A large chondroitin sulfate proteoglycan (PG-M) synthesized before chondrogenesis in the limb bud of chick embryo

Extraction of stage 22-23 chick embryo limb buds that had been metabolically labeled with [35S]sulfate yielded heparan sulfate proteoglycan, small chondroitin sulfate proteoglycan, and large chondroitin sulfate proteoglycan (designated PG-M). PG-M constituted over 60% of the total macromolecular [35S]sulfates. It was larger in hydrodynamic size, richer in protein, and contained fewer chondroitin sulfate chains as compared to the predominant proteoglycan (PG-H, Mr congruent to 1.5 X 10(6)) of chick embryo cartilage. The chondroitin sulfate chains were notable for their large size (Mr greater than or equal to 60,000) and high content of nonsulfated chondroitin units (about 20% of the total hexosamine). Hexosamine-containing chains corresponding in size to N-linked and O-linked oligosaccharides were also present. The core protein was rich in serine, glutamic acid (glutamine), and glycine which together comprised about 38% of the total amino acids. Following chondroitinase AC II (or ABC) digestion, core molecules were obtained which migrated on sodium dodecyl sulfate gel electrophoresis as a doublet of bands with approximately Mr = 550,000 (major) and 500,000, respectively. The Mr = 550,000 core glycoprotein was structurally different from the core glycoprotein (Mr congruent to 400,000) of PG-H, as ascertained by tryptic peptide mapping and immunochemical criteria. Immunofluorescent localization of PG-M showed that the intensity of PG-M staining progressively became higher in the core mesenchyme region than in the peripheral loose mesenchyme, closely following the condensation of mesenchymal cells. Since the cell condensation process has been shown to begin with the increase of fibronectin and type I collagen concentration, the similar change in PG-M distribution suggests that PG-M plays an important role in the cell condensation process by means of its interaction with fibronectin and type I collagen.     

Hemostatic properties and serum lipoprotein binding of a heparan sulfate proteoglycan from bovine aorta

The biologic properties of two major proteoglycans of bovine aorta, heparan sulfate proteoglycan and chondroitin sulfate-dermatan sulfate proteoglycan were compared. The heparan sulfate proteoglycan was isolated either by elastase digestion or by 4.0 M guanidine hydrochloride extraction, of aorta tissue, fractionated by CsCl isopycnic centrifugation and purified by chondroitinase ABC treatment. The first method resulted in considerably greater yield (about 70% of the total heparan sulfate proteoglycan of the tissue) than the second procedure (12% of total). The chondroitin sulfate-dermatan sulfate proteoglycan was obtained by 4.0 M guanidine-HCl extraction of aorta tissue followed by CsCl isopycnic centrifugation. The chemical composition of both heparan sulfate proteoglycan preparations was similar. Unlike the chondroitin sulfate-dermatan sulfate proteoglycan, which eluted in the void volume of Sepharose CL-6B column, the heparan sulfate proteoglycan preparations were each resolved into a high molecular weight fraction (kav = 0.18 and 0.13) and a low molecular weight fraction (kav = 0.47 and 0.36). The heparan sulfate proteoglycan preparations exhibited significantly more potent anticoagulant and platelet aggregation inhibitory activities than the chondroitin sulfate-dermatan sulfate proteoglycan. The protein core of the proteoglycan molecules did not seem to be essential for their hemostatic properties. The complex forming ability of the heparan sulfate proteoglycan with serum low density lipoproteins (LDL) was much less than that of chondroitin sulfate-dermatan sulfate proteoglycan in the presence and absence of Ca2+. Interaction between heparan sulfate proteoglycan and LDL was also much more sensitive to changes in the ionic strength of the medium than that of chondroitin sulfate-dermatan sulfate proteoglycan and the lipoprotein. Since the total sulfate content of both proteoglycans is almost similar, the smaller molecular size and hence the lower overall charge density of the heparan sulfate proteoglycan appears to be partly responsible for its low affinity for LDL. The differences in biologic properties of the two proteoglycans might have implications in the pathophysiology of cardiovascular diseases.     

Regulation of cell-substrate adhesion by proteoglycans immobilized on extracellular substrates

We have demonstrated previously that chick embryo fibroblasts synthesize and secrete a large chondroitin sulfate proteoglycan (designated PG-M) that binds to fibronectin. We now report the possibility that PG-M interactions with cell surfaces can modulate cell-substrate adhesion. When PG-M was added to the medium, various types of trypsinized cells failed to adhere not only to fibronectin-coated substrates but also to collagen- or vitronectin-coated substrates. Adhesion of the cells to laminin or glycyl-arginyl-glycyl-aspartyl-serine derivatized serum albumin (arginyl-glycyl-aspartic acid-containing molecules with no capacity to bind PG-M) was also inhibited by PG-M. Treatment of the proteoglycan with either proteolytic enzymes or chondroitinase abolished its inhibitory effects on the cell adhesion. These results suggest that direct binding between PG-M and fibronectin, if any, is not a cause of the inhibition by PG-M and that only the proteoglycan form is responsible for the activity. When the immobilization of added PG-M to available plastic surfaces of coated dishes was blocked by pretreating the dishes with serum albumin, the inhibitory effect of PG-M was abolished, suggesting that the immobilized fraction of PG-M can act as a cell adhesion inhibitor. In immobilized form, both cartilage chondroitin sulfate proteoglycan (designated PG-H) and chondroitin sulfate-derivatized serum albumin also inhibited cell adhesion. In contrast, heparan sulfate proteoglycan form LD and heparan sulfate-derivatized serum albumin had far lower inhibitory activities, indicating that the active site for the interaction between cells and PG-M is on the chondroitin sulfate chains.     

Isolation of a chondroitin sulfate--dermatan sulfate proteoglycan from bovine aorta.

Material containing proteoglycans was extracted from bovine aorta by the dissociative solvent 3.0 m MgCl². The proteoglycan that remained in solution at low ionic strength was purified by isopycnic CsCl centrifugation (?, 1.75 – 1.89 g/ml). From the lower third of the gradient a proteoglycan was isolated which behaved as a homogeneous material when analyzed by the ultracentrifuge and by electrophoresis on cellulose acetate. The proteoglycan contained 12% protein, 21% uronic acid, and 28% hexosamine. Analyses by hyaluronidase digestion and gas-liquid chromatography of the polysaccharide moieties of the proteoglycan showed a composition of 56% chondroitin 6-sulfate, 20% chondroitin 4-sulfate, and 7% dermatan sulfate. A copolymeric structure for the polysaccharide of the proteoglycan is proposed.     

Organization of glycosaminoglycan chains in a chondroitin sulfate-dermatan sulfate proteoglycan from bovine aorta

A chondroitin sulfate-dermatan sulfate proteoglycan was isolated from bovine aorta intima by extraction of the tissue by 4 M guanidine hydrochloride. The proteoglycan was purified by CsCl isopycnic centrifugation followed by gel filtration and ion-exchange chromatography. The proteoglycan had 21.9% protein, 22.1% uronate, 21.4% hexosamine and 10.8% sulfate. Glycosaminoglycan chains obtained from the proteoglycan by beta-elimination were resolved by gel filtration into two fractions, one containing chondroitin 6-sulfate with an approximate molecular weight of 49 000 and the other containing chondroitin 4-sulfate and dermatan sulfate in a proportion of 2:1 with an approximate molecular weight of 37 000. Digestion of the proteoglycan by chondroitinase ABC or AC yielded a protein core with similar composition and behavior in gel filtration and SDS-polyacrylamide gel electrophoresis. An approximate molecular weight of 180 000 was estimated for the core protein. Dermatan sulfate chains with an approximate molecular weight of 10 000 were observed only in the digest of chondroitinase AC. Limited trypsin hydrolysis of the proteoglycan yielded three peptide fragments containing chondroitin 6-sulfate, chondroitin 4-sulfate and dermatan sulfate in varied proportions. A tentative structure for the proteoglycan was suggested.     

Organization of glycosaminoglycan chains in a chondroitin sulfate - dermatan sulfate proteoglycan from bovine aorta

A chondroitin sulfate - dermatan sulfate proteoglycan was isolated from bovine aorta intima by extraction of the tissue by 4 M guanidine hydrochloride. The proteoglycan was purified by CsCl isopycnic centrifugation followed by gel filtration and ion-exchange chromatography. The proteoglycan had 21.9% protein, 22.1% uronate, 21.4% hexosamine and 10.8% sulfate. Glycosaminoglycan chains obtained from the proteoglycan by β-elimination were resolved by gel filtration into two fractions, one containing chondroitin 6-sulfate with an approximate molecular weight of 49 000 and the other containing chondroitin 4-sulfate and dermatan sulfate in a proportion of 2:1 with an approximate molecular weight of 37 000. Digestion of the proteoglycan by chondroitinase ABC or AC yielded a protein core with similar composition and behavior in gel filtration and SDS-polyacrylamide gel electrophoresis. An approximate molecular weight of 180 000 was estimated for the core protein. Dermatan sulfate chains with an approximate molecular weight of 10 000 were observed only in the digest of chondroitinase AC. Limited trypsin hydrolysis of the proteoglycan yielded three peptide fragments containing chondroitin 6-sulfate, chondroitin 4-sulfate and dermatan sulfate in varied proportions. A tentative structure for the proteoglycan was suggested.     

Isolation and characterization of a link protein from bovine aorta proteoglycan aggregate

Proteoglycan aggregates were isolated from bovine aorta by extraction with 0.5 M guanidine hydrochloride in the presence of proteinase inhibitors and purified by isopycnic CsCl centrifugation. The bottom two-fifths (A1) of the gradient contained 30% of proteoglycans in the aggregated form. The aggregate had 14.8% protein and 20.4% hexuronic acid with hyaluronic acid, dermatan sulfate and chondroitin sulfates in a proportion of 18:18:69. A link protein-containing fraction was isolated from the bottom two-fifths by dissociative CsCl isopycnic centrifugation. The link protein that floated to the top one-fifth of the gradient was purified by chromatography on Sephadex G-200 in the presence of 4 M guanidine hydrochloride. It moved as a single band in SDS-polyacrylamide gel electrophoresis with a molecular weight of 49 000. The amino acid composition of link protein resembled that of link protein from cartilage, but was strikingly different from that of the protein core of the proteoglycan monomer. The neutral sugar content of link protein was 3.5% of dry weight. Galactose, mannose and fucose constituted 21, 62 and 16%, respectively of the total neutral sugars. In aggregation studies the link protein was found to interact with both proteoglycan monomer and hyaluronic acid. Oligosaccharides derived from hyaluronic acid decreased the viscosity of link protein-free aggregates of proteoglycan and hyaluronic acid but not of link-stabilized aggregates, demonstrating that the link protein increases the stability of proteoglycan aggregates.     

Heparan sulfate proteoglycan from human and equine glomeruli and tubules

1. Proteoglycans were isolated from human and equine glomeruli or tubules by guanidine extraction and anion exchange chromatography. 2. These proteoglycan preparations contained about equal amounts of heparan sulfate and chondroitin sulfates. 3. During the preparation of glomerular or tubular basement membranes the main part of proteoglycans (greater than 50%) was extracted in the salt extract. Chondroitin sulfate proteoglycan was mainly found in the water and salt extracts of glomeruli and tubules, heparan sulfate proteoglycan in the deoxycholate extracts and the basement membranes. 4. The glomerular basement membrane (GBM) contains about 12% (human) or 20% (equine) of the proteoglycans of the total glomerulus. They consist of greater than 70% (equine) or 80% (human) of heparan sulfate. 5. Heparan sulfate proteoglycan was isolated from the proteoglycan preparations of human or equine glomeruli and tubules by additional treatment with nucleases and chondroitinase ABC followed by CsCl gradient centrifugation. 6. Protein accounts for about 40% (dry weight) of the heparan sulfate proteoglycans. Their amino acid composition is characterized by a high content of glycine, but 3-hydroxyproline, 4-hydroxyproline and hydroxylysine are lacking. 7. The biochemical characteristics of the heparan sulfate proteoglycan of human or equine glomeruli or tubules differ from that isolated from rat glomeruli by their higher protein content and their amino acid composition. The significance of these differences is discussed.     

Interleukin-1-induced alterations in proteoglycan metabolism and matrix assembly

In cartilage, the large chondroitin sulfate proteoglycan exists as aggregates by interacting with link protein and hyaluronic acid. In diseases associated with cartilage degeneration, the proteoglycan does not aggregate because of a defect in the hyaluronate-binding activity. Since interleukin-1 (IL-1) is a secretory product of activated macrophages and may influence the cartilage function in joints, we studied the effects of IL-1 on the synthesis and assembly of proteoglycan by rabbit articular chondrocytes in culture. IL-1-treated cells showed a modest increase in the total proteoglycan synthesis, but also showed a more pronounced decrease in the incorporation of extracellular matrix. Affinity chromatography of the conditioned media on hyaluronic acid-Sepharose revealed that all of the proteoglycan of control cells strongly bound to hyaluronate. The IL-1-treated medium contained two fractions: one that was strongly bound to the column and a second that did not bind. The results demonstrate that the IL-1-treated cells cannot incorporate proteoglycan into the matrix partly because of a defect in the proteoglycan molecules and partly due to other mechanisms regulating proteoglycan assembly.     

The cell surface proteoglycan from mouse mammary epithelial cells bears chondroitin sulfate and heparan sulfate glycosaminoglycans

The cell surface proteoglycan fraction isolated by mild trypsin treatment of NMuMG mouse mammary epithelial cells contains largely heparan sulfate, but also 15-24% chondroitin sulfate glycosaminoglycans. We conclude that this fraction contains a unique hybrid proteoglycan bearing both heparan sulfate and chondroitin sulfate glycosaminoglycans because (i) the proteoglycan behaves as a single species by sizing, ion exchange and collagen affinity chromatography, and by isopycnic centrifugation, even in the presence of 8 M urea or 4 M guanidine hydrochloride, (ii) the behavior of the chondroitin sulfate in these separation techniques is affected by heparan sulfate-specific probes and vice versa, and (iii) proteoglycan core protein bearing both heparan sulfate and chondroitin sulfate is recognized by a single monoclonal antibody. Removal of both types of glycosaminoglycan reduces the proteoglycan to a core protein of approximately 53 kDa. The proteoglycan fraction is heterogeneous in size, largely due to a variable number and/or length of the glycosaminoglycan chains. We estimate that one or two chondroitin sulfate chains (modal Mr of 17,000) exist on the proteoglycan for every four heparan sulfate chains (modal Mr of 36,000). Synthesis of these chains is reportedly initiated on an identical trisaccharide that links the chains to the same amino acid residues on the core protein. Therefore, some regulatory information, perhaps residing in the amino acid sequence of the core protein, must determine the type of chain synthesized at any given linkage site. Post-translational addition of these glycosaminoglycans to the protein may provide information affecting its ultimate localization. It is likely that the protein is directed to specific sites on the cell surface because of the ability of the glycosaminoglycans to recognize and bind extracellular components.     

Interactions between chondroitin sulfate proteoglycan, fibronectin, and collagen

A proteoglycan isolated from a rat yolk sac tumor and characterized as a chondroitin sulfate proteoglycan with a smaller amount of dermatan sulfate was studied with respect to complex formation with collagen and fibronectin. The proteoglycan co-precipitated with native collagen from neutral salt solutions at 6 degrees C and 37 degrees C. Addition of fibronectin in such precipitation mixtures resulted in incorporation of fibronectin to the precipitate. Treatment of the proteoglycan with alkali to separate the glycosaminoglycan chains from the protein part and digestion of the protein part with papain greatly reduced the capacity of the proteoglycan to precipitate collagen and fibronectin. A defined extracellular matrix as represented by the complexes of collagen, proteoglycan, and fibronectin constructed here may be useful for studies on the biological effects of extracellular matrices. The multiple interactions of matrix macromolecules exemplified by these results may play a role in the formation of extracellular matrices and in the maintenance of their integrity.     

Abnormal synthesis of cartilage-characteristic proteoglycan in azaserine-induced micromelial limbs.

Administration of azaserine (250 micrograms) to day-4 chick embryos in ovo was shown to induce micromelial limbs. In the present study, biosynthesis of cartilage-characteristic proteoglycan H (PG-H) as an index of limb chondrogenesis was examined in normal and micromelial hind limbs from day-7 chick embryos by biochemical and immunological methods. (1) Metabolic labelling of the micromelial limbs with [6-3H]-glucose and [35S]sulphate, followed by analysis of labelled proteoglycans by glycerol-density-gradient centrifugation under dissociative conditions, showed a marked reduction in PG-H synthesis. (2) PG-H synthesized by micromelial limbs differed from that synthesized by normal limbs in possessing a slower sedimenting velocity and much lower amounts of chondroitin sulphates. (3) The amount of PG-H core protein in micromelial limbs was significantly decreased to about 19% on a per limb basis and about 42% on a per DNA basis of that in normal limbs, as determined by e.l.i.s.a. (4) The transition from PG-M to PG-H during limb formation was retarded in micromelial limbs as judged by an indirect immunofluorescence technique using antibodies against PG-M and PG-H. (5) The deficiency of incorporation of labelled glucose into chondroitin sulphate chains of PG-H in micromelial limbs was partially restored by using [6-3H]-glucosamine as a precursor, suggesting that the synthesis of UDP-N-acetylhexosamine, required for chondroitin sulphate chain synthesis of PG-H in micromelial limbs, was decreased. These results suggest that the reduction in the synthesis of PG-H as well as the production of an abnormal form of PG-H during a critical period of limb morphogenesis may be important factors in explaining the micromelia induced by azaserine.     

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.     

Stimulation of proteoglycan synthesis in chick embryo sternum by serum and L-3,5,3'-triiodothyronine.

Incorporation of sulfate into alcian blue-precipitable glycosaminoglycan of 12-day-old chick embryo sterna is stimulated by addition, separately or together, of normal human serum and physiological concentrations of thyroid hormones (Audhya, T.K., and Gibson, K.D. (1975) Proc. Natl. Acad, Sci. U. S. A. 72, 604--608). We present evidence that this stimulation is due to increased synthesis of at least one proteoglycan, with minor alterations in the size and chemical composition of the glycosaminoglycans. Pulse-chase experiments showed no detectable loss of label during the chase, in control sterna or sterna incubated with serum and L-3,5,3'-triiodothyronine; thus, all incorporation was the result of synthesis of glycosaminoglycans. In double-label experiments, with 35SO4(2-) and [3H]acetate, the molar ratio of 3H and 35S incorporated into glycosaminoglycans was changed little, if at all, by addition of serum or triiodothyronine or both, at concentrations which increased incorporation up to 2-fold. Glycosaminoglycans isolated from these and other incubations gave similar elution patterns from agarose columns, and identical electrophoretic patterns on cellulose acetate. Digestion with chondroitinase ABC (chondroitin ABC lyase; EC 4.2.2.4.) showed that incorporation was into chondroitin sulfate and possibly hyaluronic acid, and that the proportions of non-sulfated, 4-sulfated, and 6-sulfated disaccharide units differed little between stimulated and unstimulated sterna. Incorporation of [3H]serine into glycosaminoglycans from papain digest of sterna paralleled incorporation of 35SO4(2-), and indicated a number average molecular weight between 21,000 and 25,000 for the newly synthesized chondroitin sulfate. This value was confirmed by gel filtration chromatography, which also showed that the average molecular weight of the newly synthesized chondroitin sulfate decreased up to 15% under conditions of 2-fold stimulation. Proteoglycans were extracted from sterna incubated with [3H]serine and 35SO4(2-) and analyzed by isopycinic centrifugation in CsCl and by zone sedimentation in a sucrose gradient. A major proteoglycan fraction could be separated by either method. Incorporation of both isotopes into this proteoglycan fraction, and into glycosaminoglycans isolated after papain digestion, was stimulated in a coordinate manner. Almost identical results were obtained with both separation techniques. The results indicate that the synthesis of the major proteoglycan, and probably also of a minor one, is stimulated by serum and triiodothyronine.     

Multiple domains of the large fibroblast proteoglycan, versican.

The primary structure of a large chondroitin sulfate proteoglycan expressed by human fibroblasts has been determined. Overlapping cDNA clones code for the entire 2389 amino acid long core protein and the 20-residue signal peptide. The sequence predicts a potential hyaluronic acid-binding domain in the amino-terminal portion. This domain contains sequences virtually identical to partial peptide sequences from a glial hyaluronate-binding protein. Putative glycosaminoglycan attachment sites are located in the middle of the protein. The carboxy-terminal portion includes two epidermal growth factor (EGF)-like repeats, a lectin-like sequence and a complement regulatory protein-like domain. The same set of binding elements has also been identified in a new class of cell adhesion molecules. Amino- and carboxy-terminal portions of the fibroblast core protein are closely related to the core protein of a large chondroitin sulfate proteoglycan of chondrosarcoma cells. However, the glycosaminoglycan attachment regions in the middle of the core proteins are different and only the fibroblast core protein contains EGF-like repeats. Based on the similarities of its domains with various binding elements of other proteins, we suggest that the large fibroblast proteoglycan, herein referred to as versican, may function in cell recognition, possibly by connecting extracellular matrix components and cell surface glycoproteins.     

Cell surface proteoglycan of mouse mammary epithelial cells is shed by cleavage of its matrix-binding ectodomain from its membrane-associated domain

The cell surface proteoglycan on normal murine mammary gland (NMuMG) epithelial cells consists of a lipophilic domain, presumably intercalated into the plasma membrane, and an ectodomain that binds via its glycosaminoglycan chains to matrix components, is released intact by proteases and is detected by monoclonal antibody 281-2. The antibody 281-2 also detects a proteoglycan in the culture medium conditioned by NMuMG cells. This immunoactive proteoglycan was purified to homogeneity using DEAE-cellulose chromatography, isopycnic centrifugation, and 281- 2 affinity chromatography. Comparison of the immunoreactive medium proteoglycan with the trypsin-released ectodomain revealed that these proteoglycans are indistinguishable by several criteria as both: (a) contain heparan sulfate and chondroitin sulfate chains; and (b) are similar in hydrodynamic size and buoyant density; (c) have the same size core protein (Mr approximately 53 kD); (d) are nonlipophilic as studied by liposomal intercalation and transfer to silicone-treated paper. Kinetic studies of the release of proteoglycan from the surface of suspended NMuMG cells are interpreted to indicate that the immunoreactive medium proteoglycan is derived directly from the cell surface proteoglycan. Suspension of the cells both augments the release and inhibits the replacement of cell surface proteoglycan. These results indicate that the cell surface proteoglycan of NMuMG cells can be shed by cleavage of its matrix-binding ectodomain from its membrane- associated domain, providing a mechanism by which the epithelial cells can loosen their proteoglycan-mediated attachment to the matrix.     

Isolation, characterization and immunological determination of basement membrane-associated heparan sulfate proteoglycan

Basement membrane-associated heparan sulfate proteoglycan (HSPG) was extracted from isolated porcine glomerular basement membranes and purified by ion-exchange chromatography. The proteogycan was characterized by specific enzymatic digestions, by amino-acid analysis, by SDS-polyacrylamide gel electrophoresis and by density gradient centrifugation. Polyclonal antibodies were raised against the purified HSPG in rabbits. Antibodies were characterized by enzyme immunoassays, immunoprecipitation and immunohistological methods. They were shown to recognize specifically the core protein of HSPG from porcine, human and rat glomerular basement membrane but did not recognize HSPG from guinea pig or rabbit kidney. The affinity-purified antibodies did not cross-react with other basement membrane proteins like laminin, fibronectin or collagen type IV nor with chondroitin sulfate-rich or keratan sulfate-rich proteoglycans from human or bovine tissue. Using these antibodies an enzyme immunoassay was developed for determination of HSPG in the range of 1-100 ng/ml. Studies with cultured porcine endothelial cells showed that subendothelial basement membrane-associated HSPG may be determined with the enzyme immunoassay.     

Chondronectin interactions with proteoglycan

We have investigated whether proteoglycans are involved in the attachment of embryonic chick chondrocytes to type II collagen. Chondroitin sulfate proteoglycan, when added exogenously, promotes the binding of chondronectin, the chondrocyte attachment factor, to type II collagen substrates and thereby stimulates chondrocyte adhesion. Blockage of endogenous proteoglycan synthesis with beta-xylosides prevents chondronectin-mediated chondrocyte attachment, confirming that proteoglycan is required. The intact proteoglycan must be present since chondroitin sulfate glycosaminoglycans did not promote chondronectin-mediated cell attachment but, rather, inhibited it in a dose-dependent manner. This inhibition, however, could be overcome with excess exogenous proteoglycan. Consequently, chondronectin interacts with proteoglycan and then the complex interacts with the collagen substrate and with the cell surface to promote cell adhesion. Further evidence for a direct interaction of chondronectin with the glycosaminoglycan portion of the proteoglycan is the selective binding of chondronectin to dextran-Sepharose, dextran having been shown to inhibit attachment to an extent similar to that of chondroitin sulfate.     

A monoclonal antibody that specifically recognizes a glucuronic acid 2-sulfate-containing determinant in intact chondroitin sulfate chain

Monoclonal antibodies produced against chick embryo limb bud proteoglycan (PG-M) were selected for their ability to recognize determinants on intact chondroitin sulfate chains. One of these monoclonal antibodies (IgM; designated MO-225) reacts with PG-M, chick embryo cartilage proteoglycans (PG-H, PG-Lb, and PG-Lt), and bovine nasal cartilage proteoglycan, but not with Swarm rat chondrosarcoma proteoglycan. The reactivity of PG-H to MO-225 is not affected by keratanase digestion but is completely abolished after chondroitinase digestion. Competitive binding analyses with various glycosaminoglycan samples indicate that the determinant recognized by MO-225 resides in a D-glucuronic acid 2-sulfate(beta 1----3)N-acetylgalactosamine 6-sulfate disaccharide unit (D-unit) common to antigenic chondroitin sulfates. A tetrasaccharide trisulfate containing D-unit at the reducing end is the smallest chondroitin sulfate fragment that can inhibit the binding of the antibody to PG-H. Decreasing the size of a D-unit-rich chondroitin sulfate by hyaluronidase digestion results in progressive reduction in its inhibitory activity. The results suggest that the epitope has a requirement for a long stretch of a disaccharide-repeating structure for a better fit to the antibody.     

Biological activity of a proteoglycan form of macrophage colony-stimulating factor and its binding to type V collagen.

Two different types of macrophage colony-stimulating factors (M-CSF) were found, one with an apparent molecular mass of 85 kDa and the other greater than 200 kDa. The high molecular mass M-CSF was identified as a proteoglycan carrying chondroitin sulfate glycosaminoglycan and was designated as the proteoglycan form of M-CSF (PG-M-CSF). In this study, we compared the biological activity of the 85-kDa M-CSF and PG-M-CSF and examined the binding properties of these two M-CSF to certain extracellular matrix proteins, i.e. types I-V collagen and fibronectin, using a modified enzyme-linked immunosorbent assay. PG-M-CSF was capable of supporting the formation of murine macrophage colonies, and pretreatment of PG-M-CSF with chondroitinase AC, which degrades chondroitin sulfate, did not alter its colony-stimulating activity. The specific activity of PG-M-CSF was similar to that of the 85-kDa M-CSF. The 85-kDa M-CSF had no apparent affinity for the extracellular matrix proteins examined, whereas PG-M-CSF had an appreciable binding capacity to type V collagen, but did not bind to types I, II, III, and IV collagen or to fibronectin. Pretreatment of PG-M-CSF with chondroitinase AC completely abolished the binding of the species to type V collagen. Addition of exogenous chondroitin sulfate inhibited the binding of PG-M-CSF to type V collagen in a dose-dependent manner. These data indicated that the interaction between PG-M-CSF and type V collagen was mediated by the chondroitin sulfate chain of PG-M-CSF. PG-M-CSF bound to type V collagen could stimulate the proliferation of bone marrow macrophages, indicating that the matrix protein-bound PG-M-CSF retained its biological activity. This interaction between PG-M-CSF and type V collagen implies that the role of PG-M-CSF may be distinct from that of 85-kDa M-CSF.