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.     

Turnover, change of composition with rate of cell growth and effect of phenylxyloside on synthesis and structure of cell surface sulfated glycosaminoglycans of normal and transformed cells

The synthesis of sulfated glycosaminoglycans was analysed in mouse fibroblasts during the transition from exponential growth to quiescent monolayers. 'Normal' Swiss 3T3 fibroblasts were compared with SV40 transformed 3T3, C6, ST1 and HeLa cells. p-Nitrophenyl-beta-D-xyloside, an artificial acceptor for glycosaminoglycans synthesis, was used as a probe. Exponentially growing 'normal' 3T3 cells synthesized both dermatan sulfate and chondroitin 4-sulfate, retaining the latter and releasing the former to the medium. Upon reaching quiescence these cells switched to retention of dermatan sulfate and release of chondroitin 4-sulfate. SV3T3 cells synthesized several fold less sulfated glycosaminoglycans than 'normal' 3T3. Even though SV3T3 cells are able to synthesize dermatan sulfate, they only retained chondroitin 4-sulfate, never switching to retention of dermatan sulfate. These results indicated that the transition from rapidly proliferating to resting G0 state in normal cells is accompanied by a switch from chondroitin-sulfate rich to dermatan-sulfate-rich cells. This switching was not observed with transformed cells, which are unable to enter the G0 state. Phenylxyloside caused a several fold increase in glycosaminoglycans released to the medium in both cell types, but it did not interfere with either growth rate or cell morphology. Particularly the phenylxyloside treatment led to an increase of more than 10-fold in production of dermatan and chondroitin sulfate by SV3T3, C6, ST1 and HeLa cells. This demonstrated that transformed cells have a high capacity for glycosaminoglycan synthesis. Analysis of enzymatic degradation products of glycosaminoglycans, synthesized in the presence of phenylxyloside, by normal and transformed cells, led to the finding of 4- and 6-sulfated iduronic and glucuronic acid-containing disaccharides. This result indicated that the xyloside causes the synthesis of a peculiar chondroitin sulfate/dermatan sulfate, in both normal and transformed cells.     

Influences of sulfated glycosaminoglycans on biosynthesis of hyaluronic acid in rabbit knee synovial membrane

The effect of various sulfated glycosaminoglycans on glycoconjugates syntheses in synovial membranes of rabbit knee joints in culture was investigated by two different approaches. In the first approach, synovial membranes isolated from rabbit knee joints were cultured in the presence of sulfated glycosaminoglycans and [14C]glucosamine. In the second approach, solutions of sulfated glycosaminoglycans were injected into rabbit knee joints and synovial membranes isolated from the joints were cultured in the presence of [14C]glucosamine. The major part of [14C]glucosamine-labeled glycoconjugates associated with the synovial membranes and secreted into culture medium was hyaluronic acid. Of the natural glycosaminoglycans tested, dermatan sulfate gave the maximum stimulation of hyaluronic acid synthesis followed by chondroitin 4- and 6-sulfate. Heparin, heparan sulfate, keratan sulfate, keratan polysulfate, and hyaluronic acid had no significant effect. Of the chemically polysulfated glycosaminoglycans, GAGPS (a persulfated derivative of chondroitin sulfate) gave high stimulation but N-acetylchitosan 3,6-disulfate had no effect. The effect of sulfated glycosaminoglycans on hyaluronic acid synthesis was the same in both experimental approaches. The increase in the amount of secreted hyaluronic acid in culture medium paralleled that in synovial membranes. The results indicate that the galactosamine-containing sulfated glycosaminoglycans have a specific stimulatory effect on hyaluronic acid synthesis. A high degree of sulfation of the molecules appeared to potentiate the stimulatory effect.     

Regulation of serum glycosaminoglycan sulfotransferase activities: inhibition by sulfated glycosaminoglycans and activation by polyamines and basic peptides including a polylysine-containing segment of the c-Ki-ras 2 protein

The regulatory mechanisms for the glycosaminoglycan sulfotransferases in fetal calf serum were investigated. The enzymes examined were those which transfer sulfate from 3'-phosphoadenosine 5'-phosphosulfate to 1) position 6 of the internal N-acetylgalactosamine units of chondroitin, 2) position 6 of galactose units of keratan sulfate, and 3) position 2 (an amino group) of glucosamine units of heparan sulfate. The former two enzymes were activated by spermidine, spermine, protamine, and poly L-lysine. All the enzymes were strongly inhibited by heparin and dextran sulfate, whereas only the chondroitin 6-O-sulfotransferase was inhibited by sulfated galactosaminoglycans. The inhibition of this enzyme by the sulfated glycosaminoglycans was abolished by polylysine, indicating that the activation by polylysine is partly due to the neutralization of endogenous acidic inhibitors, including sulfated glycosaminoglycans. Affinity chromatographic studies demonstrated that heparin specifically binds to the three enzymes, which have anionic isoelectric points, and that chondroitin 6-sulfate, spermine, and polylysine bind to the former two enzymes under physiological conditions. Thus, the activation by spermine and polylysine as well as the inhibition by sulfated glycosaminoglycans also appears to occur through their binding to the enzymes. Studies with synthetic lysine oligomers and an affinity-purified (approximately 700-fold) fraction containing the former two enzymes indicated that the pentamer is the minimum unit required for the activation. A synthetic peptide, containing six consecutive lysines at the carboxy terminus of the human c-Ki-ras 2 protein, also regulated the two enzyme activities at micromolar concentrations. The possible physiological implications of the observed effects of these regulatory substances on the glycosaminoglycan sulfotransferases are discussed in relation to glycosaminoglycan synthesis during the proliferation, differentiation, and transformation of cells. The possibility of sulfated glycosaminoglycans being enzyme regulators is also discussed.     

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

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

Sulfated glycosaminoglycans from ovary of Rhodnius prolixus

We have characterized sulfated glycosaminoglycans from ovaries of the blood-sucking insect Rhodnius prolixus, and determined parameters of their synthesis and distribution within this organ by biochemical and histochemical procedures. The major sulfated glycosaminoglycan is heparan sulfate while chondroitin 4-sulfate is a minor component. These glycosaminoglycans are concentrated in the ovarian tissue and are not found inside the oocytes. Besides this, we detected the presence of a sulfated compound distinguished from sulfated glycosaminoglycans and possibly derived from sulfated proteins. Conversely to the compartmental location of sulfated glycosaminoglycans, the unidentified sulfated compound is located in the ovarian tissue as well as inside the oocytes. Based on these and other findings, the possible roles of ovarian sulfated glycosaminoglycans on the process of oogenesis in these insects are discussed.     

Glycosaminoglycans and proteoglycans synthesized by rat limb buds during prechondrogenic and chondrogenic stages

The sulfated glycosaminoglycans synthesized in the forelimb plates of rats on days 12, 13, 14, and 15 of gestation were characterized by their susceptibility to various glycosaminoglycan lyases. On days 12 and 13, heparan sulfate accounted for approximately 65% of the newly synthesized sulfated glycosaminoglycans. Small amounts of dermatan sulfate and chondroitin sulfates were also observed. On day 14, the relative amount of chondroitin 4-sulfate began to increase, there being a compensatory decrease in the amount of heparan sulfate. 35S-Sulfate-labeled material was extracted from day-13 forelimb plates with 4 M guanidine/HCl without proteolysis. Using ultracentrifugation on a sucrose density gradient, the extract was separated into two peaks: a light peak (L) mainly composed of heparan sulfate, and a faster-sedimenting peak (M) mainly composed of chondroitin sulfate. The cartilage-type proteoglycan (H) was first detectable on day 14 of gestation, indicating that chondrogenesis in rat forelimb plates starts on day 14 of gestation. In addition to these previously identified glycosaminoglycans or proteoglycans, we isolated an unknown component in the glycosaminoglycan preparations obtained from limb plates during these developmental stages. This component was not found in glycosaminoglycan preparations obtained either from the brain or tail of rat fetuses at the same stages.     

Human venous and arterial glycosaminoglycans have similar affinity for plasma low-density lipoproteins

We compared the glycosaminoglycan content of human venous and arterial walls. The most abundant glycosaminoglycan in human veins is dermatan sulfate whereas chondroitin 4/6-sulfate is preponderant in arteries. The concentrations of chondroitin 4/6-sulfate and heparan sulfate are approximately 4.8- and approximately 2.5-fold higher in arteries than in veins whereas dermatan sulfate contents are similar in the two types of blood vessels. Normal and varicose saphenous veins do not differ in their glycosaminoglycan contents. It is known that certain glycosaminoglycan species from the arterial wall, mainly high-molecular-weight fractions of dermatan sulfate+chondroitin 4/6-sulfate have greater affinity for plasma LDL. These types of glycosaminoglycans can be identified on a LDL-affinity column. We now demonstrated that a similar population of glycosaminoglycan also occurs in veins, although with a lower concentration than in the arteries due to less chondroitin 4/6-sulfate with affinity for LDL. The concentrations of dermatan sulfate species, which interact with LDL, are similar in arteries and veins. The presence of these glycosaminoglycans with affinity to plasma LDL in veins raises interesting questions concerning the role of these molecules in the pathogenesis of atherosclerosis. Possibly, the presence of these glycosaminoglycans in the vessel wall are not sufficient to cause retention of LDL and consequently endothelial dysfunction, but may require additional intrinsic factors and/or the hydrodynamic of the blood under the arterial pressure.     

The binding of chondroitin 6-sulfate to plasma low density lipoprotein

The interaction in vitro of several sulfated glycosaminoglycans with low density lipoproteins (LDL) has been studied. Chondroitin 6-sulfate and heparin were the only ones to produce turbidity when added to LDL in presence of Ca2+. However, when these two glycosaminoglycans were applied to LDL-affinity columns in presence of Ca2+, only chondroitin 6-sulfate was retained. Partially desulfated chondroitin 6-sulfate was not retained on LDL-affinity column, indicating the relevance of sulfate groups in the binding of LDL. Since chondroitin 4-sulfate and heparin, with a sulfate content respectively equal to and greater than that of chondroitin 6-sulfate, are not retained on LDL-affinity columns, the factors relevant to the binding of LDL are probably the conformation of the glycan in solution and the orientation of its sulfate groups.     

Inhibitory effects of various 3'-deoxyribonucleotides on DNA polymerase alpha 2-primase from developing cherry salmon (Oncorhynchus masou) testes

DNA polymerase alpha 2-primase has been purified 2750 fold from developing cherry salmon (Oncorhynchus masou) testes by the following purification steps: fractional extraction, phosphocellulose (1st), ammonium sulfate fractionation, DEAE-cellulose, phosphocellulose (2nd), hydroxylapatite and single-stranded DNA-cellulose column chromatographies. Final preparation of this enzyme has a specific activity of 107,000 units/mg protein (activated salmon sperm DNA as template-primer). DNA primase activity (rGTP dependent incorporation of labelled dGMP into poly (dC) or rNTP dependent incorporation of dNMP into M13 single-stranded DNA) was tightly associated with DNA polymerase alpha activity during all stage of this purification process. Inhibition of DNA primase activity by six kinds of 3'-deoxyribonucleotides was studied by using rNTP dependent DNA synthesis on M13 DNA as template. The inhibition constants (Ki) were larger than those of DNA-dependent RNA polymerases I and II. However, Ki/Km values were very close.     

Modulation of cAMP-dependent protein kinase by derivatives of chondroitin sulfate

Here, we report investigations about the direct effect of glycosaminoglycans, such as dermatan sulfate, chondroitin 4- and 6-sulfate upon cAMP-dependent protein kinase activity. The results indicate that glycosaminoglycans strongly influence the phosphorylation activity of this enzyme against histone type IIa and [Val⁶,Ala⁷]-kemptide. While chondroitin 4-sulfate and dermatan sulfate exhibit inhibitory effects, chondroitin 6-sulfate shows a stimulating effect. In addition, the chondroitin 6-sulfate is also able to reduce the chondroitin 4-sulfate and dermatan sulfate specific inhibition.     

Divergent regulation of proteoglycan and glycosaminoglycan free chain expression in human keratinocytes and melanocytes

Summary Keratinocytes and melanocytes, which together form units of structure and function within human epidermis, are known to differ in expression of autocrine growth factors, particularly those with heparin binding affinity. Because such cytokines could be regulated by the endogenous heparinlike glycosaminoglycan, heparan sulfate, proteoglycan synthesis was compared between human keratinocytes and melanocytes cultured from a common donor. Following steady-state isotopic labeling under conditions of active growth (low density cultures) and growth inhibition (high density cultures), the sulfated polymers were isolated from conditioned media and cell extracts. We found that keratinocytes produced substantially more sulfated glycosaminoglycans than did the melanocytes. There was no evidence for hyaluronic acid synthesis by the melanocytes. The majority of [³⁵S]-sulfate labeling was in the heparan sulfates of the keratinocytes and in the chondroitin sulfates of the melanocytes. During the transition from active growth to growth inhibition, there was increased heparan sulfate proteoglycan and free chain synthesis by keratinocytes but not by melanocytes, and chondroitin sulfate proteoglycan production declined in both cell lineages. The differences may reflect divergent evolution as each cell type came to exploit those complex polysaccharides in different ways to regulate molecular pathways of growth and differentiation. The coupling of growth inhibition with augmented synthesis of heparan sulfates observed for the keratinocytes suggests a regulatory role in growth factor signaling in that cell type.     

Proteoglycan and glycosaminoglycan synthesis by cultured rat mesangial cells

The synthesis of metabolically labeled proteoglycans and glycosaminoglycans from medium, cell layer and substrate attached material by rat glomerular mesangial cells in culture was characterized. The cellular localization of the labeled proteoglycans and glycosaminoglycans was determined by treating the cells with Flavobacterial heparinase. Of the total sulfated glycosaminoglycans, 33% were heparan sulfate; 55% of the cell layer material was heparan sulfate; 80% of sulfated proteins in the medium were chondroitin sulfate/dermatan sulfate. Putative glycosaminoglycan free chains of heparan sulfate and chondroitin sulfate were found in both the medium and cell layer; 95% of total proteoglycans and most (90%) of the putative heparan sulfate free chains were removed from the cell layer by the heparinase, whereas only 50% of the chondroitin sulfate and 25% of dermatan sulfate were removed. Large amounts of hyaluronic acid labeled with 3H glucosamine were found in the cell layer. In summary, approximately 60% of total sulfated glycoproteins was in the form of putative glycosaminoglycan free chains. Thus rat mesangial cells may synthesize large amounts of putative glycosaminoglycan free chains, which may have biological functions in the glomerulus independent of proteoglycans.     

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 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 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.     

Isolation and characterization of the sulfated glycosaminoglycans of the vitreous body

Ester sulfate containing glycosaminoglycans comprising approx. 3% of the total glycosaminoglycan content, have been isolated from protease-digested bovine vitreous body by stepwise fractionation on AG-1X2(Cl^?) and gel filtration on Bio-Gel P-300. Two heparan sulfate and two chondroitin-4-sulfate fractions were isolated in nearly pure form. The heparan sulfate fractions were undersulfated and contained the same relative proportions of N- and O-sulfate (1 : 2), although the total sulfate content differed by approx. 100%. No chondroitin-6-sulfate was present in the isolates, based on evidence obtained from chondroitin ABC lyase experiments.