable formation of proteoglycan. Our results suggest that although nanomelic chondrocytes are deficient in the production of normal CSPG and its core protein, they do synthesize a smaller, immunologically related glycoprotein that does not undergo the posttranslational processing characteristic of the normal cartilage core protein.
The extracellular matrix (ECM)’ of normal cartilage functions to absorb compressive forces and toprovide a model for bone replacement during long bone growth and development. The major macromolecular constituents of the cartilage ECM, chondroitin sulfateproteoglycan (CSPG) andtype I1 collagen, provide the structural basis for these tissue functions (Hay, 1981). In particular, cartilage function is dependent upon the high degree of hydration maintained by the large, negatively charged, link protein-stabilized aggregates of hyaluronic acid and CSPG monomer. The cartilage CSPG monomer itself is very large and com*This research was supported by United States Public Health Service Grants AM-28433 and HD 22016. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. (To whom correspondence should be sent: Dept. of Cell Biology and Anatomy, University of Health Sciences/The Chicago Medical School, 3333 Green Bay Rd., North Chicago, IL 60064. The abbreviations used are: ECM, extracellular matrix; CSPG, chondroitin sulfate proteoglycan; HBSS, Hanks’ balanced salt solution; Endo H, endoglycosidase H; PMSF, phenylmetbylsulfonyl fluoride; SDS, sodium dodecyl sulfate.
plex. Chondroitin sulfate and keratansulfate glycosaminoglycan chains, and both N - and 0-linked oligosaccharides are covalently attached to the core protein (reviewed by Hascall, 1981; Heingird and Paulsson, 1984; Lohmander and Kimura, 1986; Hassell et al., 1986). Functional domains of the CSPG monomer corresponding tothe NH2-terminal, hyaluronic acid-binding region, the keratan sulfate-rich region, and the chondroitin sulfate-rich region have been identified (HeinegCd and Axelsson, 1977). The core protein accounts for only 8-10% of the 1-5 X 106-daltonCSPG monomer. Several mutations have been described which exhibit defects of cartilage tissues (Goetinck et al., 1981). The lethal avian mutant, nanomelia, and the cartilage matrix-deficient mouse are each characterized by malformed and shortened skeletal structures (Landauer, 1965; Mathews, 1967; Pennypacker and Goetinck, 1976; Kimata et al., 1981). The cartilage ECM occupies a greatly reduced volume in the affected animals (Pennypacker and Goetinck, 1976) as a result of decreased amounts of CSPG (Palmoskiand Goetinck, 1972; Pennypacker and Goetinck, 1976). Further studies suggest that the absence of detectable CSPG core protein provides the basis for the defect (Argraves et al., 1981). Recent studies have indicated that the mutant chondrocytes contain core protein mRNA of normal size but at a level of 6% that of normal chondrocytes (Stirpe et al., 1987). We recently initiated studies of nanomelic chondrocytes because of our long-standing interest in the synthesis and processing of cartilage CSPG. In this report, we identify a new glycoprotein of M, 300,000 in nanomelic chondrocytes which is immunologically related to the M, 370,000 CSPG core protein of normal cells. Our data suggest that nanomelic chondrocytes produce a glycoprotein related to CSPG core protein that does not undergo the post-translational processing characteristic of the normal cartilage core protein. EXPERIMENTALPROCEDURES
Materials-Fertile White Leghorn chicken eggs purchased from Cornel1 University (Ithaca, NY) and Sharpe Farms (Glen Ellyn, IL) were used as a source of normal chondrocytes. Fertile nanomelic eggs were generously provided by the Department of Animal Genetics a t the University of Connecticut (Storrs, CT). Trypsin, Ham’s F-12 medium, fetal calf serum, antibiotic-antimycotic mixture, and Hanks’ balanced salt solution (HBSS) were obtained from GIBCO. Testicular hyaluronidase was a product of Leo (Helsingborg, Sweden). [36S] Methionine (1150-1350 Ci/mmol), Na235S04,and EN3HANCE were purchased from DuPont-New England Nuclear. Endoglycosidase H (Endo H) was purchased from Miles Laboratory (Naperville, IL). Protein A-Sepharose CL-4B was from Pharmacia. Cell Culture-White Leghorn embryos were used for the preparation of normal chondrocytes (no; na+/na+). The embryos produced by the crossing of chickens known to be heterozygous for the nanomelic gene were separated into those with obvious nanomelic traits (na; na-/na-) and phenotypically normal offspring (mi; na+/
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na+,na+/na-). The phenotypically normal offspring of these matings consisted of homozygous normal embryos (na+/na+) and embryos that were heterozygous for the nanomelic gene (na+/na-)in an expected ratio of 1 to 2. Cartilage cells were prepared from the sternaof 15-day old chicken embryos as described by Cahn et al. (1967). Cells were cultured in suspension a t a density of 1.35 X lo6 cells/lOO-mm Petri plate in 9 ml of Ham's F-12 medium containing 10% fetal calf serum and 1% antibiotic-antimycotic mix. Cultures were incubated at 37 "C in a humidified atmosphere of 95% air and 5% CO,. Medium was changed every other day. Biosynthetic Labeling-Chondrocytes a t 2-3 days of suspension culture were washed twice with HBSS containing 10% Ham's F-12 medium a t 37 "C. Cells were pulse-labeled with 75-100 pCi of either [35S]methionineor Na2%904in 200 pl of HBSS containing 10%Ham's F-12 medium. For pulse-chase experiments, cells were pulse-labeled with 100 pCi of [35S]methionine for 6 min and chased in 5 ml of Ham's F-12 medium containing 10% fetalcalf serum, 1% antibioticantimycotic mix, and 1000-fold excess unlabeled methionine. A t indicated times, aliquots were removed, centrifuged in a Microfuge, washed with HBSS by repeated suspension and centrifugation, and treated as described below for immunoprecipitation or directly used for gel electrophoresis. Samples were sometimes separated into cell and medium fractions before immunoprecipitation. Immunoprecipitations-For immunoprecipitations, cells were suspended in 2 mM PMSF (phenylmethylsulfonyl fluoride), HBSS, 2% SDS, solubilized by boiling for 2 min, and quickly cooled on ice. Solubilized cell samples were either diluted with 4 volumes of immunoprecipitation buffer (50 mM Tris, pH 7.4, 190 mM NaCI, 6 mM EDTA, 2.5% Triton X-100) and stored a t -80 "C, or stored directly a t -80 "C and diluted with immunoprecipitation buffer before use. Preparations that required reduction and alkylation for immunoprecipitation (see"Antibodies") were suspended in 2 mM PMSF, HBSS, 2% SDS, 4 mM dithiothreitol, solubilized by boiling for 2 min, and quickly cooled on ice. After the addition of iodoacetamide to a final concentration of 8 mM, the samples were incubated for 30 min a t 37 "C, diluted with 4 volumes of immunoprecipitation buffer, and stored a t -80 "C. Indirect immunoprecipitations were performed as described by Bumol and Reisfeld (1982). Briefly, antibodies described belowwere preadsorbed to Protein A-Sepharose beads, For each immunoprecipitation reaction, 1 mgof Protein A-Sepharose was reacted with 1-3 pl of antibody. The preparation of immunoadsorbants of mouse monolonal antibody 1-C-6 (which does not react with Protein A) was performed by incubation of Protein A-Sepharose beads with goat anti-mouse IgG followed by incubation of the goat anti-mouse IgG-Protein A-Sepharose bead complexes with 1-C-6 mouse monoclonal antibody. The resultant immunoadsorbants were washed extensively with buffer (phosphate-buffered saline,0.5% Triton X-100, 0.1% ovalbumin) and incubated with labeled cell extracts overnight with shaking a t 4 "C. The immunoadsorbants with reacted product were washed 10-15 times with buffer and once with phosphate-buffered saline. Subsequently, immunoprecipitates were prepared either for gel electrophoresis or for Endo H digestion. Endo H Digestion-Immunoprecipitates were washed twice with 0.05 M sodium citrate, pH 5.5, and resuspended in 0.05 M sodium citrate, 2 mM PMSF. One half of each was incubated alone as the control and the otherhalf was incubated with 0.05 IU/ml of Endo H for 3 ha t 37 "C with shaking. Samples were centrifuged in aMicrofuge and the immunobeads with adherent immunoreactive product were processed for gel electrophoresis. Antibodies-Polyclonal rabbit antibodies directed against hyaluronidase-digested CSPG monomer from embryonic chicken epiphyses were prepared and characterized previously (Vertel and Dorfman, 1978,1979; Upholt et al., 1979,1981). These antibodies recognize cellfree translation products and all intracellular and extracellular forms of the CSPG monomer. Studies also utilized two monoclonal antibodies which recognizeepitopes of the cartilage CSPG core protein. The rat monoclonal antibody S103L has been shown to immunoprecipitate the unmodified CSPG core protein translated in cell-free reactions (Upholt et al., 1981) and therefore recognizes a protein determinant (Dorfman et al., 1980; Jenkins et al., 1981; Upholt et al., 1981). The mouse monoclonal antibody 1-C-6recognizes an epitope exposed after reduction and alkylation that is present within two NH2-terminal, globular domains of the cartilage CSPG core protein (Stevens et al., 1984; Hejna et al., 1987). One of these globular domains is located within the hyaluronic acid binding region. The 1-C-6 monoclonal antibody also recognizes the hyaluronic acid binding region of other aggregating proteoglycans (Caterson et al., 1986).
to CSPG Core Protein SDS-Polyacrylamide Gel Electrophoresis-Labeled cell samples and immunoprecipitates were solubilized in gel sample buffer (75 mM Tris-HCI, pH 6.7, 2% glycerol, 0.003% bromphenol blue, 0.1 M dithiothreitol) by heating a t 70 "C for 30 min, and electrophoresed on 35% gradient polyacrylamide slab gels containing 0.1% SDS (Neville, 1971; Vertel and Hitti, 1987). Gels were treated with EN3HANCE and dried for autoradiographic fluorography as described in Laskey and Mills (1975). Fluorographs of selected gels were analyzed quantitatively using an LKB laser scanning densitometer. In addition, bands of interest were located on fluorographed gels, excised as 3mm pieces, solubilized overnight in 750 pl of 30% H202, and quantitated by scintillation counting. Additional slices from each lane were used to determine background disintegrations/min. Dynein (340,000), myosin (200,000), and collagen (migrating as a globular protein of 130,000) were used as high molecular weight standards for determinations of apparent Mr. RESULTS
The Comparison of Narwmelic and Normal Chondrocyte Products-When normal chicken chondrocytes were incubated in Na235S04,they synthesized and secreted into the medium a labeled highM , proteoglycan which barely entered a 3-5% polyacrylamide resolving gel,as shown in Fig. 1. This product is shown immunoprecipitated from the medium by rabbit anti-CSPG antibodies (Fig. 1, Lane 2). In contrast, no immunoprecipitableproteoglycan product was detected in the medium fraction of nanomelic chondrocytes labeled with Na25S04(Fig. 1,Lane 1). Secreted products labeled with [35S]methioninewere compared in samples not immunoprecipitated, but removed directly from the culture medium (Fig. 1, Lanes 3 and 4 ) . The major product of nanomelic and normal chondrocytes that was observed was type I1 procollagen, which appeared to be equivalent qualitatively and quantitatively in both samples (Ilc, Fig. 1, Lanes 3 and 4 ) . Several other labeled bands present in the medium and appearing faintly on the exposure of the fluorograph displayed in Fig. 1, Lanes 3 and 4 corresponded as well. As expected, labeled product in the CSPG region of the gelwasobserved in the medium samples of na no na no "
4CSPG
1
1
2
3
4llc
4
FIG. 1. Comparison of the secreted products of normal nanomelic chondrocytes. Nanomelic, na-/na- (na;Lanes I and 3 ) and normal, na+/na+ (no;Lanes 2 and 4 ) chondrocytes were metabolically labeled with [35S]sulfate(Lanes l and 2) or [35S]methionine (Lanes 3 and 4 ) as described under"Experimental Procedures." Samples were removed a t 3.0 h, and medium fractions were separated from the cell pellets. The sulfate-labeled medium fractions were immunoprecipitated with rabbit anti-CSPG, electrophoresed, and shown in Lanes 1 and 2. The methionine-labeled medium fractions were solubilized directly with gel sample buffer, electrophoresed, and shown in Lanes3 and 4. For Fig. 1and all subsequent figures, samples were displayed on fluorographed, 0.1% SDS, 3-5% polyacrylamide gradient gels. Chondroitin sulfate proteoglycan (CSPG) and type I1 procollagen (IIc) are indicated. Positions of molecular weight standards are shown to the left for some, but not all figures.
Nanomelic GlycoproteinRelated to
CSPG Core Protein
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cells, which has an apparent MI of 370,000, was immunoprecipitated from the normal cartilage cells and the na+/na+, na+/na- mixture, but not from the nanomelic chondrocytes (CP, Fig. 3, Lanes 1 , 3 and 5 ) . Interestingly, however, a product of MI 300,000was immunoprecipitated from the nanomelic sample. The M , 300,000product was detected along with the MI 370,000 core protein in the immunoprecipitates of the mixed chondrocyte population, but appeared to be absent from the immunoprecipitates of normal chondrocytes ( N ,Fig. 3,Lanes 1,3 and 5 ) .The normal rabbit serum controls contained neither the MI 370,000 nor the M , 300,000 protein (Fig. 3, Lanes 2, 4 and 6). Occasionally, additional contaminating protein bands in the MI 300,000 and other regions of the gelwereobserved in core protein immunoprecipitates from normal chondrocytes. In order to determine quantitative relationships among the immunoprecipitated products of normal, nanomelic, and mixed normal/nanomelic heterozygote chondrocytes, gel bands were located by fluorography, excised, and quantitated no na mi by scintillation counting. In Table I, the distribution of radioM, -~ activity is compared for the M , 370,000 and 300,000 bands in immunoprecipitates of nanomelic, normal/nanomelic heterozygote, and normal chondrocytes. Representative data demonstrate that100% of the relative disintegrations/min of the (CP nanomelic immunoprecipitate was in the M , 300,000 glycoprotein, while in the immunoprecipitate from the mixture of normal and nanomelic heterozygotes, 67% of the disintegrations/min was in the MI 370,000 core protein band and 33% was in the M, 300,000 nanomelic glycoprotein band. The observed disintegrations/min in the M , 300,000 band of the normal/nanomelic heterozygote mixture are predicted on the 200Kbasis of simple Mendelian segregation. For immunoprecipitates of normal chondrocytes, 96% of the disintegrations/min 16OKCllC were found in the normal M , 370,000 core protein and 3.8% were found in the region of the MI 300,000 nanomelic product. 1 2 3 MI300,000 glycoprotein possibility that the FIG.2. Comparison of the biosynthetic products of nano- If weentertain the is present in normal chondrocytes, it would appear to repremelic, normal, and mixed normal/nanomelic chondrocyte populations. Nanomelic, na-/na- (nu;Lane I ) , normal na+/na+ ( n o ; sent only a minor component. Lane 2), and the normal/nanomelic mixtures, na+/na+, na+/naDeterminations of apparent MI were based on the assump(mi; Lane 3) of chondrocytes were labeled with [%]methionine for tion that electrophoretic mobility truly reflects the MI of a 45 min and solubilized with gel sample buffer. Products are displayed protein. However, this assumption should be viewed with on a 0.1% SDS,3-5% polyacrylamidegel. The M,370,000 CSPG core caution, as some proteins, notably the collagens, behave protein (CP)and type I1 procollagen (Zlc) are indicated. anomalouslyon SDS-polyacrylamidegels (Freytag et aL, 1979; Sandell and Veis, 1980),and it is possible that theCSPG core mi no protein and thenanomelic product also migrate anomalously.
normal but not of nanomelic chondrocytes (CSPG, Fig. 1, Lanes 3 and 4 ) . In order to compare biosynthetic products of nanomelic and normal chondrocytes more generally, cells were labeled with [35S]methioninefor 45 min. Products were displayed on the 0.1% SDS, 3-5% polyacrylamide gradient gel shown in Fig. 2. The CSPG core protein of M , 370,000 was produced by both normal, na+/na+ chondrocytes (no) and normal/ nanomelicheterozygote, na+/na+, na+/na- mixtures of but was absent from among the products chondrocytes (mi), of nanomelic, na-/na- chondrocytes (nu).Most other protein products were comparable among the three populations of chondrocytes. Nanomelic chondrocytes were examined further for their production of CSPG or related products by the immunoprecipitation of labeled products with rabbit anti-CSPG (Fig. 3, Lanes 1 , 3 and 5 ) or normal rabbit serum (Fig. 3, Lanes 2 , 4 and 6). The CSPG core protein characteristic of cartilage
~
4CP
