Composition of proteoglycans synthesized by rabbit aortic explants in culture and the effect of experimental atherosclerosis

The synthesis of proteoglycans by aorta explants from rabbits with diet-induced atherosclerosis and controls was studied by 35S-incorporation. Proteoglycans were isolated under dissociative conditions from incubation medium and from arterial explants. Additionally, the tissue proteoglycans that were not extracted by 4 M guanidine-HCl were solubilized by digestion of the tissue by elastase in the presence of proteinase inhibitors. The residual tissue was hydrolyzed by papain and glycosaminoglycans were isolated. The atherosclerotic aorta tissue incorporated twice the amount of 35S into proteoglycans than observed for controls; in both groups about 70% of the label incorporated into the tissue was noted in the proteoglycans extracted by guanidine-HC;, while about 30% of the total 35S-labeled proteoglycans synthesized by the explants were found in the media. Atherosclerotic tissue incorporated 35S predominantly into chondroitin sulfate proteoglycans when compared to control tissue. The chondroitinase ABC-digestable proteoglycans that were extracted by guanidine-HCl from atherosclerotic tissues were of larger molecular size than those from control tissue, but the core proteins from these preparations were similar. The heparan sulfate proteoglycan that was obtained by dissociative extraction from atherosclerotic tissue had greater amounts of N-acetyl and lesser amounts of N-sulfate ester groups than the preparation from control tissue. Digestion of the tissue by elastase yielded heparan sulfate proteoglycan as the major constituent in both groups, although atherosclerotic tissue contained relatively small amounts of this proteoglycan. The residual tissue from both groups contained chondroitin sulfate and heparan sulfate as the major glycosaminoglycans with the latter showing a decrease with atherosclerosis. Atherosclerotic tissue secreted into the medium about two-fold more 35S-labeled proteoglycans with larger molecular size than control tissue; proteoglycans of the heparan sulfate and chondroitin sulfate types were the major constituents in the culture medium of both tissues. Thus, proteoglycans undergo both quantitative and qualitative changes in atherosclerosis, reflecting the enhanced smooth muscle cell activity. These changes are potentially important in modulating lipoprotein binding and hemostatic properties, as well as fibrillogenesis of the arterial wall.     

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.     

Isolation and characterization of proteoglycans synthesized by adult human gingival fibroblasts in vitro

The proteoglycans synthesized by fibroblasts derived from healthy human gingivae were isolated and characterized. The largest medium proteoglycan was excluded from Sepharose CL-4B but not from Sepharose CL-2B; it was recovered in the most-dense density gradient fraction and identified as a chondroitin sulfate proteoglycan. The medium contained two smaller proteoglycans; one contained predominantly chondroitin sulfate proteoglycan, while the other was comprised predominantly of dermatan sulfate proteoglycan and was quantitatively the major species. The largest proteoglycan in the cell layer fraction, excluded from both Sepharose CL-2B and Sepharose CL-4B, was found in the least-dense density gradient fraction and contained heparan sulfate and chondroitin sulfate proteoglycan. It could be further dissociated by treatment with detergent, suggesting an intimate association with cell membranes. Two other proteoglycan populations of intermediate size were identified in the cell layer extracts which contained variable proportions of heparan sulfate, dermatan sulfate, or chondroitin sulfate proteoglycan. Some small molecular weight material indicative of free glycosaminoglycan chains was also associated with the cell layer fraction. Carbohydrate analysis of the proteoglycans demonstrated the glycosaminoglycan chains to have approximate average molecular weights of 25,000. In addition, N- and O-linked oligosaccharides which were associated with the proteoglycans appeared to be sulfated in varying degrees.     

Three distinct molecular species of proteoglycan synthesized by the rat limb bud at the prechondrogenic stage

To characterize proteoglycans in the prechondrogenic limb bud, proteoglycans were extracted with 4 M guanidine HCl containing a detergent and protease inhibitors from Day 13 fetal rat limb buds which had been labeled with [35S]sulfate for 3 h in vitro. About 90% of 35S-labeled proteoglycans was solubilized under the conditions used. The proteoglycan preparation was separated by DEAE-Sephacel column chromatography into three peaks; peak I eluted at 0.45 M NaCl concentration, peak II at 0.52 M, and peak III at 1.4 M. Peaks I and III were identified as proteoglycans bearing heparan sulfate side chains. The heparan sulfate proteoglycan in peak III was larger in hydrodynamic size than the proteoglycan in peak I. The heparan sulfate side chains of peak III proteoglycan were smaller in the size and more abundant in N-sulfated glucosamine than those of peak I proteoglycan. Peak II contained a chondroitin sulfate proteoglycan with a core protein of a doublet of Mr 550,000 and 500,000. The chondroitin sulfate proteoglycan was easily solubilized with a physiological salt solution and the heparan sulfate proteoglycan in peak I was partially solubilized with the physiological salt solution. The remainder of the proteoglycan in peak I and the heparan sulfate proteoglycan in peak III could be solubilized effectively only with a solution containing a detergent, such as nonanoyl-N-methylglucamide. This observation indicates the difference in the localization among these three proteoglycans in the developing rat limb bud.     

Proteoglycan synthesis by primary chick skeletal muscle during in vitro myogenesis

The proteoglycans synthesized by primary chick skeletal muscle during in vitro myogenesis were compared with those of muscle-specific fibroblasts. Cultures of skeletal muscle cells and muscle fibroblasts were separately labeled using [35S] sulfate as a precursor. The proteoglycans of the cell layer and medium were separately extracted and isolated by ion-exchange chromatography on DEAE-Sephacel followed by gel filtration chromatography on Sepharose CL-2B. Two cell layer-associated proteoglycans synthesized both by skeletal muscle cells and muscle fibroblasts were identified. The first, a high molecular weight proteoglycan, eluted from Sepharose CL-2B with a Kav of 0.07 and contained exclusively chondroitin sulfate chains with an average molecular weight greater than 50,000. The second, a relatively smaller proteoglycan, eluted from Sepharose CL-2B with a Kav of 0.61 and contained primarily heparan sulfate chains with an average molecular weight of 16,000. Two labeled proteoglycans were also found in the medium of both skeletal muscle and muscle fibroblasts. A high molecular weight proteoglycan was found with virtually identical properties to that of the high molecular weight chondroitin sulfate proteoglycan of the cell layer. A second, smaller proteoglycan had a similar monomer size (Kav of 0.63) to the cell layer heparan sulfate proteoglycan, but differed from it in that this molecule contained primarily chondroitin sulfate chains with an average molecular weight of 32,000. Studies on the distribution of these proteoglycans in muscle cells during in vitro myogenesis demonstrated that a parallel increase in the relative amounts of the smaller proteoglycans occurred in both the cell layer and medium compared to the large chondroitin sulfate proteoglycan in each compartment. In contrast, muscle-derived fibroblasts displayed a constant ratio of the small proteoglycans of the cell layer and medium fractions, compared to the larger chondroitin sulfate proteoglycan of the respective fraction as a function of cell density. Our results support the concept that proteoglycan synthesis is under developmental regulation during skeletal myogenesis.     

Heparan sulfate proteoglycans of human neuroblastoma cells: Affinity fractionation on columns of platelet factor-4+

Human neuroblastoma cells (Platt) were detached from tissue culture substrata with a Ca²⁺ chelating agent, and then the suspended cells were extracted with a sodium dodecyl sulfate (SDS)-containing buffer to maximally solubilize their sulfate-radiolabeled proteoglycans. The majority of the high-molecular-weight material in these dissociative extracts was heparan sulfate proteoglycan, which resolves into two heterodisperse size classes upon gel filtration on columns of Sepharose CL4B. After removal of SDS from these extracts by hydrophobic chromatography on Sep-Pak C₁₈ cartridges, extracts were further fractionated on various affinity matrices. All of the sulfate-radiolabeled material eluted as one peak from DEAE-Sephadex ion-exchange columns. In contrast, affinity fractionation on Sepharose columns derivatized with the heparan sulfate-binding protein, platelet factor-4, resolved three major and one minor subsets of these components. The nonbinding fraction contained some heparan sulfate proteoglycan and some chondroitin sulfate. The weak-binding fraction contained principally heparan sulfate proteoglycan, as well as a small amount of chondroitin sulfate proteoglycan; the gel-filtration properties of these proteoglycans before or after alkaline borohydride treatment indicated that they were small in size, containing perhaps 2 to 4 glycosaminoglycan chains. The high-affinity fraction eluted from platelet factor 4-Sepharose was composed entirely of “singlechain” heparan sulfate. A portion of the heparan sulfate proteoglycan of the original extract bound to the hydrophobic affinity matrix, octyl-Sepharose, and this hydrophobic proteoglycan partitioned into the nonbinding and weak-binding fractions of the platelet factor 4-Sepharose affinity columns. These studies reveal that the majority of the proteoglycan made by these neuronal cells in culture is of the heparan sulfate class, is small in size when compared to other characterized proteoglycans, and can be resolved into several overlapping subsets when fractionated on affinity matrices.     

Modification of proteoglycans during maturation of fibroblast substratum adhesion sites

Newly formed adhesion sites, left bound to the tissue culture substratum after [ethylenebis(oxyethylenenitrilo)] tetraacetic acid mediated detachment of simian virus 40 transformed Balb/c 3T3 cells, have been extracted with 0.5 M guanidine hydrochloride or Zwittergent (3-12), extractions which identify different subfractions of proteoglycans in these sites. The compositions of these extracts were then compared to similar extracts of "maturing" adhesion sites in an effort to identify structural and metabolic changes which may occur with time and which may play a role in altering adhesion during cell movement. Guanidine hydrochloride (0.5 M) extracts both hyaluronate and chondroitin sulfate proteoglycan from newly formed sites (but which are not complexed in an aggregate similar to that found in cartilage) but only hyaluronate from fully matured sites, indicating that the chondroitin sulfate proteoglycans somehow become resistant to extraction with time. Both high and low molecular weight forms of hyaluronate also accumulate in sites with time. Zwittergent 3-12 solubilizes free chains of heparan sulfate but not heparan sulfate proteoglycan from either class of sites. Most of the heparan sulfate in newly formed sites occurs as a large proteoglycan excludable from Sepharose CL-6B columns under stringent dissociative conditions; however, as adhesion sites "mature", a portion of this proteoglycan appears to be converted by some unknown mechanism to free heparan sulfate chains. This process may very well weaken the close adhesive contacts between the cell and substratum mediated by fibronectin's binding to the highly multivalent heparan sulfate proteoglycans. These studies further indicate that there is considerable metabolism and changing intermolecular associations of proteoglycans within these sites during movement of fibroblasts over this model extracellular matrix.     

Partial characterization of newly synthesized proteoglycans isolated from the glomerular basement membrane

Kidneys were perfused with [35S]sulfate at 4 h in vitro to radiolabel sulfated proteoglycans. Glomeruli were isolated from the labeled kidneys, and purified fractions of glomerular basement membrane (GBM) were prepared therefrom. Proteoglycans were extracted from GBM fractions by use of 4 M guanidine-HCl at 4 degrees C in the presence of protease inhibitors. The efficiency of extraction was approximately 55% based on 35S radioactivity. The extracted proteoglycans were characterized by gel-filtration chromatography (before and after degradative treatments) and by their behavior in dissociative CsCl gradients. A single peak of proteoglycans with an Mr of 130,000 (based on cartilage proteoglycan standards) was obtained on Sepharose CL-4B or CL-6B. Approximately 85% of the total proteoglycans were susceptible to nitrous acid oxidation (which degrades heparan sulfates), and approximately 15% were susceptible to digestion with chondroitinase ABC (degrades chondroitin-4 and -6 sulfates and dermatan sulfate). The released glycosaminoglycan (GAG) chains had an Mr of approximately 26,000. Density gradient centrifugation resulted in the partial separation of the extracted proteoglycans into two types with different densities: a heparan sulfate proteoglycan that was enriched in the heavier fraction (p greater than 1.43 g/ml), and a chondroitin sulfate proteoglycan that was concentrated in the lighter fractions (p less than 1.41). The results indicate that two types of proteoglycans are synthesized and incorporated into the GBM that are similar in size and consist of four to five GAG chains (based on cartilage proteoglycan standards). The chromatographic behavior of the extracted proteoglycans and the derived GAG, together with the fact that the two types of proteoglycans can be partially separated into the density gradient, suggest that the heparan sulfate and chondroitin sulfate(s) are located on different core proteins.     

Aortic endothelial cells synthesize a large chondroitin sulphate proteoglycan capable of binding to hyaluronate.

Confluent cultures of mouse aortic endothelial (END-D) were incubated with either [35S]methionine or 35SO4 2-, and the radiolabelled proteoglycans in media and cell layers were analysed for their hyaluronate-binding activity. The proteoglycan subfraction which bound to hyaluronate accounted for about 18% (media) and 10% (cell layers) of the total 35S radioactivity of each proteoglycan fraction. The bound proteoglycan molecules could be dissociated from the aggregates either by digestion with hyaluronate lyase or by treatment with hyaluronate decasaccharides. Digestion of [methionine-35S]proteoglycans with chondroitinase and/or heparitinase, followed by SDS/polyacrylamide-gel electrophoresis, indicated that the medium and cell layer contain at least three chondroitin sulphate proteoglycans, one dermatan sulphate proteoglycan, and two heparan sulphate proteoglycans which differ from one another in the size of core molecules. Among these, only the hydrodynamically large chondroitin sulphate species with an Mr 550,000 core molecule was shown to bind to hyaluronate. A very similar chondroitin sulphate proteoglycan capable of binding to hyaluronate was also found in cultures of calf pulmonary arterial endothelial cells (A.T.C.C. CCL 209). These observations, together with the known effects of hyaluronate on various cellular activities, suggest the existence of possible specialized functions of this proteoglycan subspecies in cellular processes characteristic of vascular development and diseases.     

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.     

Biosynthesis of proteoglycans by isolated rabbit glomeruli

Isolated rabbit glomeruli were incubated in vitro with 35SO4 in order to analyze the proteoglycans synthesized. Proteoglycans extracted with 4 M guanidine HCl from whole isolated glomeruli and from purified glomerular basement membrane (GBM) were analyzed by gel filtration chromatography. Two types of sulfated proteoglycans were found to be synthesized by rabbit glomeruli and these contained either heparan sulfate or chondroitin/dermatan sulfate glycosaminoglycan chains. These glycosaminoglycans were characterized by their sensitivity to selective degradation by nitrous acid or chondroitinase ABC, respectively. The major proteoglycan extracted from the whole glomeruli was a chondroitin/dermatan sulfate species (75%), while purified GBM contained mostly heparan sulfate (70%). The glycosaminoglycan chains were estimated to be about 12,000 molecular weight which is consistent with previous estimates for similar molecules extracted from the rat GBM.     

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.     

Structural Properties of the Heparan Sulfate Proteoglycans of Brain

The heparan sulfate proteoglycans present in a deoxycholate extract of rat brain were purified by ion exchange chromatography, affinity chromatography on lipoprotein lipase agarose, and gel filtration. Heparitinase treatment of the heparan sulfate proteoglycan fraction (containing 86% heparan sulfate and 10% chondroitin sulfate) that was eluted from the lipoprotein lipase affinity column with 1 M NaCl led to the appearance of a major protein core with a molecular size of 55,000 daltons, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the effects of heparinase and heparitinase treatment revealed that the heparan sulfate proteoglycans of brain contain a significant proportion of relatively short N-sulfoglucosaminyl 6-O-sulfate [or N-sulfoglucosaminyl](alpha 1-4)iduronosyl 2-O-sulfate(alpha 1-4) repeating units and that the portions of the heparan sulfate chains in the vicinity of the carbohydrate-protein linkage region are characterized by the presence of D-glucuronic acid rather than L-iduronic acid. After chondroitinase treatment of a proteoglycan fraction that contained 62% chondroitin sulfate and 21% heparan sulfate (eluted from lipoprotein lipase with 0.4 M NaCl), the charge and density of a portion of the heparan sulfate-containing proteoglycans decreased significantly. These results indicate that a population of "hybrid" brain proteoglycans exists that contain both chondroitin sulfate and heparan sulfate chains covalently linked to a common protein core.     

Isolation and characterization of the heparan sulfate proteoglycans of brain. Use of affinity chromatography on lipoprotein lipase-agarose

Heparan sulfate proteoglycans were extracted from rat brain microsomal membranes or whole forebrain with deoxycholate and purified from accompanying chondroitin sulfate proteoglycans and membrane glycoproteins by ion-exchange chromatography, affinity chromatography on lipoprotein lipase-Sepharose, and gel filtration. The proteoglycan has a molecular size of approximately 220,000, containing glycosaminoglycan chains of Mr = 14,000-15,000. In [3H]glucosamine-labeled heparan sulfate proteoglycans, approximately 22% of the radioactivity is present in glycoprotein oligosaccharides, consisting predominantly of N-glycosidically linked tri- and tetraantennary complex oligosaccharides (60%, some of which are sulfated) and O-glycosidic oligosaccharides (33%). Small amounts of chondroitin sulfate (4-6% of the total glycosaminoglycans) copurified with the heparan sulfate proteoglycan through a variety of fractionation procedures. Incubation of [35S]sulfate-labeled microsomes with heparin or 2 M NaCl released approximately 21 and 13%, respectively, of the total heparan sulfate, as compared to the 8-9% released by buffered saline or chondroitin sulfate and the 82% which is extracted by 0.2% deoxycholate. It therefore appears that there are at least two distinct types of association of heparan sulfate proteoglycans with brain membranes.     

Isolation of the major chondroitin sulfate/dermatan sulfate and heparan sulfate proteoglycans from embryonic chicken retina

A technique is presented for the preparation of three major proteoglycans from 14-day embryonic chicken retinas following their culture overnight with [35S]sulfate and either [3H]glucosamine or [3H]serine. Homogenization of the tissue in saline permitted extraction of heterogeneous soluble proteoglycans separately from most of the heparan sulfate proteoglycans. The latter were extracted from the 140,000g pellet with 0.5% Triton X-100 in 8 M urea. The medium plus the saline and urea-detergent extracts were separated from low-molecular-weight contaminants, and fractionated into two peaks of radioactivity on Sephacryl S-300 in saline with 3 M urea and 0.5% Triton X-100. The proteoglycans were isolated directly from these fractions on DEAE-Sephacel, and subjected to ultrafiltration concentration and then further purification on cesium chloride density gradient centrifugation in 4 M guanidine hydrochloride. A further step involving cetylpyridinium chloride precipitation was examined, but it resulted in essentially no further purification. The fractionations separated a large chondroitin sulfate/dermatan sulfate proteoglycan from the culture medium that was excluded from S-300 and of low buoyant density; a large heparan sulfate proteoglycan from the urea-detergent extract that was also excluded from S-300 and of low buoyant density; and two smaller and possibly related heparan sulfate proteoglycans. One was found in the medium and showed low to intermediate buoyant density; the other was isolated from the urea-detergent extract and showed a significantly higher buoyant density, associated with a lower protein content. The saline extract contained both of the two larger proteoglycans and only minor amounts of the smaller molecules.     

Chondroitin sulphate proteoglycan in the substratum adhesion sites of Balb/c 3T3 cells. Fractionation on various ion-exchange and affinity columns.

Proteoglycans on the cell surface play critical roles in the adhesion of fibroblasts to a fibronectin-containing extracellular matrix, including the model mouse cell line Balb/c 3T3. In order to evaluate the biochemistry of these processes, long-term [35S]sulphate-labelled proteoglycans were extracted quantitatively from the adhesion sites of 3T3 cells, after their EGTA-mediated detachment from the substratum, by using an extractant containing 1% octyl glucoside, 1 M-NaCl and 0.5 M-guanidinium chloride (GdnHCl) in buffer with many proteinase inhibitors. Greater than 90% of the material was identified as a large chondroitin sulphate proteoglycan (Kav. = 0.4 on a Sepharose CL2B column), and the remainder was identified as a smaller heparan sulphate proteoglycan; only small amounts of free chains of glycosaminoglycan were observed in these sites. These extracts were fractionated on DEAE-Sepharose columns under two different sets of elution conditions: with acetate buffer (termed DEAE-I) or with acetate buffer supplemented with 8 M-urea (termed DEAE-II). Under DEAE-I conditions about one-half of the material was eluted as a single peak and the remainder required 4 M-GdnHCl in order to recover it from the column; in contrast, greater than 90% of the material was eluted as a single peak from DEAE-II columns. Comparison of the elution of [35S]sulphate-labelled proteoglycan with that of 3H-labelled proteins from these two columns, as well as mixing experiments, indicated that the GdnHCl-sensitive proteoglycans were trapped at the top of columns, partially as a consequence of their association with proteins in these adhesion-site extracts. Affinity chromatography of these proteoglycans on columns of either immobilized platelet factor 4 or immobilized plasma fibronectin revealed that most of the chondroitin sulphate proteoglycan and the heparan sulphate proteoglycan bound to platelet factor 4 but that only the heparan sulphate proteoglycan bound to fibronectin, providing a ready means of separating the two proteoglycan classes. Affinity chromatography on octyl-Sepharose columns to test for hydrophobic domains in their core proteins demonstrated that a high proportion of the heparan sulphate proteoglycan but none of the chondroitin sulphate proteoglycan bound to the hydrophobic matrix. These results are discussed in light of the possible functional importance of the chondroitin sulphate proteoglycan in the detachment of cells from extracellular matrix and in light of previous affinity fractionations of proteoglycans from the substratum-adhesion sites of simian-virus-40-transformed 3T3 cells.     

The decreased synthesis of chondroitin sulfate-containing extracellular proteoglycans by SV40 transformed Balb/c 3T3 cells.

Balb/c 3T3 cells synthesize 5--10 times more 35SO2/4- -labeled extracellular proteoglycan per cell than do Balb/c 3T3 cells transformed by SV40 (SV3T3). The extracellular 35SO2/4- -labeled proteoglycans of the Balb/c 3T3 and SV3T3 cells differ markedly in their acid mucopolysaccharide composition. Extracellular Balb/c 3T3 proteoglycans contain about 70--80% chondroitin sulfate, most of which is chondroitin 4-sulfate, and small amounts of heparan sulfate and/or heparin. On the other hand, extracellular SV3T3 proteoglycans contain 65-75% heparan sulfate and/or heparin and less than 15% chondroitin sulfate. Analysis of extracellular 35SO2/4- -labeled proteoglycan by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that Balb/c 3T3 alone synthesizes a class of proteoglycans capable of migrating in a 10% separating gel. This class of proteoglycans, designated as fraction C, accounts for up to 45% of the total extracellular Balb/c 3T3 35 SO2/4- -labeled proteoglycans and contains chondroitin sulfate extracellular SV3T3 proteoglycans. The absence of this and other classes of chondroitin sulfate-containing proteoglycans can account for the 5-10-fold decreased synthesis of 35SO2/4- -labeled proteoglycans by SV3T3 cells when compared to Balb/c 3T3 cells.     

Chondroitin sulfate and heparan sulfate proteoglycans of PC12 pheochromocytoma cells

We have isolated and characterized the cell-associated and secreted proteoglycans synthesized by a clonal line of rat adrenal medullary PC12 pheochromocytoma cells, which have been extensively employed for the study of a wide variety of neurobiological processes. Chondroitin sulfate accounts for 70-80% of the [35S] sulfate-labeled proteoglycans present in PC12 cells and secreted into the medium. Two major chondroitin sulfate proteoglycans were detected with molecular sizes of 45,000-100,000 and 120,000-190,000, comprising 14- and 105-kDa core proteins and one or two chondroitin sulfate chains with an average molecular size of 34 kDa. In contrast to the chondroitin sulfate proteoglycans, one major heparan sulfate proteoglycan accounts for most of the remaining 20-30% of the [35S] sulfate-labeled proteoglycans present in the PC12 cells and medium. It has a molecular size of 95,000-170,000, comprising a 65-kDa core protein and two to six 16-kDa heparan sulfate chains. Both the chondroitin sulfate and heparan sulfate proteoglycans also contain O-glycosidically linked oligosaccharides (25-28% of the total oligosaccharides) and predominantly tri- and tetraantennary N-glycosidic oligosaccharides. Proteoglycans produced by the original clone of PC12 cells were compared with those of two other PC12 cell lines (B2 and F3) that differ from the original clone in morphology, adhesive properties, and response to nerve growth factor. Although the F3 cells (a mutant line derived from B2 and reported to lack a cell surface heparan sulfate proteoglycan) do not contain a large molecular size heparan sulfate proteoglycan species, there was no significant difference between the B2 and F3 cells in the percentage of total heparan sulfate released by mild trypsinization, and both the B2 and F3 cells synthesized cell-associated and secreted chondroitin sulfate and heparan sulfate proteoglycans having properties very similar to those of the original PC12 cell line but with a reversed ratio (35:65) of chondroitin sulfate to heparan sulfate.     

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.