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.     

Heterogeneity of heparan sulfate chains in a proteoglycan from bovine lung

A heparan sulfate proteoglycan from bovine lung gas-exchange tissue was isolated by extraction of the tissue with 4.0 M guanidine HCl in the presence of multiple protein inhibitors. The proteoglycan was purified by precipitation with cetylpyridinium chloride in 0.5 M KCl followed by CsCl isopycnic centrifugation (po = 1.45) in 4.0 M guanidine/HCl. Further purification was achieved by gel filtration on Sepharose CL-2B and by chromatography in DEAE-Sepharose CL-6B column. The proteoglycan had 14.9% protein and 22.4% uronate. Heparan sulfate chains from the proteoglycan were isolated after beta-elimination. Fractionation of heparan sulfate chains was achieved on Dowex-1 Cl- column, eluting with a stepwise increase in the concentration of NaCl, 1.0 to 2.0 M with 0.2 M increments. Of the total heparan sulfate recovered from the column, about 10% eluted by 1.2 M NaCl, 68% by 1.4 M NaCl, 18% by 1.6 M NaCl and 4% by 1.8 M NaCl. The fractions varied in their total and N-sulfate ester contents and iduronic acid to glucuronic acid ratios. The fraction that eluted from the Dowex-1 Cl- column at 1.6 M NaCl had the highest molecular weight, 37000, and the fraction that eluted at 1.8 M NaCl had the lowest molecular weight, 12000, as determined by gel filtration method, and the greatest sulfate content. The core protein, obtained by digestion of proteoglycan by heparan sulfate lyase, showed mostly a single band in SDS-polyacrylamide gel electrophoresis. The observations indicate a heterogeneity of the composition of heparan sulfate chains in the proteoglycan. This heterogeneity likely contributes to variations in biologic properties of different heparan sulfate proteoglycan preparations.     

beta-D-xyloside-mediated alteration in the synthesis of basement membrane proteoglycan

The effect of nitrophenyl-beta-D-xyloside (xyloside), a synthetic initiator of glycosaminoglycan synthesis, on proteoglycan and glycosaminoglycan synthesis by a basement membrane producing tumor was studied. While xyloside markedly stimulated the formation of chondroitin sulfate chains, it depressed the formation of a basement membrane heparan sulfate proteoglycan and caused only little formation of free heparan sulfate chains. However, when the synthesis of the core protein of the proteoglycan was inhibited by cycloheximide, heparan sulfate chains were produced by xyloside treatment. These heparan sulfate chains had a sulfate content higher than that of heparan sulfate found on the proteoglycan. The data indicate that xyloside can substitute for the heparan sulfate initiation site on the core protein of the proteoglycan and that this initiation is enhanced in the absence of core protein. This suggests that under normal conditions the formation of heparan sulfate chains may be tightly linked to the production of the core protein.     

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.     

Multiple heparan sulfate proteoglycans synthesized by a basement membrane producing murine embryonal carcinoma cell line

The murine embryonal carcinoma derived cell line M1536-B3 secretes the basement membrane components laminin and entactin and, when grown in bacteriological dishes, produces and adheres to sacs of basement membrane components. Heparan sulfate proteoglycans have been isolated from these sacs, the cells, and the medium. At least three different heparan sulfate proteoglycans are produced by these cells as determined by proteoglycan size, glycosaminoglycan chain length, and charge density. The positions of the N- and O-sulfate groups in the glycosaminoglycan chains from each proteoglycan appear to be essentially the same despite differences in the size and culture compartment locations of the heparan sulfate proteoglycan. Additionally, small quantities of chondroitin sulfate proteoglycans are found in each fraction and copurify with each heparan sulfate proteoglycan. Because this cell line appears to synthesize at least three different heparan sulfate proteoglycans which are targeted to different final locations (basement membrane, cell surface, and medium), this will be a useful system in which to study the factors which determine final heparan sulfate proteoglycan structures and culture compartment targeting and the possible effects of the protein core(s) on heparan sulfate carbohydrate chain synthesis and secretion.     

Isolation and partial characterization of a rat hepatoma heparan sulfate proteoglycan

A proteoglycan was isolated from a Morris rat hepatoma by sequential precipitations with ammonium sulfate and cetyl pyridinium chloride followed by chromatography on Sepharose CL-4B and DEAE-cellulose. The proteoglycan has a molecular weight of about 1.5 × 10⁵ with 40,000 molecular weight glycosaminoglycan side chains, identified as heparan sulfate based on resistance to chondroitinase and susceptibility to nitrous acid treatment. Immunological studies showed that the protein core of this proteoglycan is immunologically distinct from a rat yolk sac tumor chondroitin sulfate proteoglycan (Å. Oldberg, E. G. Hayman, and E. Ruoslahti, 1981,J. Biol. Chem.256, 10847–10852), but resembles a heparan sulfate proteoglycan isolated from a basement membrane-producing mouse tumor (J. R. Hassell, P.M. Robey, H.-J. Barrach, J. Wilczek, S. R. Rennard, and G. R. Martin, 1980, Proc. Nat. Acad. Sci. USA77, 4494–4498).     

Transforming growth factor (type beta) promotes the addition of chondroitin sulfate chains to the cell surface proteoglycan (syndecan) of mouse mammary epithelia

Cultured monolayers of NMuMG mouse mammary epithelial cells have augmented amounts of cell surface chondroitin sulfate glycosaminoglycan (GAG) when cultured in transforming growth factor-beta (TGF-beta), presumably because of increased synthesis on their cell surface proteoglycan (named syndecan), previously shown to contain chondroitin sulfate and heparan sulfate GAG. This increase occurs throughout the monolayer as shown using soluble thrombospondin as a binding probe. However, comparison of staining intensity of the GAG chains and syndecan core protein suggests variability among cells in the attachment of GAG chains to the core protein. Characterization of purified syndecan confirms the enhanced addition of chondroitin sulfate in TGF-beta: (a) radiosulfate incorporation into chondroitin sulfate is increased 6.2-fold in this proteoglycan fraction and heparan sulfate is increased 1.8-fold, despite no apparent increase in amount of core protein per cell, and (b) the size and density of the proteoglycan are increased, but reduced by removal of chondroitin sulfate. This is shown in part by treatment of the cells with 0.5 mM xyloside that blocks the chondroitin sulfate addition without affecting heparan sulfate. Higher xyloside concentrations block heparan sulfate as well and syndecan appears at the cell surface as core protein without GAG chains. The enhanced amount of GAG on syndecan is partly attributed to an increase in chain length. Whereas this accounts for the additional heparan sulfate synthesis, it is insufficient to explain the total increase in chondroitin sulfate; an approximately threefold increase in chondroitin sulfate chain addition occurs as well, confirmed by assessing chondroitin sulfate ABC lyase (ABCase)-generated chondroitin sulfate linkage stubs on the core protein. One of the effects of TGF-beta during embryonic tissue interactions is likely to be the enhanced synthesis of chondroitin sulfate chains on this cell surface proteoglycan.     

Structure of low density heparan sulfate proteoglycan isolated from a mouse tumor basement membrane

A large heparan sulfate proteoglycan of low buoyant density (p = 1.32 to 1.40 g/cm3 in 6 M-guanidine.HCl) was extracted from a tumor basement membrane with denaturing solvents and purified by chromatography and CsCl gradient centrifugation. Chemical, immunological, physical and electron microscopical analyses have demonstrated a high degree of purity and have allowed us to propose a structural model for this proteoglycan. It is composed of an 80 nm long protein core formed from a single polypeptide chain (Mr about 500,000) with intrachain disulfide bonds. This core is folded into a row of six globular domains of variable size as shown by electron microscopy after rotary shadowing and negative staining. A multidomain structure was confirmed by protease digestion experiments that allowed the isolation of a single heparan sulfate-containing peptide segment representing less than 5% of the total mass of the protein core. Electron microscopy has visualized generally three heparan sulfate chains in each molecule close to each other at one pole of the protein core. The molecular mass and length (100 to 170 nm) of the heparan sulfate chains were found to vary consistently between different preparations. The mass per length ratio (350 nm-1) indicated an extended conformation for the heparan sulfate side-chains. These structural features are distinctly different from those of the high density proteoglycan, suggesting that both forms of basement membrane heparan sulfate proteoglycan are genetically distinct and not derived from a common precursor.     

Characterization of a heparan sulfate proteoglycan that copurifies with the neural cell adhesion molecule

We have demonstrated previously that the neural cell adhesion molecule (NCAM) interacts with a neuronal heparan sulfate proteoglycan. The binding of this proteoglycan(s) by NCAM appears to be required for NCAM-mediated cell adhesion, although the mechanism is unclear. In the present study we show that a heparan sulfate proteoglycan copurifies with NCAM, and provide an initial biochemical characterization of the proteoglycan. The copurification of a heparan sulfate proteoglycan with NCAM was demonstrated following immunopurification of NCAM from a detergent extract of cell membranes derived from Na2(35)SO4-labeled neural retinal cells. A large-molecular-weight, 35SO4-labeled molecule copurified with NCAM isolated from these neural cell cultures, and was resistant to chondroitinase ABC treatment, but degraded completely by nitrous acid treatment. These results indicate that the molecule is a heparan sulfate proteoglycan. Although this proteoglycan copurifies with NCAM, it is not detected when the neuron-glia cell adhesion molecule (NgCAM) is immunopurified using the 8D9 monoclonal antibody. The heparan sulfate proteoglycan may also be a membrane-associated proteoglycan since it interacts with phenyl-Sepharose. Molecular weight characterization of the proteoglycan by gel filtration chromatography indicates a molecular weight of 400-520 kDa. The heparan sulfate glycosaminoglycan chains were shown to have an average molecular weight of approximately 40 kDa, and the polypeptide backbone was estimated to be 120 kDa by polyacrylamide gel electrophoresis. These data therefore demonstrate that a neuronal heparan sulfate proteoglycan copurifies with NCAM.     

Turnover of heparan sulfate proteoglycans from substratum adhesion sites of murine fibroblasts

Substratum adhesion sites from murine Balb/c SVT2 fibroblasts are enriched in heparan sulfate proteoglycans which have been implicated in mediating adhesion of these cells to a fibronectin-adsorbed tissue culture substratum. Most of the heparan sulfate isolated from newly formed adhesion sites is found covalently attached to protein as proteoglycan while a significant portion of heparan sulfate from older sites has been identified as a single-chain species. This observation suggests that there may be catabolism of the heparan sulfate proteoglycan during the "maturation" of these adhesion sites at the cell's undersurface. Zwittergent 3-12 selectively extracts the single-chain class of heparan sulfate from either newly formed or "mature" adhesion sites while leaving the proteoglycan firmly bound in these sites. In an effort to further characterize the metabolism of these proteoglycans, substratum adhesion sites were isolated at various times after the cells had been pulse-radiolabeled using radioactive sulfate and subsequently chased. Greater than 80% of the sulfate-radiolabeled material is lost from the substratum-attached material within 24-48 h. Characterization of both the Zwittergent-soluble and -resistant heparan sulfate indicated that there was an initial accumulation followed by a rapid loss of a portion of the radiolabeled heparan sulfate as the single-chain Zwittergent-soluble class. However, most of the heparan sulfate proteoglycan was lost from the adhesion sites following approximately a 4-h time lag during the chase period without going through a smaller molecular weight intermediate. The turnover properties of the heparan sulfate proteoglycan in the EGTA-detachable cells were different from those in the substratum-attached fraction of the cell. The significance of these two different mechanisms of turnover of heparan sulfate proteoglycan in adhesion sites is discussed in relation to the role of this proteoglycan in mediating adhesion processes.     

Membrane-anchored proteoglycans of mouse macrophages: P388D1 cells express a syndecan-4–like heparan sulfate proteoglycan and a distinct chondroitin sulfate form

Proteoglycan accumulation by thioglycollate-elicited mouse peritoneal macrophages and a panel of murine monocyte-macrophage cell lines has been examined to determine whether these cells express plasma membrane-anchored heparan sulfate proteoglycans. Initially, cells were screened for heparan sulfate and chondroitin sulfate glycosaminoglycans after metabolic labeling with radiosulfate. Chondroitin sulfate is secreted to a variable extent by every cell type examined. In contrast, heparan sulfate is all but absent from immature pre-monocytes and is associated predominantly with the cell layer of mature macrophage-like cells. In the P388D1 cell line, the cell-associated chondroitin sulfate is largely present as a plasma membrane-anchored proteoglycan containing a 55 kD core protein moiety, which appears to be unique. In contrast, the cell-associated heparan sulfate is composed of a proteoglycan fraction and protein-free glycosaminoglycan chains, which accumulate intracellularly. A fraction of the heparan sulfate proteoglycan contains a lipophilic domain and can be released from cells following mild treatment with trypsin, suggesting that it is anchored in the plasma membrane. Isolation of this proteoglycan indicates that it is likely syndecan-4: it is expressed as a heparan sulfate proteoglycan at the cell surface, it is cleaved from the plasma membrane by low concentrations of trypsin, and it consists of a single 37 kD core protein moiety that co-migrates with syndecan-4 isolated from NMuMG mouse mammary epithelial cells. Northern analysis reveals that a panel of macrophage-like cell lines accumulate similar amounts of syndecan-4 mRNA, demonstrating that this proteoglycan is expressed by a variety of mature macrophage-like cells. Syndecan-1 mRNA is present only in a subset of these cells, suggesting that the expression of this heparan sulfate proteoglycan may be more highly regulated by these cells. © 1993 Wiley-Liss, Inc.     

The core protein of the matrix-associated heparan sulfate proteoglycan binds to fibronectin

The extracellular matrix of cultured human lung fibroblasts contains one major heparan sulfate proteoglycan. This proteoglycan contains a 400-kDa core protein and is structurally and immunochemically identical or closely related to the heparan sulfate proteoglycans that occur in basement membranes. Because heparitinase does not release the core protein from the matrix of cultured cells, we investigated the binding interactions of this heparan sulfate proteoglycan with other components of the fibroblast extracellular matrix. Both the intact proteoglycan and the heparitinase-resistant core protein were found to bind to fibronectin. The binding of 125I-labeled core protein to immobilized fibronectin was inhibited by soluble fibronectin and by soluble cold core protein but not by albumin or gelatin. A Scatchard plot indicates a Kd of about 2 x 10(-9) M. Binding of the core protein was also inhibited by high concentrations of heparin, heparan sulfate, or chrondroitin sulfate and was sensitive to high salt concentrations. Thermolysin fragmentation of the 125I-labeled proteoglycan yielded glycosamino-glycan-free core protein fragments of approximately 110 and 62 kDa which bound to both fibronectin and heparin columns. The core protein-binding capacity of fibronectin was very sensitive to proteolysis. Analysis of thermolytic and alpha-chymotryptic fragments of fibronectin showed binding of the intact proteoglycan and of its isolated core protein to a protease-sensitive fragment of 56 kDa which carried the gelatin-binding domain of fibronectin and to a protease-sensitive heparin-binding fragment of 140 kDa. Based on the NH2-terminal amino acid sequence analyses of the 56- and 140-kDa fragments, the core protein-binding domain in fibronectin was tentatively mapped in the area of overlap of the two fragments, carboxyl-terminally from the gelatin-binding domain, possibly in the second type III repeat of fibronectin. These data document a specific and high affinity interaction between fibronectin and the core protein of the matrix heparan sulfate proteoglycan which may anchor the proteoglycan in the matrix.     

Expression of human perlecan domain I as a recombinant heparan sulfate proteoglycan with 20-kDa glycosaminoglycan chains

Recombinant forms of human perlecan domain I were secreted as proteoglycans by stably transfected human 293 cells. A recombinant domain I-only proteoglycan spanned the 95- to 265-kDa region in SDS-PAGE and appeared to be 160 kDa in denaturing gel filtration. Its glycosaminoglycan (GAG) content was approximately 67% heparan sulfate, and its average GAG chain size of 20 kDa suggested that the true molecular mass of the proteoglycan was 90 kDa. Domain I with enhanced green fluorescent protein fused to its C-terminus had an apparent molecular mass of 210-220 kDa and contained approximately 100% heparan sulfate. Its average GAG chain size (also 20 kDa) suggested a true molecular mass of 117 kDa for this proteoglycan. Its sulfate content of 53-77 mol SO2-4 per mole of protein indicated the presence of one sulfate group per 4-7 GAG sugar residues.     

Characterization of a heparan sulfate and a peculiar chondroitin 4-sulfate proteoglycan from platelets. Inhibition of the aggregation process by platelet chondroitin sulfate proteoglycan

A high molecular weight chondroitin sulfate proteoglycan (Mr 240,000) is released from platelet surface during aggregation induced by several pharmacological agents. Some details on the structure of this compound are reported. beta-Elimination with alkali and borohydride produces chondroitin sulfate chains with a molecular weight of 40,000. The combined results indicate a proteoglycan molecule containing 5-6 chondroitin sulfate chains and a protein core rich in serine and glycine residues. Degradation with chondroitinase AC shows that a 4-sulfated disaccharide is the only disaccharide released from this chondroitin sulfate, characterizing it as a chondroitin 4-sulfate homopolymer. It is shown that this proteoglycan inhibits the aggregation of platelets induced by ADP. Analysis of the sulfated glycosaminoglycans not released during aggregation revealed the presence of a heparan sulfate in the platelets. Degradation by heparitinases I and II yielded the four disaccharide units of heparan sulfates: N,O-disulfated disaccharide, N-sulfated disaccharide, N-acetylated 6-sulfated disaccharide, and N-acetylated disaccharide. The possible role of the sulfated glycosaminoglycans on cell-cell interaction is discussed in view of the present findings.     

Self-assembly of a high molecular weight basement membrane heparan sulfate proteoglycan into dimers and oligomers

A high molecular weight basement membrane heparan sulfate proteoglycan, isolated from murine Englebreth-Holm-Swarm tumor, is seen in platinum replicas as an elongated flexible core (Mr = 450,000) consisting of a series of tandem globular domains from which extend, at one end, two to three heparan sulfate chains (average Mr = 80,000 each). This macromolecule will self-assemble into dimers and lesser amounts of oligomers when incubated in neutral isotonic buffer. These molecular species can be separated by zonal velocity sedimentation and assembly is seen to be time- and concentration-dependent. In rotary-shadowed platinum replicas the binding region is found at or near the end of the core at the pole opposite the origin of the heparan sulfate chains. Dimers are double-length structures and oligomers are seen as stellate clusters: in both, the heparan sulfate chains appear peripherally oriented. While isolated cores self-assemble, isolated heparan sulfate chains do not bind intact proteoglycans. Furthermore, proteolytic removal of a non-heparan sulfate containing core moiety destroys the ability of the proteoglycan monomer to form larger species or bind intact proteoglycan, further supporting the binding topography determined morphologically. These negatively charged macromolecular complexes may be important contributors to basement membrane structure and function.     

The heparan sulfates of Swiss mouse 3T3 cells. The effect of transformation.

Three major pools of heparan sulfate have been isolated from cultures of Swiss mouse 3T3 and SV40-transformed 3T3 cells: cell-surface, medium, and intracellular heparan sulfates. The cell-surface heparan sulfate is a high molecular weight proteoglycan which is partially degraded by pronase. Before pronase treatment, it has a peak molecular weight (as estimated by gel filtration) of approx. 7.2 . 10(5) in contrast to only 2.4 . 10(5) after pronase treatment. The medium heparan sulfate appears to be similar in structure to the cell-surface heparan sulfate, since they coelute on Bio-Gel A-15m and DEAE-cellulose, and are both proteoglycans. In contrast, the intracellular heparan sulfate has a low molecular weight (6.0 . 10(3)) and has little if any attached protein. Both the medium and intracellular heparan sulfate exhibit the transformation-associated change in structure reported earlier for cell-surface heparan sulfate (Underhill, C.B. and Keller, J.M. )1975) Biochem. Biophys. Res. Commun. 63, 448--454). This transformation-associated change, detected by DEAE-cellulose chromatography is not the result of changes in either molecular weight or protein core. Cellulose acetate electrophoresis of the cell-surface heparan sulfate at pH 1 suggests that the transformation-associated change in structure is due to a difference in sulfate content. Both types of heparan sulfate are produced in mixed cultures of 3T3 and SV3T3 cells, indicating that neither serum factors in the culture medium nor secreted cell products are responsible for the transformation-associated change in heparan sulfate structure. The presented data are discussed with respect to the postulated role of heparan sulfate in cell social behavior.     

Immunological evidence for two distinct chondroitin sulfate proteoglycan core proteins: Differential expression in cartilage matrix deficient mice

The expression and core protein structure of two proteoglycans, the major cartilage proteoglycan isolated from a rat chondrosarcoma and a small molecular weight chondroitin sulfate proteoglycan isolated from a rat yolk sac tumor, have been compared. The cartilage proteoglycan was not detectable in the cartilage tissue of cartilage matrix deficient ($\text{cmd}\text{cmd}$) neonatal mice by immunofluorescence, but the cmd cartilage did react with antibodies against the core protein of the yolk sac tumor proteoglycan. Radioimmunoassays showed that the core proteins of these proteoglycans are not cross-reactive with each other. Analysis of the core proteins by sodium dodecyl sulfate/polyacrylamide gel electrophoresis after chondroitinase ABC treatment of the proteoglycan revealed a large difference in their sizes. The cartilage proteoglycan core protein had a molecular weight of about 200,000 while the yolk sac tumor proteoglycan core protein migrated with an apparent molecular weight of about 20,000. In addition, the cultured yolk sac tumor cells that make the small proteoglycan did not react with antiserum against the cartilage proteoglycan. These results indicate that the proteoglycan isolated from the yolk sac tumor is similar to the small chondroitin sulfate proteoglycan species found in cartilage and support the existence of at least two dissimilar and genetically independent chondroitin sulfate proteoglycan core proteins.     

Shedding of heparan sulfate proteoglycan by stimulated endothelial cells: Evidence for proteolysis of cell-surface molecules

Activation of endothelial cells by cytokines and endotoxin causes procoagulant and pro-inflammatory changes over a period of hours. We postulated that the same functional state might be achieved more rapidly by changes in the metabolism of heparan sulfate, which supports many of the normal functions of endothelial cells. We previously found that binding of anti-endothelial cell antibodies and activation of complement on endothelial cells causes the rapid shedding of endothelial cell heparan sulfate. Here we report the biochemical mechanism responsible for the release of the heparan sulfate. Stimulation of endothelial cells by anti-endothelial cell antibodies and complement resulted in the release of ³⁵S-heparan sulfate proteoglycan and partially degraded ³⁵S-heparan sulfate chains. Degradation of the ³⁵S-heparan sulfate chains was not necessary for release since heparin and suramin prevented cleavage of the heparan sulfate but did not inhibit release from stimulated endothelial cells. The ³⁵S-heparan sulfate proteoglycan released from endothelial cells originated from the cell surface and had a core protein similar in size (70.5 kD) to syndecan-1. Release was due to proteolytic cleavage of the protein core by serine and/or cysteine proteinases since the release of heparan sulfate was inhibited 87% by antipain and 53% by leupeptin. Release of heparan sulfate coincided with a decrease of ∼︁7 kD in the mass of the protein core and with a loss of hydrophobicity of the proteoglycan, consistent with the loss of the hydrophobic transmembrane domain. The cleavage and release of cell-surface ³⁵S-heparan sulfate proteoglycan might be a novel mechanism by which endothelial cells may rapidly acquire the functional properties of activated endothelial cells. © 1996 Wiley-Liss, Inc.     

Distribution of keratan sulfate in cartilage proteoglycans.

After chondroitinase digestion of bovine nasal and tracheal cartilage proteoglycans, subsequent treatment with trypsin or trypsin followed by chymotrypsin yielded two major types of polypeptide-glycosaminoglycan fragments which could be separated by Sepharose 6B chromatography. One fragment, located close to the hyaluronic acid-binding region of the protein core, had a high relative keratan sulfate content. This fragment contained about 60% of the total keratan sulfate, but less than 10% of the total chondroitin sulfate present in the original proteoglycan preparation. The weight average molecular weight of the keratan sulfate-enriched fragment was 122,000, as determined by sedimentation equilibrium centrifugation. The chemical and physical data indicate that this fragment contains an average of 10 to 15 keratan sulfate chains, if the average molecular weight of individual chains is assumed to be about 8,000, and about 5 chondroitin sulfate chains attached to a peptide of about 20,000 daltons. The other population of fragments was derived from the other end of the proteoglycan molecule, the chondroitin sulfate-enriched region, and contained mainly chondroitin sulfate chains. About 90% of the total chondroitin sulfate, but only 20 to 30% of the total keratan sulfate was recovered in these fragments. On the average, approximately 5 chondroitin sulfate chains and 1 keratan sulfate chain could be linked to the same peptide. Another 10 to 20% of the total keratan sulfate, originally found in or near the hyaluronic acid-binding region, was not separated from the chondroitin sulfate-enriched fragments. Hydroxylamine could be used to liberate a large molecular size, chondroitin sulfate-enriched fragment (Kav 0.54 on Sepharose 2B) from the proteoglycan aggregates. The remainder of the protein core, containing the keratan sulfate-enriched region, was bound to hyaluronic acid with the link proteins and recovered in the void volume on the Sepharose 2B column.