Characterization of the tissue-specific proteoglycans synthesized by chondrocytes from nanomelic chick embryos.

Cartilage from the avian mutant nanomelia has been reported to synthesize cartilage-specific proteoglycans, PGS(SC)-I, at 1-2% of normal values [McKeown & Goetinck (1979) Dev. Biol. 71, 203-215]. Proteoglycans were endogenously labelled with [35S]sulphate and extracted from cartilage in 4 M-guanidine hydrochloride and chromatographed on controlled-pore glass 1400. PGS(SC)-I was obtained from the void volume of these columns. Dissociative sucrose-density-gradient analysis revealed a greater than normal polydispersity in the nanomelic PGS(SC)-I. Fractions from both the controlled-pore glass 1400 void volume and sucrose gradients were tested for their ability to bind specific antibody against cartilage proteoglycan monomer. In all instances, binding of normal fractions was greater than 90%, whereas binding to nanomelic fractions ranged from 20 to 65%. Chromatography of PGS(SC)-I on controlled-pore glass 2500 resulted in 70% of the normal and 25% of the mutant proteoglycans eluting as aggregates. Chondroitin sulphate chains from mutant PGS(SC)-I appeared slightly larger than normal when chromatographed on controlled-pore glass 500. In addition, PGS(SC)-I from nanomelic cartilage is more susceptible to proteolysis in vitro than the PGS(SC)-I from normal cartilage. This evidence suggests that the small amount of cartilage-specific proteoglycan synthesized by nanomelic cartilage is not normal.     

Aggrecan core protein is expressed in membranous bone of the chick embryo. Molecular and biomechanical studies of normal and nanomelia embryos.

The recessive mutation nanomelia blocks the synthesis of a large aggregating proteoglycan (aggrecan) by avian embryo chondrocytes. Lack of aggrecan is associated with short stature, multiple morphological defects in cartilage, and embryo lethality. Bony defects have also been described, but were assumed to be a secondary consequence of the cartilage defect. However, two lines of evidence presented in this paper indicate that the aggrecan deficiency directly affects intramembranous bone. First, the morphology (i.e. projected area and shape) of certain membranous bones of nanomelia embryos was abnormal. Second, membranous bone from nanomelia embryos proved to be significantly stiffer in biomechanical tests that measured functional properties of the extracellular matrix. These findings were unexpected because intramembranous bones normally develop from mesenchyme and not from a cartilage intermediate, and they prompted a search for evidence of aggrecan expression in the bone of normal chick embryos. We report that: 1) aggrecan mRNA was identified by PCR analysis of total RNA isolated from day-13 chick embryo calvarium, 2) the PCR method successfully amplified aggrecan mRNA from primary chick embryo osteoblasts in culture, 3) in situ hybridization of membranous bone tissue sections demonstrated aggrecan expression by chick embryo osteoblasts in vivo, and 4) the aggrecan message was identified in Northern blots of calvarial mRNA probed at high stringency. The results of the molecular and biomechanical studies provide evidence that aggrecan is indeed expressed in membranous bone as well as cartilage. Altogether, these results suggest that aggrecan may contribute to the functional properties and the normal growth and development of avian membranous bone.     

The expression of NG2 proteoglycan in the developing rat limb

NG2 is a chondroitin sulfate proteoglycan previously found to be expressed by glial progenitor cells of the O2A lineage. We have examined the expression of NG2 in the developing rat limb by immunohistochemistry and northern blot analysis. Staining of embryonic day 14 (E14) rat limb bud sections with polyclonal and monoclonal anti-NG2 antibodies reveals reactivity in the precartilaginous mesenchymal condensation. The staining intensity increases with the differentiation of chondrocytes until E16. NG2 staining is not detected in the mature hypertrophic chondrocytes of E17 and postnatal day 3 (P3) limbs even after treatment of the sections with hyaluronidase or collagenase. Immuno-precipitations with anti-NG2 antibody using 125I-labeled limb cells in culture showed a 400 to 800 x 10(3) Mr proteoglycan species with a core protein size of 300 x 10(3) Mr, comparable to NG2 from O2A cells and neural cell lines. Northern blot analysis reveals the expression of an 8.9 kb mRNA in E16 limbs and at a lower level in P1 cartilage. The northern blot analyses also show that NG2 is distinct from the large aggregating proteoglycan of the cartilage. Our results indicate that in the developing limb cartilage, as in the differentiating oligodendrocytes, NG2 is present on immature cells in the process of differentiating, but its expression is downregulated as terminal differentiation of chondrocytes takes place.     

Deglycosylation of chondroitin sulfate proteoglycan and derived peptides

In order to define the domain structure of proteoglycans as well as identify primary amino acid sequences specific for attachment of the various carbohydrate substituents, reliable techniques for deglycosylating proteoglycans are required. In this study, deglycosylation of cartilage chondroitin sulfate proteoglycan (CSPG) with minimal core protein cleavage was accomplished by digestion with chondroitinase ABC and keratanase, followed by treatment with anhydrous HF in pyridine. Nearly complete deglycosylation of secreted proteoglycan was verified within 45 min of HF treatment by loss of incorporated [3H]glucosamine label from the proteoglycan as a function of time of treatment, as well as by direct analysis of carbohydrate content and xylosyltransferase acceptor activity of unlabeled core protein preparations. The deglycosylated CSPG preparations were homogeneous and of high molecular weight (approximately 370,000). Comparison of the intact deglycosylated core protein preparations with newly synthesized unprocessed precursors (apparent Mr approximately 360,000) suggested that extensive proteolytic cleavage of the core protein did not occur during normal intracellular processing. Furthermore, peptide patterns generated after clostripain digestion of core protein precursor and of deglycosylated secreted proteoglycan were comparable. With the use of the clostripain digestion procedure, peptides were produced from unlabeled proteoglycan, and two predominant peptides from the most highly glycosylated regions (the chondroitin sulfate rich regions of the proteoglycan) were isolated, characterized, and deglycosylated. These peptides were found to follow similar kinetics of deglycosylation and to acquire xylose acceptor activity comparable to the intact core protein.     

Proteoglycan biosynthesis in chondrocytes: protein A-gold localization of proteoglycan protein core and chondroitin sulfate within Golgi subcompartments

The intracellular pathway of cartilage proteoglycan biosynthesis was investigated in isolated chondrocytes using a protein A-gold electron microscopy immunolocalization procedure. Proteoglycans contain a protein core to which chondroitin sulfate and keratan sulfate chains and oligosaccharides are added in posttranslational processing. Specific antibodies have been used in this study to determine separately the distribution of the protein core and chondroitin sulfate components. In normal chondrocytes, proteoglycan protein core was readily localized only in smooth-membraned vesicles which co-labeled with ricin, indicating them to be galactose-rich medial/trans-Golgi cisternae, whereas there was only a low level of labeling in the rough endoplasmic reticulum. Chondroitin sulfate was also localized in medial/trans-Golgi cisternae of control chondrocytes but was not detected in other cellular compartments. In cells treated with monensin (up to 1.0 microM), which strongly inhibits proteoglycan secretion (Burditt, L.J., A. Ratcliffe, P. R. Fryer, and T. Hardingham, 1985, Biochim. Biophys. Acta., 844:247-255), there was greatly increased intracellular localization of proteoglycan protein core in both ricin-positive vesicles, and in ricin-negative vesicles (derived from cis-Golgi stacks) and in the distended rough endoplasmic reticulum. Chondroitin sulfate also increased in abundance after monensin treatment, but continued to be localized only in ricin-positive vesicles. The results suggested that the synthesis of chondroitin sulfate on proteoglycan only occurs in medial/trans-Golgi cisternae as a late event in proteoglycan biosynthesis. This also suggests that glycosaminoglycan synthesis on proteoglycans takes place in a compartment in common with events in the biosynthesis of both O-linked and N-linked oligosaccharides on other secretory glycoproteins.     

Extracellular matrix formation by chondrocytes in monolayer culture

In previous studies were have reported on the secretion and extracellular deposition of type II collagen and fibronectin (Dessau et al., 1978, J. Cell Biol., 79:342-355) and chondroitin sulfate proteoglycan (CSPG) (Vertel and Dorfman, 1979, Proc. Natl. Acad. Sci. U. S. A. 76:1261-1264) in chondrocyte cultures. This study describes a combined effort to compare sequence and pattern of secretion and deposition of all three macromolecules in the same chondrocyte culture experiment. By immunofluorescence labeling experiments, we demonstrate that type II collagen, fibronectin, and CSPG reappear on the cell surface after enzymatic release of chondrocytes from embryonic chick cartilage but develop different patterns in the pericellular matrix. When chondrocytes spread on the culture dish, CSPG is deposited in the extracellular space as an amorphous mass and fibronectin forms fine, intercellular strands, whereas type II collagen disappears from the chondrocyte surface and remains absent from the extracellular space in early cultures. Only after cells in the center of chondrocyte colonies shape reassume spherical shape does the immunofluorescence reveal type II collagen in the refractile matrix characteristic of differentiated cartilage. By immunofluorescence double staining of the newly formed cartilage matrix, we demonstrate that CSPG spreads farther out into the extracellular space that type II collagen. Fibronectin finally disappears from the cartilage matrix.     

Arterial chondroitin sulfate proteoglycan: localization with a monoclonal antibody

We generated a monoclonal antibody (Mab) against a large chondroitin sulfate proteoglycan (CSPG) isolated from bovine aorta. This Mab (941) immunoprecipitates a CSPG synthesized by cultured monkey arterial smooth muscle cells. The immunoprecipitated CSPG is totally susceptible to chondroitinase ABC digestion and possesses a core glycoprotein of Mr approximately 400-500 KD. By use of immunofluorescence light microscopy and immunogold electron microscopy, the PG recognized by this Mab was shown to be deposited in the extracellular matrix of monkey arterial smooth muscle cell cultures in clusters which were not part of other fibrous matrix components and not associated with the cell's plasma membrane. With similar immunolocalization techniques, the CSPG antigen was found enriched in the intima and present in the medial portions of normal blood vessels, as well as in the interstitial matrix of thickened intimal lesions of atherosclerotic vessels. Immunoelectron microscopy revealed that this CSPG was confined principally to the space within the extracellular matrix not occupied by other matrix components, such as collagen and elastic fibers. These results indicate that this particular proteoglycan has a specific but restricted distribution in the extracellular matrix of arterial tissue.     

Cell-free translation of messenger RNA for chondroitin sulphate proteoglycan core protein in rat cartilage.

Total RNA was extracted from the cartilage tissues rat Swarm chondrosarcoma, neonatal-rat breastplate and embryonic-chicken sterna and translated in wheat-germ cell-free reactions. The core protein of the chondroitin sulphate proteoglycan subunit was identified among translation products of rat mRNA by its apparent Mr of 330 000 and by its immunoprecipitation with specific antisera prepared against rat or chicken proteoglycan antigens. The apparent Mr of the rat proteoglycan core protein is 8000-10000 less than that of the equivalent chicken cartilage core-protein product.     

Stimulation of chondroitin sulfate synthesis by beta-D-xyloside in chondrocytes of the proteoglycan deficient mutant nanomelia.

The potential of nanomelic chondrocytes to synthesize chondroitin sulfate was investigated by providing the mutant cells with p-nitrophenyl-beta-D-xyloside, a compound which acts as an artificial acceptor for glycosaminoglycan synthesis. Under these conditions the synthesis of chondroitin sulfate in nanomelic and normal chondrocytes is comparable. The chondroitin sulfate synthesized by the mutant is indistinguishable in molecular size and composition from that synthesized by similarly treated normal chondrocytes.     

Immunological characterization of a basement membrane-specific chondroitin sulfate proteoglycan

Reichert's membrane, an extraembryonic membrane present in developing rodents, has been proposed as an in vivo model for the study of basement membranes. We have used this membrane as a source for isolation of basement membrane proteoglycans. Reichert's membranes were extracted in a guanidine/3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate buffer followed by cesium chloride density-gradient ultracentrifugation under dissociative conditions. The proteoglycans were subsequently purified from the two most dense fractions (greater than 1.3 g/ml) by ion-exchange chromatography. Mice were immunized with the proteoglycan preparation and four mAbs recognizing the core protein of a high-density, buoyant chondroitin sulfate proteoglycan were raised. Confirmation of antibody specificity was carried out by the preparation of affinity columns made from each of the mAbs. Chondroitin sulfate proteoglycans (CSPGs) were purified from both supernatant and tissue fractions of Reichert's membranes incubated in short-term organ culture in the presence of radiolabel. The resultant affinity-purified proteoglycan samples were examined by gel filtration, SDS-PAGE, and immunoblotting. This proteoglycan is of high molecular weight (Mr = 5-6 x 10(5)), with a core protein of Mr = approximately 1.5-1.6 x 10(5) and composed exclusively of chondroitin sulfate chains with an average Mr = 1.6-1.8 x 10(4). In addition, a CSPG was purified from adult rat kidney, whose core protein was also Mr = 1.6 x 10(5). The proteoglycan and its core protein were also recognized by all four mAbs. Indirect immunofluorescence of rat tissue sections stained with these antibodies reveal a widespread distribution of this proteoglycan, localized specifically to Reichert's membrane and nearly all basement membranes of rat tissues. In addition to heparan sulfate proteoglycans, it therefore appears that at least one CSPG is a widespread basement membrane component.     

Two immunologically and developmentally distinct chondroitin sulfate proteolglycans in embryonic chick brain.

Two different chondroitin sulfate proteoglycans (CSPG) in embryonic chick brain were distinguished by immunoreactivity either with S103L, a rat monoclonal antibody which reacts specifically with an 11-amino-acid region in the chondroitin sulfate domain of the core protein of chick cartilage CSPG (Krueger, R. C., Jr., Fields, T. A., Mensch, J. R., and Schwartz, N. B. (1990) J. Biol. Chem. 265, 12088-12097), or with HNK-1, a mouse monoclonal antibody which reacts with a 3-sulfoglucuronic acid residue on neural glycolipids and glycoproteins (Chou, D. K. H., Ilyas, A., Evans, J. E. Costello, C., Quarles, R. H., and Jungawala, F. B. (1986) J. Biol. Chem. 261, 11717-11725) but not with both antibodies. This specific immunoreactivity was used to separate the two CSPGs for further characterization. The S103L reactive brain proteoglycan had a core protein of similar size to cartilage CSPG (370 kDa) but exhibited a smaller hydrodynamic size (K(av) of 0.308). It was substituted predominantly with chondroitin sulfate chains and virtually no keratan sulfate chains. The HNK-1 reactive CSPG had a smaller core protein (340 kDa), an even smaller hydrodynamic size (K(av) of 0.564), and was substituted with both chondroitin sulfate and keratan sulfate chains. Glycosidase digestion patterns with endo-beta-galactosidase, N-glycosidase F, neuraminidase, and O-glycosidase, and reactivity with an antibody to the hyaluronate binding region also showed significant differences between the two brain CSPGs. Expression of the S103L reactive brain CSPG was developmentally regulated from embryonic day 7 through 19 with a peak in core protein on day 13, and in mRNA expression at day 10. In contrast the HNK-1 reactive brain CSPG was constitutively present from day 7 through hatching. These data suggest that these two distinct core proteins are immunologically and biochemically unique translation products of two different CSPG genes.     

The folded modules of aggrecan G3 domain exert two separable functions in glycosaminoglycan modification and product secretion.

Aggrecan is the major proteoglycan in the extracellular matrix of cartilage. A notable exception is nanomelic cartilage, which lacks aggrecan in its matrix. The example of nanomelia and other evidence leads us to believe that the G3 domain plays an important role in aggrecan processing, and it has indeed been confirmed that G3 allows glycosaminoglycan (GAG) chain attachment and product secretion. However, it is not clear how G3, which contains at least a carbohydrate recognition domain (CRD) and a complement binding protein (CBP) motif, plays these two functional roles. The present study was designed to dissect the mechanisms of this phenomenon and specially 1) to determine the effects of various cysteine residues in GAG modification and product secretion as well as 2) to investigate which of the two processing events is the critical step in the product processing. Our studies demonstrated that removal of the two amino-terminal cysteines in the CRD motif and the single cysteine in the amino terminus of CBP inhibited secretion of CRD and CBP. Use of the double mutant CRD construct also allowed us to observe a deviation from the usual strict coupling of GAG modification and product secretion steps. The presence of a small chondroitin sulfate fragment overcame the secretion-inhibitory effects once the small chondroitin sulfate fragment was modified by GAG.     

Chondroitin 6-sulfate oligosaccharides as immunological determinants of chick proteoglycans

Monoclonal antibodies to hyaluronidase-treated chondroitin sulfate proteoglycan (CSPG) were used to study the immunological determinants of chick cartilage proteoglycan. The determinants recognized by the antibodies were studied by a radioimmune inhibition assay utilizing hyaluronidase-treated [35S]CSPG. Hyaluronidase-treated CSPG inhibits the reaction of four clonal antibodies, S54C, S103L, S11D, and P100D, with [35S]CSPG, but to varying degrees. Only the reaction of S103L is inhibited to a considerable extent by undigested CSPG, indicating that hyaluronidase treatment exposes determinants specific for the other three antibodies. These findings are consistent with the earlier conclusion that S103L is specific for a protein determinant (Dorfman et al., 1980). Only the reaction of S54C is not significantly inhibited by chondroitinase ABC-digested CSPG. This result indicates that chondroitinase ABC digestion can also expose determinants recognized by S11D and P100D but that such digestion removes the determinant recognized by S54C. Of the four antibodies tested, only the reaction of S54C with hyaluronidase-treated [35S]CSPG is significantly inhibited by chondroitin-6-SO4 tetra- and hexasaccharide (59 and 43% inhibition, respectively, at a concentration of 1333 microM). The reaction of S54C is inhibited to a lesser extent by chondroitin tetra- and hexasaccharide (28 and 26% inhibition, respectively, at a concentration of 1333 microM). In contrast, chondroitin-4-SO4 oligosaccharides do not inhibit the reactions of any of the clonal antibodies. These result suggest that S54C recognizes a determinant that contains chondroitin-6-SO4 oligosaccharide, attached via the linkage oligosaccharide to core protein.     

Proteoglycans of developing bone

We purified and characterized the bone proteoglycans from fetal calves, growing rats, and human fetuses. The major proteoglycan is part of the mineralized tissue matrix and only 10-20% can be extracted prior to demineralization. This bone proteoglycan is a small glycoconjugate (Mr = 80,000-120,000) containing approximately 20-30% protein and either one or two chondroitin sulfate chains (Mr = 40,000) attached to a relatively monodisperse protein core (Mr = 38,000). "O"-linked and "N"-linked oligosaccharide units are also present. Antibodies directed against the protein core of calf bone proteoglycan do not cross-react with cartilage, skin, corneal, or basement membrane proteoglycans in immunoassays and have minimal cross-reactivity with scleral proteoglycans. Quantitative immunoassays and indirect immunofluorescence were used to show that the molecule is localized to forming bone trabeculae and dentin, but not to any other tissue. Osteoblasts and osteoprogenitor cells adjacent to areas undergoing rapid osteogenesis also contain this small proteoglycan. A second proteoglycan (Mr approximately equal to 1,000,000) was extracted from newly forming bone prior to demineralization. This large proteoglycan, which was isolated from the cartilage-free areas of developing intramembranous bone, has a protein core similar to that of the cartilage aggregating proteoglycan and cross-reacts with antisera raised against these cartilage proteoglycans but not with the small mineral-entrapped proteoglycan. It contains larger (Mr = 40,000) and fewer chondroitin sulfate chains than its cartilage-derived analogue, and is localized to the soft connective tissue mesenchyme lying between growing bone trabeculae. More fully formed compact bone did not contain detectable quantities of this proteoglycan.     

Isolation and characterization of a third proteoglycan (PG-Lt) from chick embryo cartilage which contains disulfide-bonded collagenous polypeptide

Chick embryo epiphyseal cartilage has been shown to contain three different proteoglycan species (PG-H, PG-Lb, and PG-Lt). This report is concerned with the purification and characterization of the third proteoglycan, PG-Lt. The proteoglycan can be separated from the other two by virtue of its low buoyant density in a CsCl density gradient and further purified by consecutive ion exchange and gel chromatography. The final preparation is composed of PG-Lt monomer and PG-Lt oligomer. The amino acid composition of PG-Lt is quite different from that of PG-H and PG-Lb and rather resembles that of collagens with respect to high content of glycine and high degrees of hydroxylation of proline and lysine. PG-Lt monomer is composed of disulfide-bonded subunits of Mr congruent to 120,000 and 190,000 as demonstrated by its gel electrophoretic behavior after reduction with 2-mercaptoethanol. The latter, but not the former, contains dermatan sulfate chains with glucuronic acid/iduronic acid residues and yields a protein-enriched core molecule of Mr congruent to 100,000 after digestion with chondroitinase ABC. Both of the protein subunits are completely digestible with bacterial collagenase. Immunofluorescence microscopic examination of cartilage tissues, using an antibody against PG-Lt, shows that this proteoglycan exists in both the cartilage matrix and perichondrial noncartilagenous region. When chondrocytes are plated onto tissue culture dishes, the antibody stains strands found on the cell surfaces and in the intercellular space of substrate-attached cell layers, suggesting that PG-Lt mediates cell-to-cell and cell-to-substrate contacts.     

Scorbutic and fasted guinea pig sera contain an insulin-like growth factor I-reversible inhibitor of proteoglycan and collagen synthesis in chick embryo chondrocytes and adult human skin fibroblasts

Chick embryo chondrocytes cultured in sera from scorbutic and fasted guinea pigs exhibited decreases in collagen and proteoglycan production to about 30-50% of control values (I. Oyamada et al., 1988, Biochem. Biophys. Res. Commun. 152, 1490-1496). Here we show by pulse-chase labeling experiments that in the chondrocyte system, as in the cartilage of scorbutic and fasted guinea pigs, decreased incorporation of precursor into collagen was due to decreased synthesis rather than to increased degradation. There was a concomitant decrease in type II procollagen mRNA to about 32% of the control level. As in scorbutic cartilage, proteoglycan synthesis by chondrocytes in scorbutic serum was blocked at the stage of glycosaminoglycan chain initiation. Scorbutic and fasted guinea pig sera also caused a 50-60% decrease in the rates of collagen and proteoglycan synthesis in adult human skin fibroblasts, which synthesize mainly type I collagen. Decreased matrix synthesis in both cell types resulted from the presence of an inhibitor in scorbutic and fasted sera. Elevated cortisol levels in these sera were not responsible for inhibition, as determined by the addition of dexamethasone to chondrocytes cultured in normal serum. Insulin-like growth factor I (IGF-I, 300-350 ng/ml) reversed the inhibition of extracellular matrix synthesis by scorbutic and fasted guinea pig sera in both cell types and prevented the decrease in type II procollagen mRNA in chondrocytes. Therefore, in addition to its established role in proteoglycan metabolism, IGF-I also regulates the synthesis of several collagen types. An increase in the circulating inhibitor of IGF-I action thus could lead to the negative regulation of collagen and cartilage proteoglycan synthesis that occurs in ascorbate-deficient and fasted guinea pigs.     

Identification of the keratan sulfate attachment sites on bovine fibromodulin

The small keratan sulfate-substituted proteoglycan (fibromodulin) from articular cartilage was shown to contain keratan sulfate linked to the core protein through N-glycosidic linkages to residues Asn-109, Asn-147, Asn-182, and Asn-272. Biosynthetic experiments with articular chondrocytes in the presence of tunicamycin, an inhibitor of N-linked oligosaccharide synthesis, demonstrated a specific inhibition of [35S]SO4 incorporation into fibromodulin. Under the same conditions no effect on the addition of keratan sulfate to the large aggregating proteoglycan was detected. Fibromodulin substituted with keratan sulfate was purified from bovine articular cartilage extracts by density gradient centrifugation, ion-exchange chromatography, and gel-permeation chromatography. Isolation of glycosylated peptides from tryptic digests of fibromodulin by ion-exchange chromatography and reversed-phase high performance liquid chromatography revealed four separate hexosamine-rich species, that were also immunoreactive with monoclonal antibody 5D4. Sequence analysis of these glycopeptides gave blank cycles at positions which corresponded to Asn followed by X-Ser/Thr in the sequence derived from cDNA (Oldberg, A., Antonsson, P., Lindblom, K., and Heinegard, D. (1989) EMBO J. 8, 2601-2604). Hence, all four Asn residues in the leucine-rich region of the fibromodulin core protein can serve as acceptor sites for keratan sulfate addition.     

Identification of chick corneal keratan sulfate proteoglycan precursor protein in whole corneas and in cultured corneal fibroblasts.

The precursor protein to the chick corneal keratan sulfate proteoglycan was identified by immunoprecipitation with antiserum to its core protein from lysates of [35S]methionine-pulsed corneas and corneal fibroblasts in cell culture. Antiserum to the keratan sulfate proteoglycan immunoprecipitated a doublet of Mr 52,000 and 50,000 and minor amounts of a Mr 40,000 protein from pulsed corneas. Pulse-chase experiments, which permitted the conversion of the precursor proteins to proteoglycans and digestion of the glycosaminoglycans on immunoprecipitated proteoglycans with keratanase or chondroitinase ABC, showed that the Mr 52,000-50,000 doublet was converted to a keratan sulfate proteoglycan and the Mr 40,000 protein was converted to a chondroitin sulfate proteoglycan. Chick corneal fibroblasts in cell culture primarily produced the smaller (Mr50,000) precursor protein, and in the presence of tunicamycin the precursor protein size was reduced to Mr35,000, which indicates that the core protein contains approximately five N-linked oligosaccharides. Pulse-chase experiments with corneal fibroblasts in culture showed that the precursor protein was processed and secreted into the medium. However, its sensitivity to endo-beta-galactosidase and resistance to keratanase indicate that the precursor protein was converted to a glycoprotein with large oligosaccharides and not to a proteoglycan. This suggests that, although the precursor protein for the proteoglycan is produced in cultured corneal fibroblasts, the sulfation enzymes for keratan sulfate may be absent.     

Butyric acid causes morphological changes in cultured chondrocytes through alterations in the extracellular matrix

Butyric acid induces characteristic changes in the morphology of chick embryo chondrocytes. Chick embryo chondrocytes when cultured in the absence of butyrate exhibit a spherical morphology and synthesize cartilage-specific chondroitin sulfate proteoglycan (CSPG). When these cultures are initiated and maintained in the presence of butyric acid, chondrocytes exhibit a mesenchymal morphology, a 90% reduction in the synthesis of CSPG, and a 75% reduction in DNA synthesis. The reduced synthesis of CSPG and DNA was shown not to be dependent on the morphological change. Chondrocytes require CSPG in order to express a spherical morphology, since including chondroitinase ABC in the culture media caused the cells to spread. In addition, the treatment of chondrocytes with purified CSPG prior to culture in media containing butyric acid resulted in spherical cells. The butyrate-induced spreading was shown to require either serum or fibronectin and could be prevented with antiserum against chick cell-surface fibronectin (cFn). Cell-surface fibronectin, which was present on both spherical and flattened chondrocytes, organized into fibrils beneath cells which spread. Increased fibronectin synthesis was not responsible for the butyrate-induced morphological change. From this evidence, it is concluded that the mechanism by which butyrate alters the morphology of these cells in culture involves inhibiting CSPG synthesis, thus preventing CSPG accumulation in the extracellular matrix (ECM). The absence of CSPG in the ECM allows fibronectin to mediate spreading of chondrocytes in culture.