Effect ofin vitro differentiation on proteoglycan structure in cultured human monocytes

Parellel toin vitro differentiation of human monocytes into macrophage-like cells, the cells change their synthesis of glycosaminoglycans from chondroitin 4-sulfate to highly sulfated chondroitin sulfate, containing 4,6-disulfatedN-acetylgalactosamine units [Kolsetet al. (1983) Biochem J 210:661–67]. After exposure of monocyte cultures to [³⁵S]sulfate for 24h either from the onset of cultivation, prior to differentiation, or from day 4, after differentiation,³⁵S-macromolecules from medium and cell-layer were isolated and characterized. The cell-layer of day 5 cultures contained both proteoglycans and free polysaccharide chains, while the³⁵S-macromolecules present in the cell-layer of day 1 cultures and in medium of both monocytes and macrophage-like cells were almost exclusively of proteoglycan nature. Proteoglycans produced by macrophage-like cells were of larger size than the monocyte proteoglycans, most likely due to an increased polysaccharide chain length. These proteoglycans, in contrast to the monocyte-derived species, also showed affinity for fibronectin at physiological ionic strength.     

Synthesis and release of chondroitin sulphate and heparan sulphate proteoglycans from mouse macrophagesin vitro

Macrophages were obtained from the mouse peritoneal cavity and culturedin vitro. The cells were exposed to³⁵S-sulphate for 20 h, and labelled proteoglycans were recovered from both medium and cell fractions by sodium dodecylsulphate solubilization. The cell fraction contained both proteoglycans and glycosaminoglycans, whereas only intact proteoglycans could be recovered from the medium fraction. ³⁵S-Glycosaminoglycans isolated from cell and medium fractions by papain digestion were shown to contain approximately 25% heparan sulphate and 75% galactosaminoglycans comprising 55% chondroitin sulphate and 20% dermatan sulphate. The galactosaminoglycans were shown by paper chromatography to contain more than 95% 4-sulphated units. Pulse-chase experiments showed that approximately 80% of the cell-associated material was released within 6 h of incubation.³⁵S-Proteoglycans released did not bind to the macrophages, but were recovered in a soluble form from the culture medium.Abbreviations CSPG chondroitin sulphate proteoglycan - HSPG heparan sulphate proteoglycan - SDS sodium dodecylsulphate - DME Dulbecco's Minimum Essential Medium - GAG glycosaminoglycan     

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.     

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     

Isolation and characterization of membrane-associated proteoglycans from normal and malignant human mammary epithelial cells

The plasma membrane-associated proteoglycans of a malignant human breast cell line (MDA-MB-231) were compared with the corresponding proteoglycans from a normal cell line (HBL-100). The labeled proteoglycans were isolated from the plasma membranes of cells grown in the presence of [³H]glucosamine and [³⁵S]Na₂SO₄ by extraction with guanidine hydrochloride and subsequently purified by DEAE-ion exchange chromatography. Their structural properties were established by treatment with nitrous acid, heparitinase and chondroitinase ABC, and by gel filtration before and after alkaline -elimination. About 18% of the proteoglycans synthesized by these cell lines were associated with the plasma membranes. The HBL plasma membranes contained 80% heparan sulfate and 20% chondroitin sulfate proteoglycans whereas MDA plasma membranes had 50% heparan sulfate and 50% chondroitin sulfate proteoglycans. The MDA plasma membrane contained two heparan sulfate proteoglycans, both having nearly the same molecular size as the two species secreted into the medium by these cells. The HBL plasma membrane also contained two hydrodynamic size heparan sulfate proteoglycans. The larger hydrodynamic size species has a slightly lower molecular size than that secreted into the medium, and the smaller hydrodynamic size species was not detectable in the medium. Even though the major chondroitin sulfate proteoglycans from MDA plasma membranes were smaller in size than those from HBL plasma membrane, a larger proportion of the glycosaminoglycan chains of the former were bigger than those from the latter.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate - Di-OS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-d-galactose - Di-4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-4-O-sulfo-d-galactose - Di-6S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-6-O-sulfo-d-galactose - Gdn-HCl guanidine hydrochloride - WGA wheat germ agglutinin     

Isolation of <Emphasis Type="BoldItalic">Serratia marcescens</Emphasis> as a chondroitinase-producing bacterium and purification of a novel chondroitinase AC

A strain of Serratia marcescens that produced chondroitinase was isolated from soil. It produced a novel chondroitinase AC, which was purified to homogeneity. The enzyme was composed of two identical subunits of 35 kDa as revealed by SDS-PAGE and gel filtration. The isoelectric point for the chondroitinase AC was 7.19. Its optimal activity was at pH 7.5 and 40 °C. The purified enzyme was active on chondroitin sulfates A and C and hyaluronic acid, but was not with chondroitin sulfate B (dermatan sulfate), heparin or heparan sulfate. The apparent K_m and V_max of the chondroitinase AC for chondroitin sulfate A were 0.4 mg ml^–1 and 85 mmol min^–1 mg^–1, respectively, and for chondroitin sulfate C, 0.5 mg ml^–1 and 103 mmol min^–1 mg^–1, respectively.     

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 ³⁵SO₄^2?-labeled extracellular proteoglycan per cell than do Balb/c 3T3 cells transformed by SV40 (SV3T3). The extracellular ³⁵SO₄^2?-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 ³⁵SO₄^2?-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 ³⁵SO₄^2?-labeled proteoglycans and contains chondroitin sulfate exclusively. It is altogether absent in the 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 ³⁵SO₄^2?-labeled proteoglycans by SV3T3 cells when compared to Balb/c 3T3 cells.     

Synthesis of chondroitin sulfate D and heparin proteoglycans in murine lymph node-derived mast cells. The dependence on fibroblasts

Proteoglycans synthesized in cultured mast cells derived from horse serum-immunized lymph node cells were analyzed. Treatment of the 35S-proteoglycans extracted from these cells with either chondroitinase ABC or AC resulted in 95% +/- 7% and 84% +/- 7%, respectively (mean +/- S.E., n = 3), of the radioactivity associated with disaccharides eluting in the included volume of PD-10. The 35S-proteoglycans were not hydrolyzed by nitrous acid elimination treatment. The chondroitinase ABC-generated disaccharides were analyzed by aminocyano high performance liquid chromatography. 35S-Disaccharides eluted in a major peak at a retention time of 8.1 min, corresponding to the disaccharide of chondroitin 4-sulfate proteoglycan (delta Di-4S), and a second peak at 12 min, corresponding to the disaccharide of chondroitin sulfate D proteoglycan (delta Di-diSD). Further treatment with chondro-4-sulfatase did not affect the retention time of the disaccharide corresponding to delta Di-diSD whereas this peak disappeared after the digested proteoglycan was treated either by chondro-6-sulfatase or by both sulfatases. Therefore, this disaccharide was identified as chondroitin sulfate D. Quantification of the radiolabeled disaccharides showed that delta Di-diSD contributed 20% +/- 2% (n = 3) of the total sulfated disaccharides of the chondroitin sulfate of these cultured cells. The role of fibroblasts in inducing the shift of chondroitin sulfate D into heparin proteoglycan in these mast cells was also investigated by using three types of monolayers: mouse embryonic skin fibroblasts (MESF), rat embryonic skin fibroblasts (RESF), and 3T3 fibroblasts. 35S-Proteoglycans that were extracted from the lymph node-derived mast cells cultured for 30 days on MESF and on 3T3 fibroblast monolayers were 93% +/- 4% and 30% +/- 7% (n = 3) susceptible to nitrous acid elimination, respectively. No degradation by nitrous acid was observed in 35S-proteoglycans extracted from cells cultured on RESF monolayer. Since the MESF was found to be the most potent monolayer in the induction of heparin synthesis, the kinetics of changes in the synthesis of proteoglycan types were determined in lymph node-derived mast cells cultured on MESF for up to 30 days. It was found that the synthesis of chondroitin sulfate gradually declined whereas that of heparin starting between 4 and 7 days after plating gradually increased. From the 17th day on, only the synthesis of heparin was detected.     

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.     

Biosynthesis of glycosaminoglycans and proteoglycans by the lymph node

Previous studies of hyaluronan uptake and catabolism by lymph nodes indicated that the nodes might also add some HA of low molecular weight to the unabsorbed fraction that passes through from afferent to efferent lymph vessels.The ability of lymph nodes to synthesise HA and proteoglycans was therefore examined (i) by perfusion of [³H] acetate through an afferent lymph vessel in vivo, and recovery of labeled products from the efferent lymph vessel and from the node after perfusion; and (ii) by tissue culture of lymph nodes with [³H] acetate.Perfusion of lymph nodes with [³H] acetate in situ yielded: (a), in outflowing lymph, small amounts of chondroitin/dermatan sulfate within the first hour which continued to be produced for up to 24[emsp4 ]h; heparin in the second hour and HA in the third. In the nodes removed 17 to 19[emsp4 ]h later, equal amounts of hyaluronan and chondroitin/dermatan sulfate and heparan sulfate proteoglycans were detected. In the tissue culture of lymph nodes: (1) HA, heparin and proteoglycans of heparan sulfate and chondroitin/dermatan sulfate were released into the medium but in the cell extract only heparan sulfate proteoglycan was detected; and (ii) molecular weight of the released hyaluronan ranged widely but was mostly less than 4–5×10⁵[emsp4 ]D; heparan sulfate proteoglycan was 2.8×10⁴ to 9.4×10⁵[emsp4 ]D; heparin 7.9×10⁴[emsp4 ]D and chondroitin sulfate 1.3×10⁴[emsp4 ]D, suggesting that the chondrotin sulfate were released from their proteoglycans core by enzymic degradation.It is concluded that lymph nodes can release HA, heparin, heparan sulfate and chondroitin/dermatan sulfate proteoglycans into efferent lymph but the amount of hyaluronan is likely to be small without immune or other stimulation and its molecular weight is lower than in other tissues.     

Turnover of proteoglycans by chick lens epithelial cells in cell culture and intact lens

Chick lens epithelial cells were cultured on plastic and type IV collagen substrata, and the confluent cultures were labeled continuously with [35S]sulfate for 20 h. Intact lenses were also labeled in the same way. 35S-Proteoglycans isolated from those cultures were compared for their molecular sizes and glycosaminoglycan compositions. The results have shown that: 1) Proteoglycans synthesized by cells on type IV collagen were significantly smaller than those by cells on plastic. 2) Proteoglycans of intact lens showed a broad distribution of molecular size and contained a high proportion of chondroitin sulfate in the medium fraction compared to those of the two cell cultures. In order to explain such differences between proteoglycans from cultures, label-chase experiments with [35S]sulfate were done for proteoglycans synthesized. 35S-Proteoglycans isolated at each chase time 0, 2.5, and 17 h) were compared and the following results were found: 1) The cell layers of both "plastic" and "type IV collagen" cultures contained glycosaminoglycan species predominantly at each chase time rather than proteoglycans. 2) Changes in the glycosaminoglycan compositions of medium fractions of cell cultures were observed during the chase period; in medium of the "plastic" culture, proteoheparan sulfate increased with chase time, whereas in medium of the "type IV collagen" culture, chondroitin sulfate glycosaminoglycan (not proteoglycan) increased with chase time. 3) In intact lens culture, lens capsule fraction at every chase time contained a proteoglycan unique in molecular size, which was not found in cell culture fractions. 4) All fractions from intact lens cultures contained a higher content of chondroitin sulfate at every chase time than the respective fractions from cell cultures. These results suggest that adhesion of the cells to type IV collagen or lens capsule influences the degradation and secretion of proteoglycans. In addition, they can account partially for the above-described differences in molecular sizes and glycosaminoglycan compositions between 35S-proteoglycans from various cultures continuously labeled with [35S]sulfate.     

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.     

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).     

In vitro production of proteoglycans in the articular-epiphyseal cartilage of growing pigs

The failure of cartilage mineralization in osteochondrotic cartilage may be due to an impaired proteoglycan production. Thein vitro production of proteoglycans was therefore studied in the joint cartilage of growing pigs, aged 9–18 weeks, after incubation of cartilage samples with³⁵S-sulfate. Cartilage was obtained from different areas of the femoral condyles and samples from these areas were further divided into three layers, where the superficial layer contains articular cartilage and the basal layers consist of growth cartilage. There was no significant difference in the overall amount of³⁵S-proteoglycans synthesized in different areas of the condyles. However, the total production of³⁵S-proteoglycans per mg tissue was highest in the basal layer in all areas. This was not due to a larger number of cells; the superficial layer contained more DNA per mg tissue than the basal layer. Gel chromatography on Sepharose CL-2B of the cartilage extracts, which resulted in the separation of large proteoglycans (K _av 0.4) from proteoglycans of small hydrodynamic size (K _av 0.8), showed that the relative amount of large proteoglycans increased with the distance from the articular surface. Again, no difference in the relative amounts of large and small proteoglycans were found when cartilage from different areas were compared. Osteochondrotic cartilage was detected in the pigs aged 12–18 weeks. In areas where osteochondrotic cartilage were present, the total production of³⁵S-proteoglycans was lowered and the relative amount of large proteoglycans was less than that found in the adjoining areas devoid of osteochondrotic lesions. The data available indicate that the higher relative amount of small proteoglycans in the osteochondrotic cartilage was partly caused by degradation of the large proteoglycans (aggrecan).     

Glycosaminoglycan profiles in cloned granulated lymphocytes with natural killer function and in cultured mast cells: their potential use as biochemical markers

Proteoglycans from three cloned, granulated lymphocyte cell lines with natural killer (NK) function (NKB61A2, HY-3, H-1) and one mast cell line (PT-18) were labeled with [35S]sulfate. [35S]proteoglycans were extracted in 1 M NaCl with protease inhibitors to preserve their native structure and were separated from unincorporated [35S]sulfate by Sephadex G-25 chromatography. [35S]proteoglycans from all four cell lines were chromatographed over Sepharose 4B and were found to have a similar range of m.w. The [35S]glycosaminoglycans from each cell line were then separated from parent proteoglycans by treatment with 0.5 M NaOH. The [35S]glycosaminoglycans from the three lymphocyte cell lines exhibited a similar m.w. as assessed by Sepharose 4B gel filtration, whereas the [35S]glycosaminoglycans from the mast cell line chromatographed as a smaller m.w. molecule. [35S )glycosaminoglycan charge characteristics were evaluated with DEAE C1-6B ion exchange chromatography. The consistency of the elution patterns was determined by using [35S]glycosaminoglycans obtained from radiolabelings of each cell line separated by 6 mo in culture. Each NK lymphocyte cell line reproducibly produced two distinct [35S]glycosaminoglycan chains that eluted in two regions well before the commercial heparin marker. The proportions of each chain were dependent upon the specific cell line. The mast cell line produced a single [35S]glycosaminoglycan chain, which eluted overlapping the internal commercial heparin marker, consistent with its higher charge characteristics. [35S]glycosaminoglycans from all cell lines were identified as chondroitin sulfates with the use of specific polysaccharidases. The NK lymphocyte glycosaminoglycans contained chondroitin 4-sulfate disaccharides. The mast cell glycosaminoglycans contained oversulfated disaccharides and chondroitin 4-sulfate disaccharides. Thus, each granulated NK lymphocyte cell line produced chondroitin sulfate glycosaminoglycans that were characteristic of that cell line and of different composition and less charge than those produced by cultured mast cells. These findings demonstrate that glycosaminoglycan profiles are useful biochemical markers in the characterization of diverse granulated cell lines including NK lymphocytes and mast cells.     

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.     

Evidence for an interaction between canine synovial cell proteoglycans and link proteins

Link proteins are glycoproteins which stabilize aggregates of proteoglycans and hyaluronic acid in cartilage. We recently identified link proteins in canine synovial cell cultures. We now find that link proteins and proteoglycans extracted from these cells under dissociative conditions sediment in the high-buoyant-density fractions of an associative cesium chloride density gradient, suggesting that link proteins interact with high-bouyant-density proteoglycans. In gradients containing [³⁵S]sulfate-labeled synovial cell extracts, 76% of the labeled sulfate and 54% of the uronic acid is found in the high-buoyant-density fractions. Under associative conditions, Sepharose 2B elution profiles of the crude synovial cell extract, synovial cell high-buoyant-density fractions, and culture medium indicate that synovial cell proteoglycans are present in monomeric form, rather than in aggregates. Synovial cell link proteins co-elute with the [³⁵S]sulfate-labeled material under the same conditions. These proteoglycans do not interact in vitro with exogenous hyaluronic acid. Dermatan sulfate, chondroitin sulfate and heparan sulfate are the major cell-associated sulfated glycosaminoglycans synthesized by cultured canine synovial cells, while hyaluronic acid is found in the culture medium. Although the proteoglycans synthesized by cultured synovial cells interact with link proteins, these data indicate that they do not interact with hyaluronic acid to form aggregates.     

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.     

A sensitive GLC-method for component sugars andO-glycosidic linkage monosaccharides of cartilage proteoglycans

A method is described for the measurement ofN-acetylgalactosamine,N-acetylglucosamine, galactose, mannose and xylose present in the different carbohydrate chains of cartilage proteoglycans (PG). Bovine articular cartilage PG samples corresponding to the minimum of 1 nmol of each monosaccharide were reproducibly quantified following hydrolysis with 2 M HCl and derivatization into alditol acetates. An on-column injection mode and an OV-1701 fused silica capillary column were used for chromatography.Alkaline borohydride treatment of the PG was exploited to reduce the acid labile xylose in the base of the chondroitin sulphate chain into more stable xylitol, allowing the assay of chondroitin sulfate chain length as anN-acetylgalactosamine/xylose ratio. A novel procedure is described for the measurement of the galactosaminitol evolving from the protein linkage of oligosaccharides and of keratan sulphate.     

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.