Co-sedimentation of chondroitin sulfate A glycosaminoglycans and proteoglycans with the cytolytic secretory granules of rat large granular lymphocyte (LGL) tumor cells, and identification of a mRNA in normal and transformed LGL that encodes proteoglycans

Rat large granular lymphocyte (LGL) tumor cell lines were analyzed for the presence of proteoglycans and glycosaminoglycans in their cytolytic secretory granules. When isolated rat LGL tumor cells were incubated in vitro for 1 to 3 hr with [35S]sulfate, and the 35S-labeled macromolecules were purified by density-gradient centrifugation, they filtered on Sepharose CL-4B columns predominantly as approximately 500,000 m.w. macromolecules. After 19 hr of incubation with [35S]sulfate, however, an 85,000 m.w. species predominated. Pulse-chase experiments revealed that the larger macromolecules were proteoglycans that with time were processed to glycosaminoglycan-sized macromolecules. As assessed by their susceptibility to chemical and enzymatic degradation and by high pressure liquid chromatography of the chondroitinase ABC-generated unsaturated disaccharides, the cell-associated rat LGL tumor cell proteoglycans bore almost exclusively chondroitin sulfate A glycosaminoglycans. Northern blot analysis using a gene-specific probe revealed that both normal peripheral blood and transformed rat LGL expressed the same approximately 1.3-kb mRNA that encodes the peptide core of the proteoglycans in the secretory granules of rat and mouse mast cells. In vivo radiolabeling of rat LGL tumor cells and isolation of their intact granules after nitrogen cavitation and density sedimentation established that glycosaminoglycans compartmentalized with cytolytic activity. Thus these negatively charged macromolecules may play a role in the regulation of the packaging and delivery of the cytolysins and basically charged serine proteases that have been identified in the cytolytic secretory granules of LGL.     

Biochemical and morphological characterization of basophilic leukocytes from two patients with myelogenous leukemia

Basophilic leukocytes from two patients with myelogenous leukemia were enriched to a purity of 10 to 45% by density gradient centrifugation. Ultrastructurally, these basophilic leukocytes contained segmented nuclei and granules with reticular patterns resembling those of normal basophils, and other granules with scroll and grating patterns resembling those of normal connective tissue mast cells. The 35S-labeled macromolecules isolated from these cells were approximately 140,000 m.w. Pronase-resistant proteoglycans bearing approximately 15,000 m.w. glycosaminoglycans. On incubation with chondroitinase ABC, nitrous acid, and heparinase, the 35S-labeled proteoglycans were degraded 50 to 84%, 16 to 43%, and 8 to 37%, respectively, indicating the presence of both chondroitin sulfate and heparin. As assessed by high performance liquid chromatography, the 35S-labeled chondroitin sulfate disaccharides liberated by chondroitinase ABC treatment were approximately 95% monosulfated chondroitin sulfate A and approximately 5% disulfated chondroitin sulfate E. The presence of heparin was confirmed by two-dimensional cellulose acetate electrophoresis of the 35S-labeled glycosaminoglycans. Cell preparations, enriched to 75% basophilic leukocytes by sorting for IgE+ cells, also synthesized 35S-labeled proteoglycans containing chondroitin sulfate and heparin. In one experiment, treatment of the cells with 1 microM calcium ionophore A23187 resulted in a 12% net release of both chondroitin sulfate and heparin containing 35S-labeled proteoglycans, a 57% net release of histamine, and the de novo generation of 8, 8, and 0.16 ng of immunoreactive equivalents of prostaglandin D2, leukotriene C4, and leukotriene B4, respectively, per 10(6) cells. Because only mast cells have been found to contain Pronase-resistant heparin proteoglycans, to generate PGD2 on cell activation, and to contain granules with scroll and grating patterns, these findings indicate that in some patients with myelogenous leukemia there are basophilic cells that possess properties of tissue mast cells.     

Immortalization of murine connective tissue-type mast cells at multiple stages of their differentiation by coculture of splenocytes with fibroblasts that produce Kirsten sarcoma virus

Mature connective tissue mast cells (CTMC) have not been previously available as a cell line from any species. Here we describe 15 novel mast cell lines (KiSV-MC) that were derived by coculturing murine splenocytes with fibroblasts that produce a Ki-ras-containing murine sarcoma virus. Some of the KiSV-MC lines are similar to CTMC in that they synthesize predominantly heparin proteoglycans, and contain up to 35 micrograms of histamine and 2.2 units of carboxypeptidase A/10(6) cells in secretory granules which stain red with Safranin. Other cell lines display phenotypic characteristics intermediate to CTMC and mucosal-like mast cells in being predominantly Safranin-, having lower amounts of histamine and carboxypeptidase A, and in synthesizing chondroitin sulfate E proteoglycans in preference to heparin proteoglycans. When the individual KiSV-MC lines were compared, a linear relationship was found between the number of Safranin+ granules, the cellular contents of histamine and carboxypeptidase A, and the biosynthesis of heparin relative to chondroitin sulfate E proteoglycans. Upon sensitization with monoclonal IgE and exposure to hapten-specific antigen, the cells exocytose the contents of their secretory granules. Thus, these immortalized cells provide the first source of CTMC-like lines for chemical and functional analysis and illustrate that murine mast cells can express a continuum of phenotypes.     

Mast cell exocytosis: evidence that granule proteoglycan processing is not coupled to degranulation

It has been hypothesized that the dissolution of mast cell granules at the time of degranulation results from proteoglycan cleavage coupled to exocytosis. To address this hypothesis, we studied granule proteoglycan before and after exocytosis in dog mastocytoma cells, which solubilize granule contents during exocytosis. 35S-labeled proteoglycans were extracted from unstimulated whole cells and cell degranulation supernatant. Sequential anion-exchange and gel filtration chromatography, followed by specific glycosaminoglycan digestion, identified chondroitin sulfate and heparin glycosaminoglycan and proteoglycan in unstimulated cells and degranulated material alike. Glycosaminoglycan type and charge density in degranulation supernatant were unchanged compared with unstimulated cells. There was no decrease in proteoglycan size with cell activation and exocytosis. Thus, granule release and solubilization does not appear to require exocytosis-coupled degradation of granule proteoglycans. Release in association with high-m.w. proteoglycans may serve to limit rates of diffusion and activity of proteases and other mast cell mediators.     

Carboxypeptidase A in mouse mast cells. Identification, characterization, and use as a differentiation marker

By using a conventional spectrophotometric assay with hippuryl-L-phenylalanine as the substrate, 10(6) BALB/c mouse serosal mast cells possessed 1.5 +/- 0.43 U (mean +/- SE, n = 5, range = 0.48 to 2.5) of carboxypeptidase A activity, while T cell factor-dependent, mouse bone marrow-derived mast cells (BMMC) had barely detectable levels of 0.01 +/- 0.001 U/10(6) cells (mean +/- SE, n = 3). In order to characterize the carboxypeptidase A present in the BMMC, a sensitive assay was developed that used angiotensin I as the substrate and reverse phase-high performance liquid chromatography to separate and quantify production of the cleavage product des-leu-angiotensin I. Using this assay, mouse BMMC carboxypeptidase A had a neutral to basic pH optimum and hydrolyzed angiotensin I with a Km of 0.78 mM. The antigen-induced net percent release of carboxypeptidase A from IgE-sensitized BMMC was proportional to that of the secretory granule component beta-hexosaminidase which indicates a secretory granule location for the exopeptidase. As defined by exclusion during Sepharose CL-2B chromatography, carboxypeptidase A was exocytosed as a greater than 1 X 10(7) m.w. complex bound to proteoglycans. Because BMMC cocultured with mouse skin-derived 3T3 fibroblasts are known to undergo an increase in histamine content and biosynthesis of 35S-labeled heparin proteoglycans, carboxypeptidase A activity was measured during BMMC/fibroblast coculture for 0 to 28 days. The carboxypeptidase A activity increased progressively during 28 days of co-culture from 0.004 +/- 0.002 U/10(6) starting BMMC (mean +/- SE, n = 3) to 0.36 +/- 0.10 U/10(6) co-cultured mast cells. These findings indicate that carboxypeptidase A, a neutral protease, is exocytosed from the secretory granules of mouse mast cells bound to proteoglycan and is increased during the in vitro differentiation of mouse BMMC from mucosal-like mast cells to serosal-like mast cells.     

Presence of sulfated proteoglycans in prolactin secretory granules isolated from the rat pituitary gland.

The composition of the segregated content of rat prolactin granules was investigated taking advantage of the fact that these organelles, isolated as a pure fraction, retain their structural organization after solubilization of their limiting membrane by mild detergent treatment. We found that these membraneless granules contain not only the hormone, but also a number of minor macromolecular components including sulfated glycosaminoglycans, which are labeled when pituitary slices are incubated in vitro with [35S] sulfate. In order to characterize the latter components, the isolated radioactive granules were solubilized (by treatment with either a high ionic strength solution orNaOH) and 35S-labeled acidic glycosaminoglycans precipitated by complexing with cetylpirydinium chloride. A high degree of heterogeneity was observed when the ensuing precipitates were analyzed by cellulose acetate electrophoresis: different components were found to co-migrate with authentic heparin and chondroitin sulfate A and C standards. Another component, which accounts for approx. 50% of the glycosaminoglycan-bound radioactivity, might be heparin sulfate. These acidic glycosaminoglycans are linked to peptide moieties to form proteoglycans.     

Intestinal mucosal mast cells from rats infected with Nippostrongylus brasiliensis contain protease-resistant chondroitin sulfate di-B proteoglycans

Rats infected with the helminth Nippostrongylus brasiliensis were injected i.p. with 2 mCi of [35S] sulfate on days 13, 15, 17, and 19 after infection. The intestines were removed from animals on day 20 or 21 after infection, the intestinal cells were obtained by collagenase treatment and mechanical dispersion of the tissue, and the 35S-labeled mucosal mast cells (MMC) were enriched to 60 to 65% purity by Percoll centrifugation. The cell-associated 35S-labeled proteoglycans were extracted from the MMC-enriched cell preparation by the addition of detergent and 4 M guanidine HCl and were partially purified by density gradient centrifugation. The isolated proteoglycans were of approximately 150,000 m.w., were resistant to pronase degradation, and contained highly sulfated chondroitin sulfate side chains. Analysis by high-performance liquid chromatography of chondroitinase ABC-treated 35S-labeled proteoglycans from these rat MMC revealed that the chondroitin sulfate chains consisted predominantly of disaccharides with the disulfated di-B structure (IdUA-2SO4----GalNAc-4SO4) and disaccharides with the monosulfated A structure (G1cUA----GalNAc-4SO4). The ratio of disaccharides of the di-B to A structure ranged from 0.4 to 1.6 in three experiments. Small amounts of chondroitin sulfate E disaccharides (GlcUA----GalNAc-4,6-diSO4) were also detected in the chondroitinase ABC digests of the purified rat MMC proteoglycans, but no nitrous acid-susceptible heparin/heparan sulfate glycosaminoglycans were detected. The presence in normal mammalian cells of chondroitin sulfate proteoglycans that contain such a high percentage of the unusual disulfated di-B disaccharide has not been previously reported. The rat intestinal MMC proteoglycans are the first chondroitin sulfate proteoglycans that have been isolated from an enriched population of normal mast cells. They are homologous to the chondroitin sulfate-rich proteoglycans of the transformed rat basophilic leukemia-1 cell and the cultured interleukin 3-dependent mouse bone marrow-derived mast cell, in that these chondroitin sulfate proteoglycans as well as rat serosal mast cell heparin proteoglycans are all highly sulfated, protease-resistant proteoglycans.     

Characterization of a human eosinophil proteoglycan, and augmentation of its biosynthesis and size by interleukin 3, interleukin 5, and granulocyte/macrophage colony stimulating factor

Human eosinophils were cultured for up to 7 days in enriched medium in the absence or presence of recombinant human interleukin (IL) 3, mouse IL 5, or recombinant human granulocyte/macrophage colony stimulating factor (GM-CSF) and then were radiolabeled with [35S]sulfate to characterize their cell-associated proteoglycans. Freshly isolated eosinophils that were not exposed to any of these cytokines synthesized Mr approximately 80,000 Pronase-resistant 35S-labeled proteoglycans which contained Mr approximately 80,000 glycosaminoglycans. RNA blot analysis of total eosinophil RNA, probed with a cDNA that encodes a proteoglycan peptide core of the promyelocytic leukemia HL-60 cell, revealed that the mRNA which encodes the analogous molecule in eosinophils was approximately 1.3 kilobases, like that in HL-60 cells. When eosinophils were cultured for 1 day or longer in the presence of 10 pM IL 3, 1 pM IL 5, or 10 pM GM-CSF, the rates of [35S]sulfate incorporation were increased approximately 2-fold, and the cells synthesized Mr approximately 300,000 Pronase-resistant 35S-labeled proteoglycans which contained Mr approximately 30,000 35S-labeled glycosaminoglycans. Approximately 93% of the 35S-labeled glycosaminoglycans bound to the proteoglycans synthesized by noncytokine- and cytokine-treated eosinophils were susceptible to degradation by chondroitinase ABC. As assessed by high performance liquid chromatography, 6-16% of these chondroitinase ABC-generated 35S-labeled disaccharides were disulfated disaccharides derived from chondroitin sulfate E; the remainder were monosulfated disaccharides derived from chondroitin sulfate A. Utilizing GM-CSF as a model of the cytokines, it was demonstrated that the GM-CSF-treated cells synthesized larger glycosaminoglycans onto beta-D-xyloside than the noncytokine-treated cells. Thus, IL 3, IL 5, and GM-CSF induce human eosinophils to augment proteoglycan biosynthesis by increasing the size of the newly synthesized proteoglycans and their individual chondroitin sulfate chains.     

Complexes of heparin proteoglycans, chondroitin sulfate E proteoglycans, and [3H]diisopropyl fluorophosphate-binding proteins are exocytosed from activated mouse bone marrow-derived mast cells

The predominant [3H]diisopropyl fluorophosphate (DFP)-binding proteins that are released from the secretory granules of activated mouse bone marrow-derived mast cells (BMMC) are demonstrated to have an isoelectric point of approximately 9.1 and to be complexed to proteoglycans. Upon Sepharose CL-2B chromatography of the supernatants of calcium ionophore-activated BMMC, 67-78% of the total exocytosed [3H]DFP-binding proteins co-eluted in the excluded volume of the column as a greater than 1 X 10(7) Mr complex bound to 4-7% of the total exocytosed proteoglycans. The remainder of the exocytosed proteoglycans, which filtered in the included volume of the gel filtration column with a Kav of 0.66, contained chondroitin sulfate E glycosaminoglycans. After dissociation of the large Mr complexes of [3H]DFP-binding proteins-proteoglycans with 5 M NaCl and removal of the proteins via phenyl-Sepharose chromatography, the proteoglycans filtered from the Sepharose CL-2B column as a single peak with a Kav of 0.66. The susceptibility of 24-59% and 36-76% of the glycosaminoglycans in the large Mr complex to degradation by nitrous acid and chondroitinase ABC, respectively, indicated the presence of proteoglycans that contained heparin and chondroitin sulfate glycosaminoglycans. Disaccharide analysis revealed that the chondroitin sulfate in the high Mr complex was chondroitin sulfate E. Following chondroitinase ABC treatment of the large Mr complex, the residual heparin proteoglycans filtered on Sepharose CL-4B under dissociative conditions with the same Kav as the original, untreated proteoglycans. Thus, the protein-proteoglycan complexes that are exocytosed from activated mouse BMMC contain approximately equal amounts of proteoglycans of comparable size that bear either predominantly heparin or predominantly chondroitin sulfate E glycosaminoglycans. The demonstration of these secreted complexes indicates that the intragranular protease-resistant heparin and chondroitin sulfate E proteoglycans in the T cell factor-dependent BMMC bind serine proteases throughout the activation-secretion response.     

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.     

The isolation, identification and characterization of sulfated glycosaminoglycans synthesized in vitro by human eosinophils

Human eosinophils were purified to greater than 92% using 16-30% metrizamide gradients, and these cells cultured for up to 72 h in vitro to label sulfated glycosaminoglycans. Over 90% of the sulfated glycosaminoglycan-containing material was extracted in 4 M guanidine HCl and had a hydrodynamic size similar to a glycosaminoglycan marker with an approximate average molecular weight of 60,000. Treatment of this salt-extracted 35S-labeled glycosaminoglycan-containing material with 0.5 M NaOH resulted in a change in mass to approx. 20,000 daltons, suggesting that the larger molecules were proteoglycans with side chains with an approximate molecular weight of 20,000. These salt extracted presumptive 35S-labeled proteoglycans were protease insensitive and behaved in a highly charged fashion on DEAE-cellulose. The composition of 35S-labeled glycosaminoglycans from human eosinophils as identified using selected polysaccharides was 70-81% chondroitin 4-sulfate, 9-12% chondroitin 6-sulfate, and 5-12% dermatan sulfate. The predominance of chondroitin 4-sulfate in human eosinophils is similar to the predominance of chondroitin 4-sulfate in human neutrophils and human platelets.     

Identification of chondroitin sulfate E in human lung mast cells

Human lung mast cells (HLMC) enriched up to 99% purity by counter current elutriation and density gradient centrifugation were labeled with 35S-sulfate to determine cell-associated proteoglycans. The 35S-labeled proteoglycans were extracted by the addition of detergent and 4 M guanidine-HCl, and separated from unincorporated precursor by Sephadex G-50 chromatography. 35S-Proteoglycans chromatographed over Sepharose 4B with a Kav of 0.48. 35S-Glycosaminoglycans separated from the parent 35S-proteoglycans by beta-elimination and chromatographed over Sepharose 4B with a Kav of 0.63. Characterization of 35S-proteoglycans by chondroitin ABC lyase treatment revealed approximately 36% of the proteoglycan to be composed of chondroitin sulfates. Analysis by HPLC of component disaccharides liberated by chondroitin ABC lyase using an amino-cyano-substituted silica column indicated that the chondroitin sulfates consisted of the monosulfated A disaccharide (GlcUA----GaINAc4SO4) (75%) and the over-sulfated E disaccharide (GlcUA----GaINAc4,6-diSO4) (25%). Nitrous acid/heparinase-susceptible heparin proteoglycans accounted for approximately 62% of the total 35S-proteoglycans present in the HLMC. Proteoglycans remaining after exposure of the original proteoglycan extract to either heparinase or chondroitin ABC lyase were of similar size, suggesting that the majority of heparin and chondroitin sulfate glycosaminoglycans were on separate protein cores. Proteoglycans extracted from HLMC were protease insensitive. Hence, in addition to heparin proteoglycans, HLMC synthesize a hitherto unrecognized quantity of chondroitin sulfate E proteoglycans.     

Mast Cell Proteoglycans

Mast cells are versatile effector cells of the immune system, contributing to both innate and adaptive immunity toward pathogens but also having profound detrimental activities in the context of inflammatory disease. A hallmark morphological feature of mast cells is their large content of cytoplasmic secretory granules, filled with numerous secretory compounds, including highly negatively charged heparin or chondroitin sulfate proteoglycans of serglycin type. These anionic proteoglycans provide the basis for the strong metachromatic staining properties of mast cells seen when applying various cationic dyes. Functionally, the mast cell proteoglycans have been shown to have an essential role in promoting the storage of other granule-contained compounds, including bioactive monoamines and different mast cell-specific proteases. Moreover, granule proteoglycans have been shown to regulate the enzymatic activities of mast cell proteases and to promote apoptosis. Here, the current knowledge of mast cell proteoglycans is reviewed.     

Alteration of rat liver proteoglycans during regeneration.

Sepharose CL-6B column chromatography of crude extracts from the slices of regenerating rat livers after partial hepatectomy and sham-operated controls labeled with [35S]sulfuric acid revealed an enhancement of [35S]sulfate incorporation into proteoglycan fractions during regeneration. The 35S-labeled proteoglycans contained heparan sulfate (more than 80% of the total) and chondroitin/dermatan sulfate. The 35S-incorporation into both glycosaminoglycans increased to maxima 3-5 days after partial hepatectomy and decreased thereafter toward the respective control levels. When [35S]sulfuric acid was replaced by [3H]glucosamine, similar results were obtained. These results suggest that the maximal stimulation of proteoglycan synthesis in regenerating rat liver follows the maximal mitosis of hepatic cells 1-2 days after partial hepatectomy. The 35S-labeled proteoglycans from regenerating liver 3 days after partial hepatectomy and control were analyzed further. They were similar in chromatographic behavior on a gel filtration or an anion-exchange column and in glycosaminoglycan composition. Their glycosaminoglycans were indistinguishable in electrophoretic mobility. However, these proteoglycans were slightly but significantly different in their affinity to octyl-Sepharose and in the molecular-weight distribution of their glycosaminoglycans.     

Intermediates in the constitutive and regulated secretory pathways released in vitro from semi-intact cells

Regulated secretory cells have two pathways that transport secreted proteins from the Golgi complex to the cell surface. To identify carrier vesicles involved in regulated and constitutive secretion, PC12 pheochromocytoma cells were labeled with [35S]sulfate to identify markers for the two secretory pathways, then mechanically permeabilized and incubated in vitro. Small constitutive secretory vesicles, containing mostly sulfated proteoglycans, accumulated during an in vitro incubation with ATP. In the presence of GTP gamma S, the constitutive vesicles became significantly more dense, suggesting that a coated intermediate was stabilized. Larger immature regulated secretory granules, enriched in sulfated secretogranin II, also escaped from the permeabilized cells in vitro. During granule maturation, their density increased and the amount of cofractionating proteoglycans diminished. The data suggest that sorting continues during secretory granule maturation.     

The expression of proteoglycans in phorbol ester induced U-937 cells

U-937 monoblastic cells were differentiated into macrophage-like cells in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA). Control cells and differentiated cells were labeled with³⁵S-sulfate and were both found to produce exclusively chondroitin sulfate proteoglycan. No differences in glycosaminoglycan structure or macromolecular properties of the proteoglycans produced in the two different cell systems could be observed. However, the differentiated cells were found to have a lower capacity for chondroitin sulfate proteoglycan synthesis, both under ordinary experimental conditions, and when exposed to stimulators of glycosaminoglycan biosynthesis such as -d-xylosides.Abbreviations SDS sodium dodecyl sulfate - TPA 12-O-tetradecanoylphorbol-13-acetate - PG proteoglycan - GAG glycoaminoglycan - CS chondroitin sulfate - CSPG chondroitin sulfate proteoglycan - NASDAE naphthol AS-D acetate esterase     

Glycosaminoglycans synthesized by cultured bovine corneal endothelial cells

Bovine corneal endothelial (BCE) cells seeded and grown on plastic dishes were labeled with 35S-sulfate or 3H-glucosamine for 48 h at various phases of growth of the cultures. Newly synthesized proteoglycans were isolated from the culture medium and from the extracellular matrix (ECM) produced by the BCE cells, and the glycosaminoglycan (GAG) component of the proteoglycans was analyzed. Cells actively proliferating on plastic surfaces secreted an ECM that contained heparan sulfate as the major 35S-labeled GAG (86%) and dermatan sulfate as a minor component (13%). Upon reaching confluence, the BCE cells incorporated 35S-labeled chondroitin sulfate (20%), as well as heparan sulfate (66%) and dermatan sulfate (14%), into the EC. Seven-day postconfluent cells incorporated newly synthesized heparan sulfate and dermatan sulfate into the matrix in approximately equal proportions. Dermatan sulfate was the main 35S-labeled GAG (60-65%) in the medium of both confluent and postconfluent cultures. 35S-Labeled chondroitin sulfate (20-25%) and heparan sulfate (15%) were also secreted into the culture medium. The type of GAG incorporated into newly synthesized ECM was affected when BCE cells were seeded onto ECM-coated dishes instead of plastic. BCE cells actively proliferating on ECM-coated dishes incorporated newly synthesized heparan sulfate and dermatan sulfate into the ECM in a ratio that was very similar to the ratio of these GAGs in the underlying ECM. Addition of mitogens such as fibroblast growth factor (FGF) to the culture medium altered the type of GAG synthesized and incorporated into the ECM by BCE cells seeded onto ECM-coated dishes if the cells were actively growing, but had no effect on postconfluent cultures.     

In vivo turnover of the basement membrane and other heparan sulfate proteoglycans of rat glomerulus

The metabolic turnover of rat glomerular proteoglycans in vivo was investigated. Newly synthesized proteoglycans were labeled during a 7-h period after injecting sodium [35S]sulfate intraperitoneally. At the end of the labeling period a chase dose of sodium sulfate was given. Subsequently at defined times (0-163 h) the kidneys were perfused in situ with 0.01% cetylpyridinium chloride in phosphate-buffered saline to maximize the recovery of 35S-proteoglycans. Glomeruli were isolated from the renal cortex and analyzed for 35S-proteoglycans by autoradiographic, biochemical, and immunochemical methods. Grain counting of autoradiographs revealed a complex turnover pattern of 35S-labeled macromolecules, commencing with a rapid phase followed by a slower phase. Biochemical analysis confirmed the biphasic pattern and showed that the total population of [35S]heparan sulfate proteoglycans had a metabolic half-life (t1/2) of 20 and 60 h in the early and late phases, respectively. Heparan sulfate proteoglycans accounted for 80% of total 35S-proteoglycans, the remainder being chondroitin/dermatan sulfate proteoglycans. Whole glomeruli were extracted with 4% 3-[(cholamidopropyl)dimethy-lammonio]-1-propanesulfonate-4 M guanidine hydrochloride, a procedure which solubilized greater than 95% of the 35S-labeled macromolecules. Of these 11-13% was immunoprecipitated by an antiserum against heparan sulfate proteoglycan which, in immunolocalization experiments, showed specificity for staining the basement membrane of rat glomeruli. Autoradiographic analysis showed that 18% of total radioactivity present at the end of the labeling period was associated with the glomerular basement membrane. The glomerular basement membrane [35S]heparan sulfate proteoglycans, identified by immunoprecipitation, have a very rapid turnover with an initial phase, t1/2 = 5 h, and a later phase t1/2 = 20 h.     

Characterization of the sulfated glycosaminoglycan on the surface and in the storage granules of rabbit platelets.

Rabbit platelets were labeled in vivo with 35S for characterization of platelet sulfated glycosaminoglycan. When rabbit platelets were aggregated by ADP, sulfated proteoglycan was lost from the platelet surface although no release of granule contents occurred. The sulfated proteoglycan contained in the granules of platelets pretreated with ADP was subsequently released by treatment with thrombin. The 35S-labeled proteoglycan from both sources was isolated by gel filtration and the glycosaminoglycan portion of the proteoglycan was characterized as chondroitin 4-sulfate by examining the products of digestion with hyaluronidase, chondroitinase AC and ABC, and chondro-4- and 6-sulfatases; by identification of the hexosamine as N-acetylgalactosamine; by determination of a 1 : 1 : 1 molar ratio of N-acetylgalactosamine, uronic acid and inorganic sulfate; and by cetylpyridinium chloride cellulose chromatography. In these studies, the use of 35S-labeled proteoglycan made possible detection and quantification of much smaller amounts of material than would be possible with unlabeled material. Chondroitin 4-sulfate was the only sulfated glycosaminoglycan identified in the proteoglycan lost from the platelet surface during ADP-induced aggregation and in the proteoglycan released from the granules when the platelets were exposed to thrombin.     

Partial characterization of proteoglycans synthesized by human glomerular epithelial cells in culture

Confluent adult and fetal human glomerular epithelial cells were incubated for 24 h in the presence of [3H]-amino acids and [35S]sulfate. Two heparan-35SO4 proteoglycans were released into the culture medium. These 35S-labeled proteoglycans eluted as a single peak from anion exchange chromatographic columns, but were separable by gel filtration on Sepharose CL-6B columns. The larger heparan-35SO4 proteoglycan eluted with the column void volume and at a Kav of 0.26 from Sepharose CL-4B columns. The most abundant medium heparan-35SO4 proteoglycan was a high buoyant density proteoglycan similar in hydrodynamic size (Sepharose CL-6B Kav 0.23) to those previously described in glomerular basement membranes and isolated glomeruli. Heparan-35SO4 chains from both proteoglycans were 36 kDa. A smaller proportion of Sepharose CL-6B excluded dermatan-35SO4 proteoglycan was also synthesized by these cells. The predominant protein cores of both medium heparan-35SO4 proteoglycans were approximately 230 and 180 kDa. A hybrid chondroitin/dermatan-heparan-35SO4 proteoglycan with an 80-kDa protein core copurified with the smaller medium heparan-35SO4 proteoglycan. This 35S-labeled proteoglycan appeared as a diffuse, chondroitinase ABC sensitive 155-kDa fluorographic band in sodium dodecyl sulfate-polyacrylamide gels after the Sepharose CL-6B Kav 0.23 35S-labeled proteoglycan fraction was digested with heparitinase. The heparitinase generated heparan sulfate proteoglycan protein cores and the 155-kDa hybrid proteoglycan fragment had molecular weights similar to those previously identified in rat glomerular basement membrane and glomeruli using antibodies against a basement membrane tumor proteoglycan precursor (Klein et al. J. Cell Biol. 106, 963-970, 1988). Thus, human glomerular epithelial cells in culture are capable of synthesizing, processing, and releasing heparan sulfate proteoglycans which are similar to those synthesized in vivo and found in the glomerular basement membrane. These proteoglycans may belong to a family of related basement membrane proteoglycans.