Acetylcholine receptor clustering associates with proteoglycan biosynthesis in C2 variant and heterkaryon muscle cells

Several lines of evidence have suggested roles for proteoglycans (PGs) in acetylcholine receptor (AChR) clustering on muscle cells. One line of evidence comes from the correlation between a defect in the biosynthesis of glycosaminoglycans (GAGs), the defining carbohydrates of PGs, and the failure of spontaneous AChR clustering in the S27 cell line, a genetic variant of the C2 muscle cell line. Two approaches were used in the present study to investigate whether GAG and AChR clustering defects are causally linked. First, the formation of AChR clusters was examined in two more variant lines, S11 and S26, also isolated from the C2 muscle cell line on the basis of deficiencies in GAG biosynthesis. S11 and S26, like S27, are also defective in AChR clustering. Ion exchange analysis of the GAGs made by the S11, S26, and S27 lines revealed that the defects in GAG biosynthesis differ between the three lines. Second, heterokaryon myotubes formed between pairs of the GAG defective variants were tested for complementation in both AChR clustering and GAG biosynthesis. AChR clusters were conspicuous on individual heterokaryon myotubes, and GAG biosynthesis was restored to near wild type levels in the heterokaryon cultures. Complementation in GAG biosynthesis corroborates the biochemical data that the relevant mutations in the genetic variants are in different genes and establishes that the defects are not dominant. The consistent correlation between GAG defects and the failure of AChR clustering across three independent genetic variants and the complementary association of GAG biosynthesis with AChR clustering in heterokaryon myotubes argues against a chance association of the two phenotypes and for a causal relationship between PGs and AChR clustering. A prominent chondroitin sulfate peak correlated with AChR clustering in the heterokaryon cultures. This is consistent with earlier results suggesting that chondroitin sulfate in general is required for the spontaneous clustering of AChRs in C2 cultures and further suggests that a particular chondroitin sulfate proteoglycan may be essential for the clustering process. © 1996 John Wiley & Sons, Inc.     

Stimulation of human arterial smooth muscle cell chondroitin sulfate proteoglycan synthesis by transforming growth factor-beta

Summary Human platelet-derived transforming growth factor-beta (TGF-beta) is a cell-type specific promotor of proteoglycan synthesis in human adult arterial cells. Cultured human adult arterial smooth muscle cells synthesized chondroitin sulfate, dermatan sulfate, and heparan sulfate proteoglycans, and the percent composition of these three proteoglycan subclasses varied to some extent from cell strain to cell strain. However, TGF-beta consistently stimulated the synthesis of chondroitin sulfate proteoglycan. Both chondroitin 4- and chondroitin 6-sulfate were stimulated by TGF-beta to the same extent. TGF-beta had no stimulatory effect on either class of [³⁵S]sulfate-labeled proteoglycans which appeared in an approximately 1:1 and 2:1 ratio of heparan sulfate to dermatan sulfate of the medium and cell layers, respectively, of arterial endothelial cells. Human adult arterial endothelial cells synthesized little or no chondroitin sulfate proteoglycan. Pulse-chase labeling revealed that the appearance of smooth muscle cell proteoglycans into the medium over a 36-h period equaled the disappearance of labeled proteoglycans from the cell layer, independent of TGF-beta. Inhibitors of RNA synthesis blocked TGF-beta-stimulated proteoglycan synthesis in the smooth muscle cells. The incorporation of [³⁵S]methionine into chondroitin sulfate proteoglycan core proteins was stimulated by TGF-beta. Taken together, the results presented indicate that TGF-beta stimulates chondroitin sulfate proteoglycan synthesis in human adult arterial smooth muscle cells by promoting the core protein synthesis. Supported in part by grants from the Public Health Service, U.S. Department of Health and Human Services, Washington, DC (CA 37589 and HL 33842), RJR Nabisco, Inc., and Chang Gung Biomedical Research Foundation (CMRP 291).     

Tyrosine O-sulfate ester in proteoglycans

Tyrosine O-sulfate residues were detected in the protein core of sulfated proteoglycans. When cultured skin fibroblasts and arterial smooth muscle cells were incubated in the presence of [35S]sulfate, dermatan sulfate proteoglycan and chondroitin sulfate proteoglycan isolated from the culture medium contained tyrosine [35S]sulfate ester which accounted for 0.03%-0.82% of total 35S radioactivity incorporated into the sulfated proteoglycans. This corresponds to a tyrosine sulfation of every second (fibroblasts) and every 10th (smooth muscle cells) dermatan sulfate proteoglycan molecule. [3H]Tyrosine labeling of fibroblast dermatan sulfate proteoglycan gave a similar stoichiometry. However, the relative proportion of tyrosine [35S]sulfate in proteoglycans from arterial tissue was about 10 times higher than in that from cultured arterial cells. Pulse chase experiments with [35S]sulfate revealed that tyrosine sulfation is a late event in the biosynthesis of dermatan sulfate proteoglycan from fibroblasts and occurs immediately prior to secretion. Cultured skin fibroblasts from a patient with a progeroid variant (Kresse et al. 1987, Am. J. Hum. Gen. 41, 436-453) which exhibit a partial deficiency to synthesize dermatan sulfate proteoglycan were shown to form and to secrete a tyrosine-sulfated but glycosaminoglycan-free protein core, thus confirming a selective and independent [35S]sulfate labeling of the protein core.     

Proliferation-dependent changes of proteoglycan metabolism in arterial smooth muscle cells

Cultured arterial smooth muscle cells synthesize and secrete two types of sulfated proteoglycans designated as proteoglycan A and proteoglycan B. Proteoglycan A has been characterized as chondroitin sulfate-rich, whereas proteoglycan B was found to be dermatan sulfate-rich [Schmidt, A. & Buddecke, E. (1985) Eur. J. Biochem. 153, 260-273]. During the logarithmic growth phase, arterial smooth muscle cells incorporated about 3 times more [35S]sulfate into the total proteoglycans secreted into the culture medium than did non-dividing cells. When arterial smooth muscle cells stopped proliferating the ratio of [35S]proteoglycan A/B increased. No differences were detected in the respective molecular and chemical characteristics of purified proteoglycans A and B isolated from both proliferating and non-dividing cells. Regardless of the growth phase proteoglycan A had a molecular mass of about 280 kDa and contained 8-9 chondroitin sulfate-rich side chains. Proteoglycan B had a molecular mass of about 180 kDa and contained 6-7 dermatan sulfate-rich side chains. The [35S]methionine-labelled protein cores of proteoglycan A and B had a molecular mass of about 48 kDa, but were distinguishable by their specific reactions to monospecific antibodies. Proliferating cells endocytosed proteoglycan B at a rate up to 100% higher than that of non-dividing cells. In all growth phases proteoglycan A was endocytosed at a 10-fold lower rate than proteoglycan B.     

Proteoglycans in primate arteries. III. Characterization of the proteoglycans synthesized by arterial smooth muscle cells in culture

Near confluent monolayers of arterial smooth muscle cells derived from Macaca nemestrina were labeled with Na2[35S]O4 and the newly synthesized proteoglycans present in the culture medium and cell layer were extracted with either 4 M guanidine HCl (dissociative solvent) or 0.5 M guanidine HCl (associative solvent) in the presence of protease inhibitors. The proteoglycans in both compartments were further purified by cesium chloride density gradient ultracentrifugation. Two size classes of proteoglycans were observed in the medium as determined by chromatography on Sepharose CL-2B. The large population (Kav = 0.31) contained predominantly chondroitin sulfate chains with Mr = approximately 40,000. The smaller population (Kav = 0.61) contained dermatan sulfate chains of similar Mr (approximately 40,000). When tested for their ability to aggregate, only proteoglycans in the large-sized population were able to aggregate. A chondroitin sulfate containing proteoglycan with identical properties was isolated from the cell layer. In addition, the cell layer contained a dermatan sulfate component which eluted later on Sepharose CL-2B (Kav = 0.78) than the dermatan sulfate proteoglycan present in the medium. Electron microscopy of the purified proteoglycans revealed a bottlebrush structure containing a central core averaging 140 nm in length with an average of 8 to 10 side projections. The length of the side projections varied but averaged between 70 and 75 nm. Similar bottlebrush structures were observed in the intercellular matrix of the smooth muscle cell cultures after staining with Safranin 0. This culture system provides a model to investigate parameters involved in the regulation of synthesis and degradation of arterial proteoglycans.     

Isolation and partial characterization of high-buoyant-density proteoglycans synthesized in ovo by embryonic chick skeletal muscle and heart

Proteoglycans, a major component of the extracellular matrix, are produced in many tissues. A report from this laboratory describes the proteoglycans synthesized in culture by chick embryonic skeletal muscle myotubes. To extend this study to in vivo conditions, chick embryos were radiolabeled in ovo and the newly synthesized high-buoyant-density proteoglycans from skeletal muscle analyzed. In both leg muscle and pectoral muscle, three major high-density proteoglycans are synthesized. One is small and is similar to the proteoglycans synthesized in culture by muscle fibroblasts. The other two proteoglycans are large. The larger of these shares structural features with the proteoglycan synthesized by skeletal muscle cells in culture. It has large chondroitin sulfate chains (estimated molecular weight of 70,000) with a high proportion of chondroitin 6-sulfate (approximately 90%). The smaller of the two large proteoglycans is distinct (chondroitin sulfate of estimated molecular weight 24,000 and approximately 60% 6-sulfated disaccharides) and is not detected in muscle cultures; evidence suggests it is not made by myoblasts. Whole hearts synthesize proteoglycans with some structural similarities, and also differences, to those made in skeletal muscle. These data indicate that the proteoglycans synthesized in muscle cultures are likewise made in developing muscle in ovo but that another distinct strictly in ovo proteoglycan is also produced.     

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.     

Proteoglycans synthesized by cultured fibroblasts derived from normal and inflamed human gingiva

Summary The in vitro proliferations rates and ptoteoglycans synthesized by adult human gingival fibroblasts derived from six age- and sex-matched donors of healthy and chronically inflamed gingiva were analyzed. Fibroblasts from inflamed gingiva demonstrated a slower growth rate than cells from healthy tissue. The rate of incorporation of [³⁵S]sulfate into cell layer-associated proteoglycans and the release of these macromolecules into the culture medium did not differ appreciably between the two groups of cells. Similarly, no detectable differences in the overall charge of the proteoglycans synthesized by normal and inflamed gingival fibroblasts, as assessed by their elution from DEAE-Sephacel, were noted. However, sepharose CL-4B chromatography revealed that the medium-associated proteoglycans made by the inflamed tissue fibroblasts were depleted in one species of chondroitin sulfate proteoglycans and contained more dermatan sulfate than did control cells. In addition, the intracellular proteoglycan pool was found to be greatly diminished in the inflamed tissue fibroblast cell layers. Glycosaminoglycan analysis of the proteoglycans confirmed these observations. Compared to normal gingival fibroblasts, the inflamed tissue fibroblasts released less heparan sulfate into the medium. Additionally, increased levels of dermatan sulfate and depleted amounts of chondroitin sulfate in the medium of inflamed gingival cells were noted. The observed changes were stable through several transfers in culture and indicate that chronically inflamed tissue may contain fibroblasts mainfesting a heritable phenotype differing from fibroblasts in normal connective tissue. P. Mark Bartold was supported by a C. J. Martin Fellowship for the National Health and Medical Research Council of Australia. This work was also supported by grants DE-03301 and DE-02600 from the National Institutes of Health, Bethesda, MD.     

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.     

Acetylcholine receptor clustering in C2 muscle cells requires chondroitin sulfate

Proteoglycans have been implicated in the clustering of acetylcholine receptors (AChRs) on cultured myotubes and at the neuromuscular junction. We report that the presence of chondroitin sulfate is associated with the ability of cultured myotubes to form spontaneous clusters of AChRs. Three experimental manipulations of wild type C2 cells in culture were found to affect both glycosaminoglycans (GAGs) and AChR clustering in concert. Chlorate was found to have dose-dependent negative effects both on GAG sulfation and on the frequency of AChR clusters. When extracellular calcium was raised from 1.8 to 6.8 mM in cultures of wild-type C2 myotubes, increases were observed both in the level of cell layer-associated chondroitin sulfate and in the frequency of AChR clusters. Culture of wild-type C2 myotubes in the presence of chondroitinase ABC eliminated cell layer-associated chondroitin sulfate while leaving heparan sulfate intact and simultaneously prevented the formation of AChR clusters. Treatment with either chlorate or chondroitinase inhibited AChR clustering only if begun prior to the spontaneous formation of clusters. We propose that chondroitin sulfate plays an essential role in the initiation of AChR clustering and in the early events of synapse formation on muscle. © 1995 John Wiley & Sons, Inc.     

Transforming growth factor beta regulates the expression and structure of extracellular matrix chondroitin/dermatan sulfate proteoglycans

Transforming growth factor beta (TGF-beta) increases up to 20-fold the expression of various forms of chondroitin/dermatan sulfate proteoglycan, the major type of sulfated proteoglycan present in the extracellular matrix and culture medium of various human, rodent, and mink cell types including kidney and lung fibroblasts, lung epithelial cells, preadipocytes, and skeletal muscle myoblasts. TGF-beta regulates the level and molecular size of these proteoglycans by acting simultaneously at two levels: it elevates the biosynthetic rate of the 45-kDa proteoglycan core protein in a cycloheximide- and actinomycin D-sensitive manner, and it induces an increase in the molecular mass of the glycosaminoglycan chains. These cellular responses correlate with occupancy of type III TGF-beta receptors by TGF-beta 1 and TGF-beta 2 and are not induced by other growth factors tested. The parameters of this effect of TGF-beta in kidney fibroblasts and myoblasts are ED50 = 5-10 pM TGF-beta 1 or TGF-beta 2, and t 1/2 = 6-8 h. These results identify the chondroitin/dermatan sulfate proteoglycans as a major component of mammalian mesenchymal and epithelial extracellular matrices whose expression and structure are regulated by TGF-beta.     

Increased activity of chondroitin sulfate-synthesizing enzymes during proliferation of arterial smooth muscle cells

Cultured arterial smooth muscle cells incorporate [35S]sulfate into the extracellular chondroitin sulfate/dermatan sulfate containing proteoglycans at a higher rate in the phase of logarithmic growth than do non-dividing cells. The cell growth-dependent decrease in 35S incorporation with increasing cell density is accompanied by a decrease in the activity of chondroitin sulfate-synthesizing enzymes. The specific activity of xylosyl transferase, N-acetylgalactosaminyl transferase I and chondroitin sulfotransferase declines as the cells proceed from low to high densities. The corresponding correlation coefficients are 0.86, 0.91 and 0.89. The ratio of C-6OH/C-4OH sulfation of chondroitin shows a cell proliferation-dependent decrease indicating an inverse correlation of chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase activity. The observed changes in the expression of enzyme activities are thought to have some implications in the pathogenesis of arteriosclerosis, the initial stages of which are characterized by proliferation of arterial smooth muscle 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.     

The proteoglycan decorin is synthesized and secreted by differentiated myotubes

Proteoglycans (PGs) are important components of the skeletal muscle extracellular matrix (ECM). Skeletal muscles are composed of muscle fibers and mononucleated cells. The latter are known to synthesize and secrete several PGs. Rat skeletal muscle ECM contains a chondrotin/dermatan sulfate PG which was immunoprecipitated by antibodies against rat decorin. The synthesis and secretion of PGs by a mouse cell line was analyzed during in vitro differentiation. PGs were characterized by biochemical and immunological techniques including immunocytolocalization experiments. At least three different PGs are synthesized and secreted by differentiated myotubes: a 220 to 460 kDa heparan sulfate, a 250 to 310 kDa chondroitin/dermatan sulfate, and a 75 to 130 kDa chondroitin/dermatan sulfate. This latter PG was specifically immunoprecipitated with antibodies against rat fibroblast decorin. Indirect immunocytolocalization analysis revealed that decorin was localized inside the cells, with a strong reaction around the nuclei. During differentiation the relative proportions of some PGs changed. Thus, a decrease in the relative proportion of the heparan sulfate PG was observed, whereas a significant increase in the relative proportion of decorin was detected. No change in the large chondroitin/dermatan PG was seen during the differentiation process. The possible cell sources of decorin found in rat skeletal muscle ECM are discussed.     

Major chondroitin sulfate proteoglycans identified in L6J1 myoblast culture

The major proteoglycans from L6J1 rat myoblast culture were identified. The proteoglycans were isolated from different constituents of cell culture: culture medium, extracellular matrix (ECM), and myoblasts. To identify their core proteins, the proteoglycans were treated with enzymes specifically digesting chondroitin/dermatan sulfates or chondroitin sulfates. Subsequent electrophoresis and mass spectrometry revealed versican, collagen XII, and inter-α-trypsin inhibitor classified as chondroitin sulfate proteoglycans and biglycan known to be chondroitin/dermatan sulfate proteoglycan. Versican was identified in ECM and the other proteoglycans in the culture medium. Such difference in localization is likely to be a consequence of different biological functions. Versican, collagen XII, and biglycan are synthesized by myoblasts and inter-α-trypsin inhibitor originates from fetal bovine serum (a culture medium component).     

Dog mastocytoma proteoglycans: occurrence of heparin and oversulfated chondroitin sulfates, containing trisulfated disaccharides, in three cell lines

The cell-associated proteoglycans synthesized by three dog mastocytoma cell lines were isolated and their structural features compared. The lines were propagated as subcutaneous tumors in athymic mice for over 25 generations. In primary cell culture, all three lines incorporated [35S]sulfate into high molecular weight proteoglycans which were heterogeneous in size and glycosaminoglycan content. Two lines, BR and G, synthesized both a heparin proteoglycan (HPG) and a chondroitin sulfate proteoglycan (ChSPG) in different proportions. The third line, C2, synthesized predominantly a ChSPG with little or no detectable heparin. Gel filtration of the 35S-labeled HPG and ChSPG from the BR line on Sepharose CL-4B in dissociative conditions (4 M guanidine, Triton X-100) yielded a major polydisperse peak (Kav = 0.22) accounting for 70% of 35S activity. Under aggregating conditions (0.1 M sodium acetate) on Sepharose CL-4B, the BR proteoglycans eluted in the excluded volume. Proteoglycans from lines G and C2 also eluted in the void volume under nondissociative conditions, however the C2 line yielded additional fractions of smaller hydrodynamic size (Kav = 0.81) suggesting the presence of intracellular proteoglycan cleavage products or incompletely processed proteoglycans. As assessed by dissociative chromatography on Sepharose CL-4B, proteoglycans from the BR line were resistant to proteinase cleavage under conditions which degraded a rat chondrosarcoma proteoglycan. For all lines, glycosaminoglycans released by pronase/alkaline-borohydride had molecular weights ranging from 20,000 to 50,000 on gel filtration. For line BR, 75% of 35S-labeled glycosaminoglycans were degraded to oligosaccharides by nitrous acid, and the remaining 25% were degraded by chondroitinase ABC. Corresponding percentages for line G were 89% and 11%, and for line C2, 2% and 98%. Paper chromatography of the chondroitinase digestion products from lines BR and C2 showed products corresponding to unsaturated standards delta Di-diSB and delta Di-diSE, derived from the disaccharides IdoUA-2-SO4----GalNAc-4-SO4 and GlcUA----GalNAc-4,6-diSO4 respectively, in addition to smaller amounts of monosulfated disaccharides. Glycans from lines C2 and BR contained small quantities of a trisulfated disaccharide which was degraded to delta Di-diSB upon incubation with chondro-6-sulfatase. The results demonstrate the simultaneous presence of heparin and polysulfated chondroitin sulfate in dog mast cells of clonal origin.     

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.     

Glycosaminoglycan variants in the C2 muscle cell line

Using a replica technique, we have isolated and characterized five genetic variants of the C2 mouse muscle cell line that are defective in incorporation of radiolabeled sulfate into glycosaminoglycans (GAGs). The variants incorporate free sulfate into GAGs at 5-20% of wild-type levels. None of the variants is defective in sulfate transport across the cell membrane, and in no case could the deficit in incorporation of sulfate be reversed by addition of an artificial initiator of GAG biosynthesis, p-nitrophenyl beta-D-xyloside. Analysis of the incorporation of [3H]glucosamine into GAGs by the variants revealed three different patterns: one variant incorporated [3H]glucosamine at the wild-type level; one, S27, at a severely reduced level; and three at intermediate levels. Four of the five variants showed marked deficits in their ability to differentiate and fuse. The remaining variant, S27, formed multinucleated myotubes and expressed acetylcholine receptor with a normal time course. Differentiation of the first four variants could not be restored by addition of exogenous GAGs or extracellular matrix. Because of the important roles that GAGs and proteoglycans are thought to play in the differentiation of muscle, these genetic variants should serve as useful tools in functional analyses of these molecules.     

Proteoglycan synthesis by normal and neoplastic human transitional epithelial cells

Metabolically 35S-labeled proteoglycans were isolated from cell-associated matrices and media of confluent cultures of human normal transitional epithelial cells and HCV-29T transitional carcinoma cells. On Sepharose CL-4B columns, the cell-associated proteoglycans synthesized from both cell types separated into three identical size classes, termed CI, CII, and CIII. Normal epithelial cell C-fractions eluted in a 22:34:45 proportion and contained 64%, 64%, and 72% heparan sulfate, whereas corresponding HCV-29T fractions eluted in a 29:11:60 proportion, and contained 91%, 77%, and 70% heparan sulfate, respectively. Medium proteoglycans from normal cells separated into two size classes in a proportion of 6:94 and were composed of 35% and 50% heparan sulfate. HCV-29T medium contained only one size class of proteoglycans consisting of 23% heparan sulfate. The remaining percentages were accounted for by chondroitin/dermatan sulfate. On isopycnic CsCl gradients, proteoglycan fractions from normal cells had buoyant densities that were higher than the corresponding fractions from HCV-29T cells. DEAE-Sephacel chromatography showed that cell and medium associated heparan sulfate from HCV-29T cells was consistently of lower charge density (undersulfated) than that from normal epithelial cells. In contrast, the chondroitin/dermatan sulfate of HCV-29T was of a charge density similar to that of normal cells. These as well as other structural and compositional differences in the proteoglycan may account, at least in part, for the altered behavioral traits of highly invasive carcinoma cells.