Sulfated mucopolysaccharide synthesis during the development of Rana pipiens

Sulfated mucopolysaccharide (MPS) synthesis during the development of Rana pipiens was studied autoradiographically and biochemically following injection of embryos with ³⁵S-sulfate. ³⁵S-sulfate incorporation can be detected in unfertilized and fertilized eggs. The sulfate-incorporating material accumulates along the periphery of yolk platelets of eggs. During cleavage, the ³⁵S-sulfate-incorporating material accumulates on cell surfaces as well as along the periphery of yolk platelets. Biochemical analysis utilizing the enzymes chondroitinase ABC and AC and nitrous acid degradation indicates that the MPS synthesized during cleavage is approximately 82% heparin and/or heparan sulfate and 18% chondroitin 4-sulfate. During gastrulation, a greatly enhanced incorporation of ³⁵S-sulfate is observed in the invaginating chordamesoderm and lateroventral mesoderm, and by the end of gastrulation enhanced incorporation can be detected in neural tissue. During this period, chondroitin 6-sulfate synthesis is initiated. Incorporation of ³⁵S-sulfate is observed in all tissues of the embryo from the beginning of neurulation through hatching. This ubiquitous incorporation is accompanied by an increase in the relative amount of chondroitin 6-sulfate synthesized. During the period following hatching, incorporation is suppressed in some tissues and enhanced in others so that by the late feeding tadpole stage a very high incorporation is observed only in cartilaginous tissue. These results indicate that sulfated MPS synthesis occurs in all stages of development of Rana pipiens, but that significant changes in the rate of synthesis occur in various cell types during gastrulation and after hatching. The ubiquity of sulfated MPS synthesis during the critical early stages of development and the changes in the pattern of synthesis in various cell types suggest that these molecules are involved in a number of embryonic processes.     

Comparison of proteoglycans synthesized by porcine normal and polycystic renal tubular epithelial cells in vitro

Newly synthesized porcine tubular epithelial cell proteoglycans were labeled in vitro with Na2[35S]SO4. At the beginning of the labeling period (24 h) [35S] sulfate incorporated into macromolecules was measured following PD-10 chromatography. There was a significant reduction in the amount of 35S-labeled macromolecules isolated from polycystic cells compared to that from normal cells. The distribution of recovered radiolabeled material among the medium, cell surface, and intracellular fractions was similar for both normal and polycystic cells. Analysis of the proteoglycans in polycystic cells demonstrated that 86 and 73% of 35S-labeled macromolecules were of the heparan sulfate type in polycystic and normal cells, respectively. The remainder was chondroitin sulfate. Proteoglycans were characterized using DEAE-Sephacel ion-exchange chromatography, chondroitinase ABC, heparitinase, and nitrous acid digestion followed by Sepharose CL-4B gel permeation chromatography. The majority of radiolabeled material in the medium, cell surface, and intracellular fractions eluted between 0.35 and 0.39 M NaCl. However, a second peak (peak II) that eluted at 0.25 M NaCl was found in the medium from polycystic cells. This peak accounted for 27% of the total macromolecules secreted into the medium. Proteoglycans in the major peak were susceptible to nitrous acid and chondroitinase ABC digestion. A similar proportion of peak II was degraded by chondroitinase ABC. However, the remainder was only slightly susceptible to treatment with nitrous acid or heparitase. In normal cells a small amount of material eluted at a similar low charge; the proteoglycans were the same as those found in the major peak and appeared as a shoulder on this peak.     

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.     

Thymic epithelial cells synthesize a heparan sulfate with a highly sulfated region

Epithelial cells are important components of the thymus microenvironment and are involved in thymocyte differentiation. The production and secretion of sulfated glycosaminoglycans by these cells grown in culture were investigated using labeling with radioactive ³⁵S-Na₂SO₄ and ³H-glucosamine. The major glycosaminoglycans synthesized by these cells are heparan sulfate and hyaluronic acid. The structure of the heparan sulfate was investigated by the pattern of degradation products formed by deaminative cleavage with nitrous acid. The ratio ³⁵S-sulfate/³H-glucosamine is high in the segments of the heparan sulfate released during the deaminative cleavage with nitrous acid but low in the resistant portion of the molecule. Thus, the heparan sulfate synthesized by the thymic epithelial cells contains a highly sulfated region. Digestion with heparitinase reveals that this highly sulfated region is a heparin-like segment of the molecule. The heparan sulfate is rapidly incorporated into the cell surface but its secretion to the extracellular medium requires a longer incubation period. Finally, heparin was used to mimic the possible effect of this heparan sulfate with a highly sulfated region, as ascertained by its ability to modulate thymocyte adhesion to thymic epithelial cells. Since heparin actually enhanced thymocyte adhesion, it is suggested that the heparan sulfate described herein, secreted by the thymic epithelium, may play a role upon intrathymic heterotypic cellular interactions. J Cell Physiol 178:51–62, 1999. © 1999 Wiley-Liss, Inc.     

Analysis of glycosaminoglycans in bovine retinal microvessel basement membrane

Glycosaminoglycans (GAG) were isolated from bovine retinal microvessel basement membrane (RMV-BM) and quantitatively analyzed using a recently described competitive binding assay that is specific for and sensitive to nanogram amounts of heparan and chondroitin sulfates. Treatment of osmotically lysed retinal microvessels with the ionic detergent deoxycholate (DOC), required for liberation of the extracellular matrix for plasma membrane lipoproteins and purification of the insoluble matrix, solubilized less than 5% of the GAG in the water-insoluble material. Total GAG content in the DOC-insoluble basement membranes was approx. 0.52 micrograms/mg dry weight; about 70% of the measurable GAG was resistant to both chondroitinase ABC and chondroitinase AC digestion and was sensitive to nitrous acid treatment, indicating its heparan sulfate nature. Cellulose acetate electrophoresis revealed two bands, one of which had an electrophoretic mobility similar to heparan sulfate standard and was sensitive to nitrous acid; the other migrated in the same position as chondroitin sulfate standard and was sensitive to chondroitinase ABC and chondroitinase AC digestion. These results provide evidence that RMV-BM contains chondroitin sulfate(s) as well as heparan sulfate, and offer the first quantitative analysis of GAG in this extracellular matrix.     

Characterization of proteoglycans in Alzheimer's disease fibroblasts.

Skin fibroblasts lines established from patients with Alzheimer's disease and old normal individuals were cultured with 35S-sodium sulfate and 3H-glucosamine. Proteoglycans were isolated and characterized. Sulfate incorporation into proteoglycans increased in Alzheimer's disease fibroblasts relative to normal controls. These increases changed the ratio of chondroitin sulfate to heparan sulfate proteoglycan from 1.4 to 1.7 (p = 0.0012) and decreased the ratio of cell to medium proteoglycans from 0.32 to 0.26 in normal and Alzheimer fibroblasts (p = 0.006), respectively. HPLC analysis of the disaccharides produced by chondroitinase ABC revealed no differences in composition between proteoglycans of Alzheimer and normal fibroblasts in either the cell or medium fraction. However, analysis of disaccharides produced by heparinase plus heparitinase showed differences in composition in the medium but not the cell fraction. delta UA-GlcNS was increased by 30% while delta UA-GlcNS-6S was reduced by 40% in Alzheimer's disease.     

Modulation of sulfated proteoglycan synthesis by bovine aortic endothelial cells during migration

The rates of 35S-sulfate incorporation into proteoglycan were compared in multi-scratch wounded and confluent cultures of bovine aortic endothelial cells to determine whether proteoglycan synthesis is altered as cells are stimulated to migrate and proliferate. Incorporation was found to be stimulated in a time-dependent manner, reaching maximal levels 44-50 h after wounding, as cells migrated into wounded areas of the culture dish. Quantitative autoradiography of 35S-sulfate-labeled single-scratch wounded cultures demonstrated a 2-4-fold increase in the number of silver grains over migrating cells near the wound edge when compared to cells remote from the wound edge. Furthermore, when cell proliferation was blocked by inhibition of DNA synthesis, the increase in 35S-sulfate incorporation into proteoglycan after wounding was unaffected. These data indicate that cell division is not required for the modulation of proteoglycan synthesis to occur after wounding. Characterization of the newly synthesized proteoglycan by ion-exchange and molecular sieve chromatography demonstrated that heparan sulfate proteoglycan constitutes approximately 80% of the labeled proteoglycan in postconfluent cultures, while after wounding, chondroitin sulfate proteoglycan and/or dermatan sulfate proteoglycan (CS/DSPG) increases to as much as 60% of the total labeled proteoglycan. These results suggest that CS/DSPG synthesis is stimulated concomitant with the stimulation of endothelial cell migration after wounding.     

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.     

D(+)catechin enhances heparan sulphate content in rat liver

Administration of (D+) catechin (100 mg/kg body wt) to rats resulted in an increase in the amount of total sulphated glycosaminoglycans (GAG) in liver. The increase was more pronounced in the case of heparan sulphate than chondroitin sulphate and dermatan sulphate. The liver slices prepared from catechin-treated rats showed a significant increase in the rate of incorporation of 35S-sulphate into GAG. Similarly there was a concentration-dependent increase in the rate of 35S-sulphate incorporation into GAG by normal liver slices in presence of catechin in vitro. Susceptibility to nitrous acid degradation and chondroitinase ABC digestion showed that more than 80% of the GAG labelled in vivo with 35S-sulphate, was heparan sulphate and about 10% chondroitin sulphate and dermatan sulphate. Gel filtration of the 35S-labelled material isolated from livers of normal and catechin-treated animals over sephacryl S-300 did not show any difference probably excluding the possibility of free GAG chains initiated on catechin or any of its metabolites in vivo. These results indicate that catechin stimulates the synthesis of sulphated GAG, particularly heparan sulphate in liver.     

Sulfated mucopolysaccharides of midgestation embryonic and extraembryonic tissues of the mouse

Incorporation of [³⁵S]sulfate into sulfated mucopolysaccharides has been characterized in midgestation mouse embryo, yolk sac, trophoblast, and decidua. Enzymatic analysis indicated that chondroitin sulfates contained approximately half of the label in embryo, trophoblast, and decidua, but less than 20% in yolk sac. While the labeled chondroitin sulfate fraction of trophoblast and decidua was mainly chondroitin-4-sulfate, only embryo contained a significant proportion of labeled chondroitin-6-sulfate. The relative incorporation into embryo chondroitin-6-sulfate was also substantially higher than that observed in four adult soft tissues. Labeled dermatan sulfate was absent from the embryo and yolk sac, but small amounts might have been synthesized by the placenta. Nitrous acid degradation studies revealed that essentially all the chondroitinase resistant MPS was N-sulfated, i.e., heparan sulfate and/or heparin. Electrophoretic profiles indicate that the bulk of the N-sulfated material resembles heparan sulfate rather than heparin. Electrophoretic heterogeneity and slow migration rates relative to standard markers suggest that the majority of labeled chondroitin sulfates may be undersulfated. The different mucopolysaccharide patterns in the various tissues may reflect their specialized properties and functions.     

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.     

Sulfated glycosaminoglycans in the extracellular matrix of muscle tissue in Atlantic cod (Gadus morhua) and Spotted wolffish (Anarhichas minor)

Two species of commercially important cold water fish were investigated for content of sulfated glycosaminoglycans (GAGs) in muscle tissue by use of in vivo 35S-sulfate labeling combined with different digestions (papain, chondroitinase ABC, keratanase and nitrous acid treatment), DEAE chromatography, SDS-PAGE and histology techniques. The species investigated in this study have different gaping properties. The non-gaping species, spotted wolffish (Anarhichas minor), contained 3-4 times more 35S-sulfated anionic components than the gaping species, Atlantic cod (Gadus morhua). The higher level of sulfation in wolffish was supported by light microscopy studies using Alcian blue staining with different concentrations of MgCl2 as critical electrolyte. Furthermore, the muscular connective tissue in the non-gaping species was dominated by chondroitin sulfate (CS)/dermatan sulfate (DS), whereas the gaping species was more dominated by heparan sulfate (HS). Moreover, structural differences were observed in the junctions between the myofibers, which were more pronounced in the wolffish. The histological studies revealed that the basement membrane area was rich in acidic mucopolysaccharides in both species.     

Glycosaminoglycan production by bovine aortic endothelial cells cultured in sulfate-depleted medium

Bovine aortic endothelial cells were cultured in medium containing [3H]glucosamine and concentrations of [35S]sulfate ranging from 0.01 to 0.31 mM. While the amount of [3H]hexosamine incorporated into chondroitin sulfate and heparan sulfate was constant, decreasing concentrations of sulfate resulted in lower [35S]sulfate incorporation. Sulfate concentrations greater than 0.11 mM were required for maximal [35S]sulfate incorporation. Chondroitin sulfate was particularly affected so that the sulfate to hexosamine ratio in [3H]chondroitin [35S]sulfate dropped considerably more than the sulfate to hexosamine ratio in [3H] heparan [35S]sulfate. Sulfate concentration had no effect on the ratio of chondroitin 4-sulfate to chondroitin 6-sulfate. The ratios of sulfate to hexosamine in cell-associated glycosaminoglycans were essentially identical with the ratios in media glycosaminoglycans at all sulfate concentrations. DEAE-cellulose chromatography confirmed that sulfation of chondroitin sulfate was particularly sensitive to low sulfate concentrations. While cells incubated in medium containing 0.31 mM sulfate produced chondroitin sulfate which eluted later than heparan sulfate, cells incubated in medium containing less than 0.04 mM sulfate produced chondroitin sulfate which eluted before heparan sulfate and near hyaluronic acid, indicating that many chains were essentially unsulfated. At intermediate concentrations of sulfate, chondroitin sulfate was found in very broad elution patterns suggesting that most did not fit an "all or nothing" mechanism. Heparan sulfate produced at low concentrations of sulfate eluted with narrower elution patterns than chondroitin sulfate, and there was no indication of any "all or nothing" sulfation.     

Biosynthesis of proteoglycans by proliferating and differentiating normal human keratinocytes cultured in serum-free medium

Normal human keratinocytes (NHK) were cultured in serum-free medium, containing low (0.1 mM) or high (2 mM) calcium, to obtain proliferating and differentiating cultures, respectively. Proteoglycan (PG) synthesis of proliferating and differentiating NHK was investigated. Cultures were labeled with 35S-sulfate, and the PGs were extracted from medium and cell layer. The newly synthesized PGs were isolated by ion-exchange chromatography on a column of DEAE-Sephacel. The molecular properties of the PGs and the size and composition of glycosaminoglycans (GAGs) were determined. In general, the PGs are relatively small size (Mr 70,000-120,000). The PGs of proliferating cultures are larger in molecular size than the PGs of differentiating cultures, and this is due to the degradation of the GAG chains. The molecular weight of the GAG chains of proliferating NHK ranged from 4,800 to 22,000, and the range for GAGs from differentiating cultures varied from 2,800 to 9,600. By compositional analysis, these PGs proved to contain heparan sulfate, chondroitin sulfate, and dermatan sulfate as determined by nitrous acid degradation, and chondroitinase ACII and ABC digestion. No significant differences were found in the overall GAG composition of the medium secreted PGs of proliferating and differentiating cultures. In contrast, cell-associated PGs of differentiating cells had higher levels of heparan sulfate than those of proliferating cells.     

Synthesis of complex saccharides by synchronized NIL-8 hamster cells.

Complex saccharide synthesis by synchronized NIL-8 cells was studied by metabolic labeling with [³H]glucosamine. Hyaluronic acid, a chondroitin sulfate and heparan sulfate are produced during G 1, S and G 2/M but the latter is absent or altered in media during G 2/M. Glucosamine is the sole amino sugar in cetylpyridinium bromide precipitable glycopeptides except for G 1 cell associated material; CPB-soluble glycopeptides contained label in both glucosamine and galactosamine in contrast to products of NIL-8 cells transformed by hamster sarcoma virus (HSV) in which galactosamine was absent from the glycopeptide fractions. The transformed cells synthesized hyaluronic acid, chondroitin sulfate and heparan sulfate in amounts comparable to those found in the NIL-8 line.     

Cell surface glycosaminoglycans of a tumor cell line and its DNA transfected variant differing in their lung colonizing potential

A highly lung-colonizing cell line RMS/8² was obtained by DNA transfection from a low lung-colonizing line RMS/8, a clone of a rat rhabdomyosarcoma cell line. The cells were metabolically labeled with³H-glucosamine and³⁵S-sulfate. The newly synthesized pericellular glycosaminoglycans and the ability of the cells from the two lines to degrade extracellular matrix components were studied comparatively. The following conclusions were obtained: 1) Thein vitro proliferation rate is not a determinant in the modulation of the colonizing potential of these cells; 2) The strongly colonizing RMS/8² cells release more radioactivity from the radiolabeled extracellular matrix than their weakly colonizing counterparts; 3) The cells with a high colonizing potential incorporated less radioactivity into the cell surface glycosaminoglycans, and exhibited a lower heparan sulfate to chondroitin sulfate ratio than the weakly colonizing RMS/8 line.     

Heparan sulfate and control of endothelial cell proliferation: increased synthesis during the S phase of the cell cycle and inhibition of thymidine incorporation induced by ortho-nitrophenyl-beta-D-xylose

The effect of xylosides on the synthesis of [35S]-sulfated glycosaminoglycans by endothelial cells in culture was investigated. Ortho-nitrophenyl-beta-D-xylose (10(-3)M) produces a dramatic enhancement on the synthesis of heparan sulfate and chondroitin sulfate secreted to the medium (20- and 100-fold, respectively). Para-nitrophenylxyloside, at the same concentration, produces an enhancement of only 37- and 3-fold of chondroitin sulfate and heparan sulfate, respectively. These differences of action seem to be related with the higher lipophilic character of ortho-nitrophenyl-xyloside. A lower enhancement of the synthesis of the two glycosaminoglycans is also observed with 2-naphtol beta-D-xylose and cis/trans-decahydro-2-naphtol beta-D-xylose. Besides stimulating the synthesis, O-nitrophenyl-beta-D-xylose as PMA [J. Cell. Biochem. 70 (1998) 563] also inhibits [3H]-thymidine incorporation by quiescent endothelial cells stimulated for growth by fetal calf serum (FCS). The combination of xylosides with PMA produced some cumulative effect. PMA stimulates the synthesis of heparan sulfate mainly at G1 phase whereas the highest enhancement of synthesis produced by the xylosides is in the S phase of the endothelial cell cycle.     

Purification and characterization of protease-resistant secretory granule proteoglycans containing chondroitin sulfate di-B and heparin-like glycosaminoglycans from rat basophilic leukemia cells

Proteoglycans were extracted from nuclease-digested sonicates of 10(9) rat basophilic leukemia (RBL-1) cells by the addition of 0.1% Zwittergent 3-12 and 4 M guanidine hydrochloride and were purified by sequential CsCl density gradient ultracentrifugation, DE52 ion exchange chromatography, and Sepharose CL-6B gel filtration chromatography under dissociative conditions. Between 0.3 and 0.8 mg of purified proteoglycan was obtained from approximately 1 g initial dry weight of cells with a purification of 200-800-fold. The purified proteoglycans had a hydrodynamic size range of Mr 100,000-150,000 and were resistant to degradation by a molar excess of trypsin, alpha-chymotrypsin, Pronase, papain, chymopapain, collagenase, and elastase. Amino acid analysis of the peptide core revealed a preponderance of Gly (35.4%), Ser (22.5%), and Ala (9.5%). Approximately 70% of the glycosaminoglycan side chains of RBL-1 proteoglycans were digested by chondroitinase ABC and 27% were hydrolyzed by treatment with nitrous acid. Sephadex G-200 chromatography of glycosaminoglycans liberated from the intact molecule by beta-elimination demonstrated that both the nitrous acid-resistant (chondroitin sulfate) and the chondroitinase ABC-resistant (heparin/heparan sulfate) glycosaminoglycans were of approximately Mr 12,000. Analysis of the chondroitin sulfate disaccharides in different preparations by amino-cyano high performance liquid chromatography revealed that 9-29% were the unusual disulfated disaccharide chondroitin sulfate di-B (IdUA-2-SO4----GalNAc-4-SO4); the remainder were the monosulfated disaccharide GlcUA----GalNAc-4-SO4. Subpopulations of proteoglycans in one preparation were separated by anion exchange high performance liquid chromatography and were found to contain chondroitin sulfate glycosaminoglycans whose disulfated disaccharides ranged from 9-49%. However, no segregation of subpopulations without both chondroitin sulfate di-B and heparin/heparan sulfate glycosaminoglycans was achieved, suggesting that RBL-1 proteoglycans might be hybrids containing both classes of glycosaminoglycans. Sepharose CL-6B chromatography of RBL-1 proteoglycans digested with chondroitinase ABC revealed that less than 7% of the molecules in the digest chromatographed with the hydrodynamic size of undigested proteoglycans, suggesting that at most 7% of the proteoglycans lack chondroitin sulfate glycosaminoglycans.(ABSTRACT TRUNCATED AT 400 WORDS)     

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

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

Surface glycosaminoglycans as a differentiation cofactor in neuroblastoma cell cultures.

The possible role of surface glycosaminoglycans (GAGs) in neuronal maturation occuring in neuroblastoma cultures has been investigated. GAGs of neuroblastoma cells, grown in suspension and monolayer, were labelled with 3H-glucosamine and 35S-sulfate. Neuron maturation, following cell adhesion to culture dishes, is accompanied by an increased ability of the cells to retain heparan sulfate (HS) on their surface, which is otherwise lost into the culture medium. The role of surface HS as a cofactor of cellular differentiation is discussed.