Isolation and characterization of proteoglycans secreted by normal and malignant human mammary epithelial cells

The proteoglycans secreted by a malignant human breast cell line (MDA-MB-231) were compared with the corresponding proteoglycans from a normal human breast cell line (HBL-100). The physicochemical characteristics of these proteoglycans were established by hexosamine analysis, chemical and enzymatic degradations, and dissociative cesium chloride density gradient centrifugation, and by gel filtration before and after alkaline beta-elimination. Both cell lines secreted approximately 70% of the synthesized proteoglycans, which were composed of 20% heparan sulfate and 80% chondroitin sulfate proteoglycans. The MDA cell line secreted large hydrodynamic size (major) and small hydrodynamic size heparan sulfate proteoglycan. In contrast HBL cells secreted only one species having a hydrodynamic size intermediate to the above two. The chondroitin sulfate proteoglycans from MDA medium were slightly larger than the corresponding polymers from HBL medium. All proteoglycans except the small hydrodynamic size heparan sulfate proteoglycan from MDA medium were of high buoyant density. The proteoglycans of both cell lines contained significant proportions of disulfide-linked lower molecular weight components which were more pronounced in the proteoheparan sulfate polymers, particularly those from MDA medium, than in chondroitin sulfate proteoglycans. The glycosaminoglycans of heparan sulfate proteoglycans from MDA medium were more heterogeneous than those from HBL medium. The glycosaminoglycan chains of large hydrodynamic size heparan sulfate proteoglycans from MDA medium were larger in size than those from HBL medium while small hydrodynamic size heparan sulfate proteoglycans contained shorter glycosaminoglycan chains. In contrast to the glycosaminoglycans derived from chondroitin sulfate proteoglycans of both MDA and HBL medium were comparable in size. The heparan sulfate as well as chondroitin sulfate proteoglycans of both cell lines contained both neutral (di- and tetrasaccharides) and sialylated (tri- to hexasaccharides) O-linked oligosaccharides.     

Cell-surface heparan sulfate and heparan-sulfate/chondroitin-sulfate hybrid proteoglycans of mouse mammary epithelial cells

The hydrophobic cell-surface proteoglycans of mouse mammary epithelial cells were purified by gel filtration, ion-exchange chromatography, and liposome incorporation. The size of the proteoglycans appeared to be directly proportional to the size of their heparan-sulfate chains, larger proteoglycans yielding larger chains. The chondroitin sulfate chains, in contrast, showed no size heterogeneity. Digestion of 125I-labeled proteoglycans with heparitin-sulfate lyase and chondroitin ABC lyase yielded core proteins of approximately 93 kDa, approximately 85 kDa and approximately 38 kDa. Comparison with single enzyme digestions identified the 93-kDa and 85-kDa cores as components of hybrid proteoglycans that carried both heparan-sulfate and chondroitin-sulfate chains. Immunoblotting indicated that the 93-kDa and 85-kDa cores shared the epitope defined by monoclonal antibody 281-2. The 38-kDa core, in contrast, carried only heparan-sulfate chains and lacked the 281-2 epitope. Preparations enriched in heparan sulfate or in heparan-sulfate/chondroitin-sulfate hybrid proteoglycans were obtained by N-desulfation and ion-exchange chromatography. Hybrid proteoglycans accounting for the bulk of the chondroitin-sulfate and nearly half of the heparan-sulfate residues of the proteoglycans showed a similar polydispersity of heparan-sulfate chain sizes as found in proteoglycans that carried only, or predominantly, heparan-sulfate chains. These hybrids contained heparan-sulfate and chondroitin-sulfate chains in similar molar amounts. Analysis of 125I-labeled proteoglycans suggested that typical hybrid proteoglycans were composed of a 85-kDa core protein that carries a single chondroitin-sulfate chain and a single heparan-sulfate chain of variable length. A minority of hybrids seemed characterized by the variant, but possibly structurally related, 93-kDa core protein. The other half of the hydrophobic proteoglycans were composed of the 38-kDa core and carried only heparan-sulfate chains. The significance of the co-existence of hybrid and heparan-sulfate proteoglycans at the cell surface and possible relationships between the proteoglycans need to be further clarified.     

Proteoglycans of L6J1 myoblast extracellular matrix. Characteristics and effect on myoblast adhesion

Proteoglycans were isolated from extracellular matrix of L6J1 rat myoblasts and their influence on myoblast adhesion was studied. Proteoglycan digestion with chondroitinase AC and heparinase III degrading the polysaccharide moieties revealed that chondroitin sulfate proteoglycans are the main class of myoblast extracellular matrix proteoglycans. Electrophoresis of enzymatically processed proteoglycans was used to examine their core proteins. Myoblast adhesion was suppressed by proteoglycans or the mixture of proteoglycans and fibronectin/extracellular matrix. When being processed with chondroitinase AC the combined substrate of fibronectin and proteoglycans lost the capability of myoblast adhesion suppression. Thus, as a result of presented work the proteoglycans of L6J1 rat myoblast extracellular matrix were isolated and purified. The main class of proteoglycans was chondroitin sulphate proteoglycans. Isolated proteoglycans suppressed myoblast adhesion and this effect was mediated by polysaccharide moieties of proteoglycans.     

The dermatan sulfate proteoglycans of bovine sclera and their relationship to those of articular cartilage. An immunological and biochemical study

Dermatan sulfate proteoglycans were isolated from adult bovine sclera and adult bovine articular cartilage. Their immunological relationships were studied by enzyme-linked immunosorbent assays using polyclonal antibodies raised against the large and small dermatan sulfate proteoglycans from sclera and a polyclonal and monoclonal antibody directed against the small dermatan sulfate proteoglycans from cartilage. The small dermatan sulfate proteoglycans from sclera and cartilage displayed immunological cross-reactivity while there was no convincing evidence of shared epitope(s) with the larger dermatan sulfate proteoglycans, nor did these larger proteoglycans share any common epitopes with each other. A hyaluronic acid binding region was detected immunologically on the larger scleral dermatan sulfate proteoglycan but was absent from the larger dermatan sulfate proteoglycan of cartilage and both the small dermatan sulfate proteoglycans. These antibodies were used in immunofluorescence microscopy to localize the scleral proteoglycans and molecules containing these epitopes in the eye. The large scleral dermatan sulfate proteoglycan was restricted to sclera while molecules related to the small scleral and cartilage proteoglycans were found in the sclera, anterior uveal tract, iris, and cornea. Amino acid sequencing of the amino-terminal regions of the core proteins of the small dermatan sulfate proteoglycans from sclera and articular cartilage showed that all the first 14 amino acids analyzed were identical and the same as reported earlier for the small bovine skin and tendon dermatan sulfate proteoglycans. These studies demonstrate that the larger dermatan sulfate proteoglycans of sclera and cartilage are chemically unrelated to each other and to the smaller dermatan sulfate proteoglycans isolated from these tissues. The latter have closely related core proteins and probably represent a molecule with a widespread distribution in which the degree of epimerization of glucuronic acid and iduronic acid varies between tissues.     

Proteoglycans of the intervertebral disc. Homology of structure with laryngeal proteoglycans.

The structure of the proteoglycans from normal pig nucleus pulposus and relatively normal human annulus fibrosus and nucleus pulposus was investigated in detail and the results were compared with the current structural model of proteoglycans of hyaline cartilage. Like proteoglycans of cartilage, those of intervertebral disc contain keratan sulphate and chondroitin sulphate attached to a protein core; they are able to aggregate to hyaluronic acid; the protein core likewise has three regions, one lacking glycosaminoglycans, another rich in keratan sulphate and a third region rich in chondroitin sulphate. However, disc proteoglycans contain more keratan sulphate and protein and less chondroitin sulphate and are also considerably smaller than cartilage proteoglycans. In proteoglycans of human discs, these differences appeared to be due principally to a shorter region of the core protein bearing the chondroitin sulphate chains, whereas in proteoglycans of pig discs their smaller size and relatively low uronic acid content were due to shorter chondroitin sulphate chains. There were subtle differences between proteoglycans from the nucleus and annulus of human discs. In the latter a higher proportion of proteoglycans was capable of binding to hyaluronate.     

Proteoglycans of L6J1 myoblast extracellular matrix: Properties and effect on myoblast adhesion

Proteoglycans were isolated from the extracellular matrix (ECM) of L6J1 rat myoblasts; their influence on myoblast adhesion has been studied. Proteoglycan digestion with chondroitinase AC and heparinase III, which degrade polysaccharide moieties, has revealed that chondroitin sulfate proteoglycans are a major class of myoblast extracellular matrix proteoglycans. Electrophoresis of enzymatically processed proteoglycans was used to examine their core proteins. Myoblast adhesion was suppressed by proteoglycans or a mixture of proteoglycans and a fibronectin-extracellular matrix. Myoblast adhesion to a substrate composed of fibronectin and proteoglycans is restored after the substrate was treated with chondroitinase AC. In conclusion, proteoglycans of L6J1 rat myoblast ECMs were isolated and purified. Chondroitin sulfate proteoglycans are a major class of proteoglycans. Isolated proteoglycans suppressed myoblast adhesion; the effect was mediated by polysaccharide moieties of proteoglycans.     

Biosynthesis of proteoglycans in organ cultures of developing kidney mesenchyme

The biosynthesis of proteoglycans was studied in organ cultures of differentiating metanephric mesenchymes. When triggered by a contact-mediated inductive interaction, this tissue undergoes transition from a mesenchyme to an epithelium. In the present study, proteoglycans were extracted by guanidinium hydrochloride in the presence of protease inhibitors. We found that, as a response to induction, the differentiating mesenchyme begins to synthesize large size proteoglycans with an apparent molecular weight (MW) of 1 X 10(6) D. The major glycosaminoglycans detected were chondroitin sulfates. Heparan sulfate proteoglycans were also detected, constituting 20% of the proteoglycans. An inhibitor of glucosamine synthesis, 6-diazo-5-oxo-norleucine (DON) was found to inhibit glycosaminoglycan synthesis by approx. 60%, and the size of the proteoglycans was also diminished. Our studies suggest that the transition of the mesenchyme to epithelium is associated with initiation of synthesis of large size proteoglycans.     

Proteoglycans of the human intervertebral disc. Electrophoretic heterogeneity of the aggregating proteoglycans of the nucleus pulposus.

Nuclei pulposi were dissected from lumbar discs of radiologically normal human spines of cadavers aged 17, 20 and 21 years. Proteoglycans were extracted with 4 M guanidine hydrochloride (dissociative conditions) with proteinase inhibitors and isolated as A1 fractions by associative density-gradient centrifugation. Aggregating and non-aggregating proteoglycans were separated by Sepharose 2B chromatography. Both aggregating and non-aggregating proteoglycans contained a keratan sulphate-rich region as isolated by chondroitinase/trypsin/chymotrypsin digestion and Sepharose CL-6B chromatography. Agarose/acrylamide-gel electrophoresis of individual fractions of a Bio-Gel A-50m dissociative-column separation of the aggregating proteoglycans revealed two, well-separated bands: S and F, the slower and faster migrating bands respectively. The non-aggregating proteoglycan fractions were eluted under associative conditions (0.5 M-sodium acetate, pH 6.8) and migrated as a single band in the electrophoretic system. The gel-electrophoretic heterogeneity of the aggregating proteoglycans was still evident after hydroxylamine fragmentation and removal of the hyaluronate-binding portion of the molecule. Dissociative density-gradient centrifugation of the aggregating proteoglycans partially separated the Band-S proteoglycans from the Band-F population. Subsequent dissociative chromatography of the high-buoyant-density Band F proteoglycans permitted discrimination of this band into two gel-electrophoresis-distinguishable populations (Bands F-1 and F-2). Enzyme-linked immunosorbent assays with a monoclonal antibody that recognized keratan sulphate demonstrated that the D1 fraction containing the Band F-1 proteoglycans was enriched in keratan sulphate compared with the total aggregating or non-aggregating pool of proteoglycans. The proteoglycans of young adult nucleus pulposus could then be ascribed to one of four structurally and/or electrophoretically distinct populations: (1) the non-aggregating population, which comprised about 70% of the total extractable proteoglycans; (2) the aggregating pool, comprising: (a) Band F-1 proteoglycans, which had a relatively large hydrodynamic size, uronate/protein weight ratio, were enriched in keratan sulphate and had a high buoyant density; (b) Band S proteoglycans, which migrated slower in agarose/acrylamide gels, had a smaller hydrodynamic size, lower buoyant density and a lower uronate/protein ratio than the Band F-1 population; (c) Band F-2 proteoglycans, which were lower in buoyant density, smaller in hydrodynamic size and slightly faster in electrophoretic mobility than the Band F-1 proteoglycans.     

Heparan sulfate-chondroitin sulfate hybrid proteoglycan of the cell surface and basement membrane of mouse mammary epithelial cells

Chondroitin sulfate represents approximately 15% of the 35SO4-labeled glycosaminoglycans carried by the proteoglycans of the cell surface and of the basolateral secretions of normal mouse mammary epithelial cells in culture. Evidence is provided that these chondroitin sulfate-carrying proteoglycans are hybrid proteoglycans, carrying both chondroitin sulfate and heparan sulfate chains. Complete N-desulfation but limited O-desulfation, by treatment with dimethyl sulfoxide, of the proteoglycans decreased the anionic charge of the chondroitin sulfate-carrying proteoglycans to a greater extent than it decreased the charge of their constituent chondroitin sulfate chains. Partial depolymerization of the heparan sulfate residues of the proteoglycans with nitrous acid or with heparin lyase also reduced the effective molecular radius of the chondroitin sulfate-carrying proteoglycans. The effect of heparin lyase on the chondroitin sulfate-carrying proteoglycans was prevented by treating the proteoglycan fractions with dimethyl sulfoxide, while the effect of nitrous acid on the dimethyl sulfoxide-treated proteoglycans was prevented by acetylation. This occurrence of heparan sulfate-chondroitin sulfate hybrid proteoglycans suggests that the substitution of core proteins by heparan sulfate or chondroitin sulfate chains may not solely be determined by the specific routing of these proteins through distinct chondroitin sulfate and heparan sulfate synthesizing mechanisms. Moreover, regional and temporal changes in pericellular glycosaminoglycan compositions might be due to variable postsynthetic modification of a single gene product.     

Studies on the extraction of different proteoglycan populations in bovine articular cartilage

Large proteoglycan monomers and small dermatan sulfate proteoglycans were extracted from explants of bovine articular cartilage with increasing (0-4 M) concentrations of guanidinium chloride (GuHCl). The first extractions were followed by a second extraction with 4 M GuHCl. The amount of proteoglycans extracted in the first buffer depended on the GuHCl concentration. At low concentrations of GuHCl, a relatively high amount of small proteoglycans was obtained. Fifty percent of the small proteoglycans was extracted in buffer with 0.85 M GuHCl, while 2.0-2.2 M GuHCl was needed to extract half of the large proteoglycans. Immediately after synthesis, 35S-labeled large proteoglycans were extracted much easier (50% at 1.4 M GuHCl), and those extracted at low concentrations of GuHCl were less capable of aggregation with hyaluronic acid. After 7 days of 'chase' these differences between endogenous and 35S-labeled proteoglycans had disappeared.     

Structure and properties of an under-sulfated heparan sulfate proteoglycan synthesized by a rat hepatoma cell line

A rat hepatoma cell line was shown to synthesize heparan sulfate and chondroitin sulfate proteoglycans. Unlike cultured hepatocytes, the hepatoma cells did not deposit these proteoglycans into an extracellular matrix, and most of the newly synthesized heparan sulfate proteoglycans were secreted into the culture medium. Heparan sulfate proteoglycans were also found associated with the cell surface. These proteoglycans could be solubilized by mild trypsin or detergent treatment of the cells but could not be displaced from the cells by incubation with heparin. The detergent-solubilized heparan sulfate proteoglycan had a hydrophobic segment that enabled it to bind to octyl- Sepharose. This segment could conceivably anchor the molecule in the lipid interior of the plasma membrane. The size of the hepatoma heparan sulfate proteoglycans was similar to that of proteoglycans isolated from rat liver microsomes or from primary cultures of rat hepatocytes. Ion-exchange chromatography on DEAE-Sephacel indicated that the hepatoma heparan sulfate proteoglycans had a lower average charge density than the rat liver heparan sulfate proteoglycans. The lower charge density of the hepatoma heparan sulfate can be largely attributed to a reduced number of N-sulfated glucosamine units in the polysaccharide chain compared with that of rat liver heparan sulfate. Hepatoma heparan sulfate proteoglycans purified from the culture medium had a considerably lower affinity for fibronectin-Sepharose compared with that of rat liver heparan sulfate proteoglycans. Furthermore, the hepatoma proteoglycan did not bind to the neoplastic cells, whereas heparan sulfate from normal rat liver bound to the hepatoma cells in a time-dependent reaction. The possible consequences of the reduced sulfation of the heparan sulfate proteoglycan produced by the hepatoma cells are discussed in terms of the postulated roles of heparan sulfate in the regulation of cell growth and extracellular matrix formation.     

Collagen increases the synthesis of membrane-associated proteoglycans produced by Sertoli cells.

Sertoli cells in culture produce two isoforms of proteoglycans which are found in the culture medium and associated with the cell membrane. The amount of both types of proteoglycans increased when Sertoli cells were plated on type I collagen-coated dishes as compared to uncoated dishes. The effect is due to an increase in the synthesis of proteoglycans rather than a diminished rate of degradation of these molecules. The collagen substrate also affects the distribution of these macromolecules; an increase in the amount of membrane-associated proteoglycans occurs at the expense of the proteoglycans released to the culture medium.     

Characterization and macromolecular association of proteoglycans produced by pig arterial smooth muscle cells in culture

1. Medium and cell-layer proteoglycans from pig aorta smooth muscle cells in culture were compared. In both compartments, the main proteoglycans contained chondroitin sulfate-dermatan sulfate chains of 40 kDalton. 2. However, cell-layer proteoglycans differed from those of the medium by the presence of: (a) some small-size proteoglycans; (b) a greater amount of heparan sulfate; (c) chondroitin sulfate-dermatan sulfate enriched in iduronate and in 4 sulfate- (instead of 6 sulfate-) residues. 3. During dissociation-reassociation assays of arterial proteoglycans with exogenous hyaluronate or "aggregate" proteoglycans, the in vitro formation of complexes appeared to involve inter-associations between proteoglycans molecules, in addition to aggregation with hyaluronate.     

Proteoglycans of human gingival epithelium and connective tissue.

Proteoglycans extracted from separated specimens of healthy human gingival epithelium and from connective tissue have been purified. The epithelial proteoglycans fractionated as a single included peak on Sepharose 4B-CL and contained heparan sulphate and dermatan sulphate glycosaminoglycans. The connective-tissue proteoglycans separated into three major populations on Sepharose 4B-CL, one of which was excluded from this gel under associative conditions (0.5 M-sodium acetate, pH 7.4). Subsequent fractionation of the excluded material under dissociative conditions (4 M-guanidinium chloride/0.05 M-sodium acetate, pH 7.4) revealed an absence of any aggregate formation of molecules within this population. The connective-tissue proteoglycans contained heparan sulphate, dermatan sulphate and chondroitin 4-sulphate, the proportions of which varied with the molecular size of the proteoglycans. Amino acid analysis of the protein cores of gingival-epithelial and connective-tissue proteoglycans revealed differences that were similar to the differences described between other types of proteoglycans such as those from skin.     

The ratio of protein and carbohydrate components of proteoglycans from normal and degenerative articular cartilage

The protein/uronic acid ratio in monomers and aggregates of proteoglycans in the human articular cartilage is investigated. It is shown that for the first two hours of cartilage extraction by isotonic solution proteoglycans with the low concentration of chondroitin sulphate are mainly removed; in the process of the subsequent extraction proteoglycans with a large amount of chondroitin sulphate; the quantity of chondroitin sulphate in the molecule does not effect the ability of proteoglycans to aggregation. The protein/uronic acid ratio increases in cartilage proteoglycans with aging and in the process of cartilage degeneration due to a decrease in the amount of the carbohydrate part of the molecule.     

Metabolism of sulfated glycosaminoglycans in cultivated bovine arterial cells. II. Quantitative studies on the uptake of 35SO4-labeled proteoglycans.

Cultured arterial fibroblasts were used for a quantitative study on adsorption, uptake and degradation of [35S]proteoglycans derived from secretions of cultured arterial or skin fibroblasts. The following results were obtained: 1) Proteoglycans added to the culture medium are integrated into the pool of cell membrane-associated (trypsin-removable) glycosaminoglycans by a saturable process, which depends on time and temperature. 2) Up to 17% of the added proteoglycans are taken up by the cells within 24 h. The uptake exhibits saturation kinetics, characteristic for adsorptive pinocytosis. Proteoglycan concentrations required for half-maximum uptake are higher than for half-maximum saturation of the glycosaminoglycan pool associated with the cell membrane. 3) After a lag phase, inorganic 35SO4 appears in the culture medium as a degradation product of the internalized proteoglycans. Pinocytosed proteoglycans are catabolized more rapidly than proteoglycans which remain inside the cell after their biosynthesis. 4) Pinocytosis exhibits specificity, the individual proteoglycans being internalized at different rates. The highest rate of uptake was measured for a dermatan-sulfate-rich proteoglycan. No competition of uptake between a dermatan-sulfate-rich and a heparan-sulfate-rich proteoglycan was observed. 5) Optimum pinocytosis requires an intact protein moiety and, presumably, undegraded carbohydrate chains of the proteoglycans.     

Endocytosis of sulphated proteoglycans by cultured skin fibroblasts.

1. Human skin fibroblasts internalize homologous sulphated proteoglycans by adsorptive endocytosis. Endocytosis rate is half maximal when the concentration of the proteoglycans is 0.1 nM. At saturation, a single fibroblast may endocytose up to 8 X 10(6) proteoglycan molecules/h. 2. The kinetics of prote;glycan binding to the cell surface suggest the presence of 6 X 10(5) high-affinity binding sites per cell. The bulk of sulphated proteoglycans associates to low-affinity binding sites on the cell surface. 3. Glycosaminoglycans and other anionic macromolecules inhibit endocytosis of sulphated proteoglycans non-competitively. The lack of interaction of glycosaminoglycans with the cell-surface receptors for sulphated proteoglycans suggests that the protein core of proteoglycans is essential for binding to the cell surface. 4. The effects of trypsin, cell density, serum concentration and medium pH on endocytosis and degradation of endocytosed sulphated proteoglycans is described. 5. A comparison of the number of the high-affinity binding sites and the number of molecules endocytosed with respect to time suggests a recycling of the proteoglycan receptors between the cell surface and the endocytotic vesicles and/or the lysosomes.     

Extracellular matrix and the maintenance of the differentiated state: proteoglycans synthesized by replated chondrocytes and nonchondrocytes

It has been previously shown that undifferentiated stage 23 to 24 chick limb bud mesenchymal cells can be maintained in culture under conditions which promote chondrogenesis. As the chondrocytes mature in vitro, their proteoglycan synthesis progresses through a specific and reproducible biosynthetic program. By the eighth day of culture, the chondrocytes are making proteoglycans that are similar to proteoglycans isolated from adult animal tissues. Relative to the Day 8 proteoglycans, the proteoglycans synthesized by chick limb bud chondrocytes earlier in culture have a smaller monomer size, longer chondroitin sulfate chains, shorter keratan sulfate chains, a higher ratio of chondroitin-6-sulfate to chondroitin-4-sulfate, and a decreased ability to interact with hyaluronic acid. We have reported a procedure to remove the cells from Day 8 cultures and strip away most, if not all, of the extracellular matrix. In addition, the chondrocytes can be separated from the 40-50% nonchondrocytic cells normally found in Day 8 cultures, and the two cell populations replated separately. This report describes the analysis of the proteoglycans synthesized by replated cells; this analysis demonstrates quantitative and qualitative differences between chondrocyte and nonchondrocyte proteoglycans. The overall rate of proteoglycan synthesis is fourfold higher and the rate of synthesis of high buoyant density proteoglycans 30-fold higher for replated chondrocytes relative to nonchondrocytes. Qualitatively, more newly synthesized nonchondrocyte proteoglycans partition at lower buoyant density on CsCl equilibrium density gradients than do chondrocyte proteoglycans. Nonchondrocyte proteoglycans are of two major classes: One has a monomer size slightly smaller than that of Day 8 chondrocyte proteoglycan, but has much longer glycosaminoglycan chains. The other is considerably smaller than Day 8 chondrocyte proteoglycans, but has glycosaminoglycans of slightly larger size. In contrast, replated chondrocytes synthesize, even as soon as 4.5 hr after replating, proteoglycans that are identical to Day 8 chondrocyte proteoglycan in monomer size, in glycosaminoglycan chain size, in aggregability, and in the ratio of 6-sulfated to 4-sulfated chondroitin. Since denuding mature Day 8 chondrocytes of their extracellular matrix does not cause them to recapitulate their developmentally regulated program for the biosynthesis of proteoglycans, it is concluded that the quality of mature chondrocyte proteoglycan is not altered by the absence of extracellular matrix.     

The core proteins of large and small interstitial proteoglycans from various connective tissues form distinct subgroups.

Large and small proteoglycans were separately isolated from a number of connective tissues and compared to determine the extent of structural similarity. This was studied by enzyme-linked immunosorbent assays and by the peptide patterns obtained when 125I-labelled proteoglycans were digested with trypsin. All the large proteoglycans, i.e. from tendon, sclera, cartilage and aorta, appear to contain the structure typical for the hyaluronic acid-binding region, both shown by enzyme-linked immunosorbent assay and by content of peptides unique for this region. These proteoglycans also share other structural features of the protein core, as indicated by immunological cross-reactivity and similar peptide patterns. The large proteoglycans from aorta in addition show the presence of unique structures both upon immunoassay and with regard to peptide pattern. Among the small proteoglycans two groups can be identified, although amino acid composition and protein core sizes are grossly similar. One group consists of the small proteoglycans from aorta and cartilage having similar peptide maps and showing immunological cross-reactivity in enzyme-linked immunosorbent assay. The other distinctly different group consists of the small proteoglycans from bone, cornea, sclera and tendon, which among them show identity in enzyme-linked immunosorbent assay and similar peptide patterns. Proteoglycans from the two groups, however, show partial immunological cross-reactivity.     

Characterization and biosynthesis of proteoglycans of corneal stroma from rhesus monkey.

The proteoglycans of the Rhesus monkey corneal stroma were characterized by analyzing both radiolabeled proteoglycans synthesized by corneas in organ culture and native corneal proteoglycans obtained by large scale preparations. The analyses indicate that the proteoglycans synthesized in organ culture were similar to, if not identical with, their counterparts in the stroma although they are synthesized in different prportions in vitro than they acumulate in vivo. The corneal stroma contains two proteoglycans. The chondroitin-dermatan sulfate proteoglycan consists of approximately 70% protein and has a Mr = approximately 100,000 to 150,000. It contains one chondroitin-dermatan sulfate side chain of Mr = approximately 55,000. The keratan sulfate proteoglycan consists of approximately 74% protein and has a Mr = approximately -40,000 to 70,000. It contains one or two keratan sulfate side chains with a Mr = approximately 7,000 each. Radiolabeling indicates that both proteoglycans contain glycoprotein-type oligosaccharides as part of their structure.