Effects of sulfate deprivation on the production of chondroitin/dermatan sulfate by cultures of skin fibroblasts from normal and diabetic individuals

Human skin fibroblast monolayer cultures from two normal men, three Type I diabetic men, and one Type I diabetic woman were incubated with [3H]glucosamine in the presence of diminished concentrations of sulfate. Although total synthesis of [3H]chondroitin/dermatan glycosaminoglycans varied somewhat between cell lines, glycosaminoglycan production was not affected within any line when sulfate levels were decreased from 0.3 mM to 0.06 mM to 0.01 mM to 0 added sulfate. Lowering of sulfate concentrations resulted in diminished sulfation of chondroitin/dermatan in a progressive manner, so that overall sulfation dropped to as low as 19% for one of the lines. Sulfation of chondroitin to form chondroitin 4-sulfate and chondroitin 6-sulfate was progressively and equally affected by decreasing the sulfate concentration in the culture medium. However, sulfation to form dermatan sulfate was preserved to a greater degree, so that the relative proportion of dermatan sulfate to chondroitin sulfate increased. Essentially all the nonsulfated residues were susceptible to chondroitin AC lyase, indicating that little epimerization of glucuronic acid residues to iduronic acid had occurred in the absence of sulfation. These results confirm the previously described dependency of glucuronic/iduronic epimerization on sulfation, and indicate that sulfation of the iduronic acid-containing disaccharide residues of dermatan can take place with sulfate concentrations lower than those needed for 6-sulfation and 4-sulfation of the glucuronic acid-containing disaccharide residues of chondroitin. There were considerable differences among the six fibroblast lines in susceptibility to low sulfate medium and in the proportion of chondroitin 6-sulfate, chondroitin 4-sulfate, and dermatan sulfate. However, there was no pattern of differences between normals and diabetics.     

Chlorate: a reversible inhibitor of proteoglycan sulfation

Bovine aorta endothelial cells were cultured in medium containing [3H]glucosamine, [35S]sulfate, and various concentrations of chlorate. Cell growth was not affected by 10 mM chlorate, while 30 mM chlorate had a slight inhibitory effect. Chlorate concentrations greater than 10 mM resulted in significant undersulfation of chondroitin. With 30 mM chlorate, sulfation of chondroitin was reduced to 10% and heparan to 35% of controls, but [3H]glucosamine incorporation on a per cell basis did not appear to be inhibited. Removal of chlorate from the culture medium of cells resulted in the rapid resumption of sulfation.     

Effects of brefeldin A on the synthesis of chondroitin 4-sulfate by cultures of mouse mastocytoma cells.

Mouse mastocytoma cells were cultured with brefeldin A in medium containing [35S]sulfate and [3H]glucosamine in order to determine the effects of this fungal metabolite on the formation of chondroitin 4-sulfate by these cells. There was a marked reduction in the incorporation of [35S]sulfate into the glycosaminoglycan which was approximately equal to the reduction in the incorporation of [3H]hexosamine into the same molecule. The chondroitin 4-sulfate chain size was greatly diminished, while the number of chains appeared to remain relatively constant, indicating that the brefeldin A partially disrupted the polymerizing system, but had little effect upon movement of the nascent proteochondroitin to the site for chondroitin polymerization and sulfation.     

Highly sulfated glycosaminoglycans inhibit aggrecanase degradation of aggrecan by bovine articular cartilage explant cultures.

The catabolism of 35S-labeled aggrecan and loss of tissue glycosaminoglycans was investigated using bovine articular cartilage explant cultures maintained in medium containing 10(-6) M retinoic acid or 40 ng/ml recombinant human interleukin-1alpha (rHuIL-1alpha) and varying concentrations (1-1000 microg/ml) of sulfated glycosaminoglycans (heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and keratan sulfate) and calcium pentosan polysulfate (10 microg/ml). In addition, the effect of the sulfated glycosaminoglycans and calcium pentosan polysulfate on the degradation of aggrecan by soluble aggrecanase activity present in conditioned medium was investigated. The degradation of 35S-labeled aggrecan and reduction in tissue levels of aggrecan by articular cartilage explant cultures stimulated with retinoic acid or rHuIL-1alpha was inhibited by heparin and heparan sulfate in a dose-dependent manner and by calcium pentosan polysulfate. In contrast, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and keratan sulfate did not inhibit the degradation of 35S-labeled aggrecan nor suppress the reduction in tissue levels of aggrecan by explant cultures of articular cartilage. Heparin, heparan sulfate and calcium pentosan polysulfate did not adversely affect chondrocyte metabolism as measured by lactate production, incorporation of [35S]-sulfate or [3H]-serine into macromolecules by articular cartilage explant cultures. Furthermore, heparin, heparan sulfate and calcium pentosan polysulfate inhibited the proteolytic degradation of aggrecan by soluble aggrecanase activity. These results suggest that highly sulfated glycosaminoglycans have the potential to influence aggrecan catabolism in articular cartilage and this effect occurs in part through direct inhibition of aggrecanase activity.     

Effect of monensin on the sulfation of heparan sulfate proteoglycan from endothelial cells.

Monensin is a monovalent metal ionophore that affects the intracellular translocation of secretory proteins at the level of trans-Golgi cisternae. Exposure of endothelial cells to monensin results in the synthesis of heparan sulfate and chondroitin sulfate with a lower degree of sulfation. The inhibition is dose dependent and affects the ratio [35S]-sulfate/[3H]-hexosamine of heparan sulfate from both cells and medium, with no changes in their molecular weight. By the use of several degradative enzymes (heparitinases, glycuronidase, and sulfatases) the fine structure of the heparan sulfate synthesized by control and monensin-treated cells was investigated. The results have shown that among the six heparan sulfate disaccharides there is a specific decrease of the ones bearing a sulfate ester at the 6-position of the glucosamine moiety. All other biosynthetic steps were not affected by monensin. The results are indicative that monensin affects the hexosamine C-6 sulfation, and that this sterification is the last step of the heparan sulfate biosynthesis and should occur at the trans-Golgi compartment.     

Formation of dermatan sulfate by cultured human skin fibroblasts. Effects of sulfate concentration on proportions of dermatan/chondroitin

[3H,35S]Dermatan/chondroitin sulfate glycosaminoglycans produced during culture of fibroblasts in medium containing varying concentrations of sulfate were tested for their susceptibility to chondroitin ABC lyase and chondroitin AC lyase. Chondroitin ABC lyase completely degraded [3H]hexosamine-labeled and [35S] sulfate-labeled dermatan/chondroitin sulfate to disaccharides. Chondroitin AC lyase treatment of the labeled glycosaminoglycans produced different results. With this enzyme, dermatan/chondroitin sulfate formed at high concentrations of sulfate yielded small glycosaminoglycans and larger oligosaccharides but almost no disaccharide. This indicated that the dermatan/chondroitin sulfate co-polymer contained mostly iduronic acid with only an occasional glucuronic acid. As the medium sulfate concentration was progressively lowered, there was a concomitant increase in the susceptibility to degradation by chondroitin AC lyase. Thus, the labeled glycosaminoglycans formed at the lowest concentration of sulfate yielded small oligosaccharides including substantial amounts of disaccharide. The smaller chondroitin AC lyase-resistant [3H,35S]dermatan/chondroitin sulfate oligosaccharides were analyzed by gel filtration. Results indicated that, in general, the iduronic acid-containing disaccharide residues present in the undersulfated [3H,35S]glycosaminoglycan were sulfated, whereas the glucuronic acid-containing disaccharide residues were non-sulfated. This work confirms earlier reports that there is a relationship between epimerization and sulfation. Moreover, it demonstrates that medium sulfate concentration is critical in determining the proportions of dermatan to chondroitin (iduronic/glucuronic acid) produced by cultured cells.     

Transformed mouse mammary epithelial cells synthesize undersulfated basement membrane proteoglycan

Proteoglycans deposited in the basal lamina of [14C] glucosamine-labeled normal and [3H]glucosamine-labeled transformed mouse mammary epithelial cells grown on type I-collagen gels, were extracted in 4 M guanidinium chloride and cofractionated over Sepharose CL 4B. The heparan sulfate chains carried by these proteoglycans were isolated by treatment with alkaline borohydride, protease K, chondroitinase ABC, and cetylpyridinium chloride precipitation. Heparan sulfate isolated from transformed cell cultures consistently eluted from DEAE-cellulose at lower salt concentrations and was of smaller apparent Mr when chromatographed over Sepharose CL 6B, than heparan sulfate of normal cell cultures. Experiments using doubly labeled cultures ([3H]glucosamine and [35S]sulfate) demonstrated an approximately 30% reduction in the sulfate/hexosamine ratio in heparan sulfate derived from transformed cultures. Both N- and O-sulfate were decreased. The decreased Mr and decreased sulfation of heparan sulfate upon transformation appear sufficient to explain the altered heparan sulfate/chondroitin sulfate ratios previously observed in these cells. These changes may have implications for the molecular interactions in which these proteoglycans are normally engaged during basal lamina assembly, and cause the poor basal lamina formation displayed by these transformed cells.     

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.     

Biosynthesis of chondroitin sulfate. Organization of sulfation

The potential relationship of an intact membrane organization for the synthesis of chondroitin and chondroitin 4-sulfate was examined after modification of a mouse mast cell microsomal system with the nonionic detergent, Triton X-100. The results indicated that Triton X-100 had no effect on the rate of polymerization but had a slight effect on the size of glycosaminoglycan chains. An "all or nothing" pattern of sulfation of newly formed chondroitin was obtained in both the presence and the absence of Triton X-100, and this pattern did not change whether sulfation was initiated concurrent with or subsequent to polymerization. Sulfation of exogenous [14C]chondroitin and exogenous proteo[3H]chondroitin by the microsomal system required Triton X-100 but still produced an all or nothing pattern rather than a random sulfation pattern. When a 100,000 x g supernatant fraction was utilized for sulfation of [14C]chondroitin or proteo[3H]chondroitin, Triton X-100 was not needed, and a partial sulfation pattern was obtained. However, it was similar to the all or nothing pattern in that it still produced two populations, with some chains nonsulfated and others approximately 50% sulfated. When chondroitin hexasaccharide was used with 3'-phosphoadenylylphospho[35S]sulfate, multiple GalNAc residues of the individual hexasaccharides were found to be sulfated. This was relatively independent of Triton X-100 or the concentration of the hexasaccharide acceptors. With soluble enzyme, sulfation of multiple GalNAc residues on the individual hexasaccharide molecules was even greater, so that trisulfated products were found. These results suggest that efficient sulfation of chondroitin is related to enzyme-substrate interaction more than to membrane organization.     

Altered metabolism of heparan sulfate in simian virus 40 transformed cloned mouse cells.

Glycoconjugates have been analyzed from a family of closely related mouse cells: a parent clone and three daughter subclones, two of which expressed the simian virus 40 (SV40) T-antigen. The experimental procedure involved the simultaneous comparison by DEAE-cellulose chromatography of papain-digested macromolecules from the parent, labeled with [3H]glucosamine, and one of the daughter subclones, labeled with [14C]-glucosamine. Three cultures compartments (the medium, the cell surface trypsinate, and the cells) from the paired cell lines were combined at the earliest time during the harvesting of the cells. Heparan sulfate on the surface of cells and secreted into the medium from T-antigen-positive subclones was eluted at lower salt concentrations from the anion exchange column than that from the parent clone. In the viable trypsinized cells a marked reduction of heparan sulfate was detected in the T-antigen-positive subclones. These changes were highly reproducible, were observed during both logarithmic and stationary phase of growth, and neither change was observed in the T-antigen-negative sister subclone. The elution point of heparan sulfate from Sepharose 6B was unaltered. Ratios of 35S to 3H for heparan sulfate obtained from cells doubly labeled with [35S]sulfate and [3H]glucosamine were lower in the T-antigen-positive subclones. Similar changes for the 35S to 3H ratio of chondroitin sulfate were associated with only small alterations in elution from anion exchange columns. Kinetic experiments suggested a reduced rate of incorporation of [35S]sulfate with no change in turnover rate. A substantial portion of the labeled heparan sulfate was associated with the cell surface; in contrast most of the hyaluronic acid and a large proportion of the chondroitin sulfate was apparently secreted. Quantitative changes in hyaluronic acid labeling did not correlate with expression of T-antigen. Glycosaminoglycans left on the dish after detaching cells with ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid were nearly completely released by subsequent trypsinization. Cell detachment by trypsinization left an insignificant amount of labeled glycosaminoglycan on the dish surface. The alterations in heparan sulfate metabolism correlated with the expression of T-antigen and with the cells' ability to grow to high densities in monolayer culture, but not with growth in suspension in viscous medium. Tumorigenicity of the subclones was essentially the same as that of the parent clone.     

Altered expression of glycosaminoglycans in metastatic 13762NF rat mamma adenocarcinoma cells

A difference in the expression and metabolism of sulfated glycosaminoglycans between rat mammary tumor cells derived from a primary tumor and those from its metastatic lesions has been observed. Cells from the primary tumor possessed about equal quantities of chondroitin sulfate and heparan sulfate on their cell surfaces but released fourfold more chondroitin sulfate than heparan sulfate into their medium. In contrast, cells from distal metastatic lesions expressed approximately 5 times more heparan sulfate than chondroitin sulfate in both medium and cell surface fractions. This was observed to be the result of differential synthesis of the glycosaminoglycans and not of major structural alterations of the individual glycosaminoglycans. The degree of sulfation and size of heparan sulfate were similar for all cells examined. However, chondroitin sulfate, observed to be only chondroitin 4-sulfate, from the metastases-derived cells had a smaller average molecular weight on gel filtration chromatography and showed a decreased quantity of sulfated disaccharides upon degradation with chondroitin ABC lyase compared to the primary tumor derived cells. Major qualitative or quantitative alterations were not observed for hyaluronic acid among the various 13762NF cells. The metabolism of newly synthesized sulfated glycosaminoglycans was also different between cells from primary tumor and metastases. Cells from the primary tumor continued to accumulate glycosaminoglycans in their medium over a 72-h period, while the accumulation of sulfated glycosaminoglycans in the medium of metastases-derived cells showed a plateau after 18-24 h. A pulse-chase kinetics study demonstrated that both heparan sulfate and chondroitin sulfate were degraded by the metastases-derived cells, whereas the primary tumor derived cells degraded only heparan sulfate and degraded it at a slower rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis of proteoglycans by rat embryo parietal yolk sacs in organ culture

The embryonic rat parietal yolk sac has been previously shown to synthesize a number of basement membrane glycoconjugates including type IV procollagen, laminin, and entactin. In this study, parietal yolk sacs were isolated from 14.5-day rat embryos and incubated in organ culture for 4-7 h with [35S]sulfate, [3H] glucosamine, and/or 3H-labeled amino acids, and the newly synthesized proteoglycans were characterized. The major [35S]sulfate-labeled macromolecule represented approximately 90% of the medium and 80% of the tissue radioactivity. It also represented nearly 80% of the total [3H]glucosamine-labeled glycosaminoglycans. After purification by sequential ion-exchange chromatography and isopycnic CsCI density gradient ultracentrifugation, size-exclusion high-performance liquid chromatography showed a single species with an estimated Mr of 8-9 X 10(5). The intact proteoglycan did not form aggregates in the presence of exogenous hyaluronic acid or cartilage aggregates. Alkaline borohydride treatment released glycosaminoglycan chains with Mr of 2.0 X 10(4) which were susceptible to chondroitinase AC II and chondroitinase ABC digestion. Analysis by high-performance liquid chromatography of the disaccharides generated by chondroitinase ABC digestion revealed that chondroitin 6-sulfate was the predominant isomer. The uronic acid content of the glycosaminoglycans was 92% glucuronic acid and 8% iduronic acid, and the hexosamine content was 96% galactosamine and 4% glucosamine. No significant amounts of N- or O-linked oligosaccharides were detected. Deglycosylation of the proteoglycan with chondroitinase ABC in the presence of protease inhibitors revealed a protein core with an estimated Mr of 1.25-1.35 X 10(5). These results indicated that the major proteoglycan synthesized by the 14.5-day rat embryo parietal yolk sac is a high-density chondroitin sulfate containing small amounts of copolymeric dermatan sulfate. Hyaluronic acid and minor amounts of heparan sulfate proteoglycan were also detected.     

Heparan sulfate biosynthesis by embryonic tissues and primary fibroblast populations.

Fibroblasts from cornea, heart, and skin of day 14 embryonic chicks demonstrate the ability to make heparan sulfate-like polysaccharide when examined during the 10 hr period immediately following their removal from the embryo. Both the whole tissues from which these fibroblasts are isolated and the fibroblasts grown for 2–5 weeks in vitro also synthesize heparan sulfate. During their first few days in vitro, the three fibroblast populations display increasing rates of [³⁵S]-sulfate and d-[1-³H]-Glucosamine incorporation into glycosaminoglycans and sharp fluctuations of those rates, yet the percentage of total [³⁵S]-sulfate incorporated into heparan sulfate-like polysaccharide and the distribution of this polysaccharide between cells and nutrient medium do not change significantly. During their first 48 hr in vitro, skin fibroblasts, but not those from cornea or heart, show steadily decreasing discrepancies between the proportions of [³⁵S]-sulfate and d-[1-³H]-Glucosamine incorporated into heparan sulfate, suggesting a sharp decline in the synthesis of nonsulfated glycosaminoglycans. These data support the hypothesis of Kraemer than many cell-types in vivo may normally make heparan sulfate. The data largely eliminate the hypothesis that the biosynthesis of this polysaccharide is selectively stimulated as embryonic cells adapt to growth in vitro.     

Apparent sulfation of glycosaminoglycans by ascorbic acid 2-[3 5-S] sulfate: an explanation.

The sulfation of glycosaminoglycans by ascorbic acid 2-[35S]sulfate was studied in costal cartilage and chondrocytes in vitro. Negligable (if any) sulfation of glycosaminoglycans was detected with immediately isolated ascorbic acid 2-[35S]sulfate. However, formation of [35S]glycosaminoglycans was readily detected with ascorbic acid 2-[35S]sulfate which had been stored at minus 20 degrees C for several days. The [35S]glycosaminoglycans did not result from the direct transfer of 35S from ascorbic acid 2-sulfate but rather from a decomposition product of ascorbic acid 2-[35S]sulfate. Evidence is presented to show that the sulfation pathway with the decomposition product involves exchange with inorganic sulfate, and strongly suggests that sulfation proceeds via 3'-phosphoadenosine 5'-phosphosulfate. The decomposition product appears similar to inorganic sulfate in several test systems. In view of these observations, it is suggested that previous conclusions implicating as acid 2-sulfate as a biological sulfate donor, based on the use of ascorbic acid 2-[35S]sulfate be re-evaluated.     

Metabolism of sulfated glycosaminoglycans in cultivated bovine arterial cells. I. Characterization of different pools of sulfated glycosaminoglycans.

"Fibroblast-like" cells from the intimal layer of bovine aorta were grown in culture. The formation, composition, molecular weight and turnover rate of different pools of glycosaminoglycans were investigated in cultures incubated in the presence [35S]sulfate or [14C]glucosamine. The newly synthesized glycosaminoglycans are distributed into an extracellular pool (37 - 58%), a cell-membrane associated or pericellular pool (23 - 33%), and an intracellular pool (19 - 30%), each pool exhibiting a characteristic distribution pattern of chondroitin sulfate, dermatan sulfate, heparan sulfate and hyaluronate. The distribution pattern of the extracellular glycosaminoglycans resembles closely that found in bovine aorta. A small subfraction of the pericellular pool - tentatively named "undercellular" pool--has been characterized by its high heparan sulfate content. The intracellular and pericellular [35S]glycosaminoglycan pools reach a constant radioactivity after 8-12 h and 24 h, respectively, whereas the extracellular [35S]glycosaminoglycans are secreted into the medium at a linear rate over a period of at least 6 days. The intracellular glycosaminoglycans are mainly in the process of degradation, as indicated by their low molecular weight and by their half-life of 7 h, but intracellular dermatan sulfate is degraded more rapidly (half-life 4-5 h) than intracellular chondroitin sulfate and heparan sulfate (half-life 7-8 h). Glycosaminoglycans leave the pericellular pool with a half-life of 12-14 h by 2 different routes: about 60% disappear as macromolecules into the culture medium, and the remainder is pinocytosed and degraded to a large extent. Extracellular and at least a part of the pericellular glycosaminoglycans are proteoglycans. Even under dissociative conditions (4M guanidinium chloride) their hydrodynamic volume is sufficient for partial exclusion from Sepharose 4B gel. The existence of topographically distinct glycosaminoglycan pools with varying metabolic characteristics and differing accessibility for degradation requiresa reconsideration and a more reserved interpretation of results concerning the turnover rates of glycosaminoglycans as determined in arterial tissue.     

Biosynthesis of glycosaminoglycans in the developing retina

The glycosaminoglycans of neural retinas from 5-, 7-, 10-, and 14-day chick embryos were labeled in culture with [³H]glucosamine and ³⁵SO₄, extracted, and isolated by gel filtration. The incorporation of label per retina into glycosaminoglycans increased with embryonic age, but that per cell and per unit weight of uronic acid decreased. Specific enzyme methods coupled with gel filtration and paper chromatography demonstrated that [³H]glucosamine incorporation into chondroitin sulfate increased between 5 and 14 days from 7 to 34% of the total incorporation into glycosaminoglycans. During this period, incorporation into chondroitin-4-sulfate increased relative to that into chondroitin-6-sulfate. Between 5 and 10 days, incorporation into heparan sulfate showed a relative decline from 89 to 61%. Incorporation into hyaluronic acid always represented less than 2% of the total. A twofold greater increase in galactosamine concentration than in glucosamine concentration in the glycosaminoglycan fraction between 7 and 14 days supports the conclusion that chondroitin sulfate was the most rapidly accumulating glycosaminoglycan. ECTEOLA-cellulose chromatography revealed a heterogeneity in the size and/or net charge of chondroitin sulfate and heparan sulfate. We conclude that incorporation of exogenous precursors into glycosaminoglycans in the chick retina decreases relative to cell number as differentiation progresses from a period of high mitotic activity to one of tissue specialization, and that it is accompanied by a net accumulation of glycosaminoglycan and a change in the pattern of its synthesis.     

Glycosaminoglycan biosynthesis in arterial wall. Sulfation of heparan sulfate in cell membrane of aortic media-intima

Incubation of microsomal fractions with labelled 3'-phosphoadenylyl sulfate results in incorporation of [35S]sulfate into endogenous glycosaminoglycans. Specific radioactivity observed incorporated into heparan sulfate chains is 10-fold greater than that incorporated into chondro?tin sulfate chains. This is in agreement with the results obtained for glycosylation of glycosaminoglycans in arterial wall membrane fractions. Sulfation of heparan sulfate was studied since it contains N- and O-sulfate groups in contrast with the other sulfated glycosaminoglycans which contain only O-sulfate groups. Sulfation of heparan sulfate occurs rapidly, since sulfate incorporation is detected after exposure for only 0.5 min. Heparan sulfate was identified on the basis of its resistance to hyaluronidase and chondro?tin ABC lyase, its susceptibility to heparitinase, its sensitivity to nitrous acid and the presence of glucosamine as the only hexosamine. The chemical composition of the purified heparan sulfate fractions provides evidence for the high degree of sulfation of its chains. Studies into the distribution of sulfate residues on heparan sulfate at different times of sulfation indicate that N-sulfate groups are not randomly introduced into the polymer. The relationship between the processes of N- and O-sulfation was studied. The present results demonstrate that preferential N-sulfation is obtained for incorporation of labelled precursor over a short period, the O-sulfation occurring on previously N-sulfated heparan sulfate.     

Comparison of Proteoglycans Extracted by Saline and Guanidinium Chloride from Cultured Chick Retinas

Abstract: To compare the loosely associated sulfated proteoglycans with those tightly bound to membranes, retinas from 14-day chick embryos were subjected to progressively disruptive techniques. The most easily removed proteoglycans were isolated from the medium in which the tissue was labeled with [³⁵S]sulfate. On the average, 25% of the glycosaminoglycans were in the labeling medium, 39% were in proteoglycans extracted from the tissue in the balanced salt solution, 32% were in a 4 m -guanidinium chloride (GuCl) fraction, and 4% remained unextracted. These glycosaminoglycans contained, respectively, 28, 28, 40, and 4% of the incorporated [³⁵S]sulfate. On the basis of electrophoretic mobility and TLC of chondroitinase digests, the ratio of ³⁵S in chondroitin sulfate to that in heparan sulfate was 4–7 times higher in the medium and balanced salt extracts than in the GuCl extracts. In both extracts there was more ³⁵S in chondroitin-6-sulfate than in chondroitin-4-sulfate. Dialysis of the extracts against 0.5 M-NaCl resulted in the precipitation of about 12% of the glycosaminoglycans in the saline extracts and about 40% in GuCl extract. These subfractions, which were relatively enriched in heparan sulfate, were largely soluble in dithiothreitol in 8 m -urea (DTT). Similarities between the proteoglycans in the medium and those extracted by balanced salt solutions suggest that the saline-extracted proteoglycans were for the most part loosely associated with cell surfaces or extracellular matrices, whereas the GuCl-extracted proteoglycans probably were bound to membranes.     

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.     

Glycosaminoglycan Composition of PC 12 Pheochromocytoma Cells: A Comparison with PC12D Cells, a New Subline of PC12 Cells

PC12D cells, a new subline of conventional PC12 cells, respond not only to nerve growth factor but also to cyclic AMP by extending their neurites. These cells are flat in shape and are similar in appearance to PC12 cells that have been treated with nerve growth factor for a few days. In both cell lines, we have characterized the glycosaminoglycans, the polysaccharide moieties of proteoglycans, which are believed to play an important role in cell adhesion and in cell morphology. Under the present culture conditions, only chondroitin sulfate was detected in the media from PC12 and PC12D cells, whereas both chondroitin sulfate and heparan sulfate were found in the cell layers. The levels of cell-associated heparan sulfate and chondroitin sulfate were about twofold and fourfold higher in PC12D cells than in PC12 cells, respectively. Compared to PC12 cells, the amounts of [35S]sulfate incorporated for 48 h into chondroitin sulfate were twofold lower but those into heparan sulfate were 35% higher in PC12D cells. The amount of chondroitin sulfate released by PC12D cells into the medium was about a half of that released by PC12 cells. The ratio of [35S]sulfate-labeled heparan sulfate to chondroitin sulfate was 6.2 in PC12D cells and 2.2 in PC12 cells. These results suggest that there may be some correlation between the increase in content of glycosaminoglycans and the change in cell morphology, which is followed by neurite outgrowth.