Effect of p-nitrophenyl-beta-D-xyloside on proteoglycan synthesis and extracellular matrix formation by bovine corneal endothelial cell cultures

The effect of p-nitrophenyl-beta-D-xyloside on proteoglycan synthesis and extracellular matrix (ECM) formation by cultured bovine corneal endothelial (BCE) cells was investigated. BCE cells actively proliferating on plastic dishes produced in the absence of xyloside an ECM containing various proteoglycans. Heparan sulfate was the main 35S-labeled glycosaminoglycan component (83%). Dermatan sulfate (14%) and chondroitin sulfate (3%) were also present. Exposure of actively proliferating BCE cells to xyloside totally inhibited synthesis of proteoglycans containing dermatan sulfate or chondroitin sulfate and caused an 86% inhibition of heparan sulfate proteoglycan synthesis. The heparan sulfate proteoglycans that were extracted from the ECM produced by BCE cells exposed to xyloside had a smaller size and a reduced charge density compared to their counterparts extracted from the ECM of cultures not exposed to xyloside. In contrast to the inhibitory effect of the xyloside on proteoglycan synthesis, exposure of actively proliferating BCE cells to xyloside stimulated synthesis of free chondroitin sulfate and heparan sulfate chains. All of the xyloside-initiated glycosaminoglycan chains were secreted into the culture medium. The proteoglycan-depleted matrices produced by BCE cells exposed to xyloside were used to study the effect of these matrices on proteoglycan synthesis by BCE cells. BCE cells growing on proteoglycan-depleted ECM showed a considerable increase in the rate of proteoglycan synthesis compared to BCE cells growing on normal ECM. Moreover, the pattern of glycosaminoglycan synthesis by BCE cells growing on proteoglycan-depleted ECM was changed to one which resembled that of BCE cells actively proliferating on plastic dishes. It is postulated that BCE cells are able to recognize when an ECM is depleted of proteoglycan and to respond to it by increasing their rate of proteoglycan synthesis and incorporation into the ECM.     

Effects of 4-methyl umbelliferyl-beta-D-xylopyranoside on chondrogenesis and proteoglycan synthesis in chick limb bud mesenchymal cell cultures

When chick limb bud mesenchyme cells from stage 23 to 24 embryos are plated at high density, they rapidly divide and a large proportion initiate chondrogenic expression during the first 2 to 3 days in culture. Between Days 4 and 8, the emergent chondrocytes mature and elaborate a cartilaginous matrix. The proteoglycans synthesized by the newly emergent Day 3 to 4 chondrocytes differ from those synthesized by either the prechondrogenic mesenchyme cells or the mature Day 8 chondrocytes. Cultures were grown from initial plating (Day 0) or from Day 2 in the continuous presence of 1 mM 4-methyl umbelliferyl-beta-D-xyloside, which acts intracellularly as a competitive acceptor with the endogenous core protein of proteoglycans for chondroitin sulfate synthesis. The proteoglycans synthesized by Day 8 cultures which had been maintained on xyloside or to which xyloside was added only 1 h prior to labeling were essentially identical. They were able to form aggregates, and they contained the same number of keratan sulfate chains, but only about 40% as many chondroitin sulfate chains, as normal. Additionally, both the chondroitin sulfate and keratan sulfate chains were 25% shorter than in the normal proteoglycans. The proteoglycans synthesized by cells in a culture maintained on xyloside until Day 8, and then switched to medium with no xyloside 1 h prior to labeling, were characteristic of those synthesized by normal mature Day 8 chondrocytes. These data suggest that stage 23 to 24 mesenchyme cells undergo normal chondrogenic maturation in culture in the presence of xylosides even though (a) most of the polysaccharides are synthesized onto the exogenously supplied xyloside substrate and released into the medium, (b) the proteoglycans that are synthesized are greatly reduced in polysaccharide content, and (c) the extracellular matrix as a consequence is greatly depleted in chondroitin sulfate content and, therefore, is abnormal in general morphology.     

Secretion of proteoglycans by chondrocytes. Influence of colchicine, cytochalasin B, and beta-D-xyloside.

Chondrocytes obtained from epiphyseal cartilage of fetal guinea pigs or ear cartilage of young rabbits were cultured in monolayer. The influence of colchicine, cytochalasin B, and p-nitrophenyl-β-d-xylopyranoside on secretion of proteoglycans was investigated. Radioactive sulfate was used as a precursor. As observed previously in other systems, β-d-xylosides initiated the synthesis of free chondroitin sulfate chains, competing with the endogenous proteoglycan core protein acceptor. The molecular weights of the chondroitin sulfate chains synthesized both on the xyloside and on the core-protein acceptor in maximally stimulated cells were similar and significantly lower than in proteoglycans synthesized in the absence of xyloside. The size of the chondroitin sulfate chains synthesized on the xyloside was inversely related to the concentration of this compound. This finding suggests that the chain length is dependent on the ratio between available acceptor and chain-lengthening enzymes or precursors. Cytochalasin B, a microfilament-modifying agent, inhibited proteoglycan synthesis, without any effect on secretion. Cells treated with cytochalasin B could be stimulated with β-d-xyloside to synthesize free chondroitin sulfate chains to the same relative degree as cells with intact microfilaments. Colchicine, an antimicrotubular agent, partially inhibited synthesis and secretion of proteoglycan. However, cells treated with colchicine could be stimulated with β-d-xyloside to synthesize and secrete free chondroitin sulfate chains to about the same relative degree as cells with intact microtubules. The data suggest that microtubules may have a facilitatory rather than an obligatory role in the secretion of proteoglycans and that at least part of the effect of colchicine is located at or after the site of glycosaminoglycan synthesis.     

beta-D-xyloside-mediated alteration in the synthesis of basement membrane proteoglycan

The effect of nitrophenyl-beta-D-xyloside (xyloside), a synthetic initiator of glycosaminoglycan synthesis, on proteoglycan and glycosaminoglycan synthesis by a basement membrane producing tumor was studied. While xyloside markedly stimulated the formation of chondroitin sulfate chains, it depressed the formation of a basement membrane heparan sulfate proteoglycan and caused only little formation of free heparan sulfate chains. However, when the synthesis of the core protein of the proteoglycan was inhibited by cycloheximide, heparan sulfate chains were produced by xyloside treatment. These heparan sulfate chains had a sulfate content higher than that of heparan sulfate found on the proteoglycan. The data indicate that xyloside can substitute for the heparan sulfate initiation site on the core protein of the proteoglycan and that this initiation is enhanced in the absence of core protein. This suggests that under normal conditions the formation of heparan sulfate chains may be tightly linked to the production of the core protein.     

Transforming growth factor (type beta) promotes the addition of chondroitin sulfate chains to the cell surface proteoglycan (syndecan) of mouse mammary epithelia

Cultured monolayers of NMuMG mouse mammary epithelial cells have augmented amounts of cell surface chondroitin sulfate glycosaminoglycan (GAG) when cultured in transforming growth factor-beta (TGF-beta), presumably because of increased synthesis on their cell surface proteoglycan (named syndecan), previously shown to contain chondroitin sulfate and heparan sulfate GAG. This increase occurs throughout the monolayer as shown using soluble thrombospondin as a binding probe. However, comparison of staining intensity of the GAG chains and syndecan core protein suggests variability among cells in the attachment of GAG chains to the core protein. Characterization of purified syndecan confirms the enhanced addition of chondroitin sulfate in TGF-beta: (a) radiosulfate incorporation into chondroitin sulfate is increased 6.2-fold in this proteoglycan fraction and heparan sulfate is increased 1.8-fold, despite no apparent increase in amount of core protein per cell, and (b) the size and density of the proteoglycan are increased, but reduced by removal of chondroitin sulfate. This is shown in part by treatment of the cells with 0.5 mM xyloside that blocks the chondroitin sulfate addition without affecting heparan sulfate. Higher xyloside concentrations block heparan sulfate as well and syndecan appears at the cell surface as core protein without GAG chains. The enhanced amount of GAG on syndecan is partly attributed to an increase in chain length. Whereas this accounts for the additional heparan sulfate synthesis, it is insufficient to explain the total increase in chondroitin sulfate; an approximately threefold increase in chondroitin sulfate chain addition occurs as well, confirmed by assessing chondroitin sulfate ABC lyase (ABCase)-generated chondroitin sulfate linkage stubs on the core protein. One of the effects of TGF-beta during embryonic tissue interactions is likely to be the enhanced synthesis of chondroitin sulfate chains on this cell surface proteoglycan.     

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.     

Tumor attenuation by 2(6-hydroxynaphthyl)-beta-D-xylopyranoside requires priming of heparan sulfate and nuclear targeting of the products

We have previously reported that the heparan sulfate-priming glycoside 2-(6-hydroxynaphthyl)-beta-D-xylopyranoside selectively inhibits growth of transformed or tumor-derived cells. To investigate the specificity of this xyloside various analogs were synthesized and tested in vitro. Selective growth inhibition was dependent on the presence of a free 6-hydroxyl in the aglycon. Because cells deficient in heparan sulfate synthesis were insensitive to the xyloside, we conclude that priming of heparan sulfate synthesis was required for growth inhibition. In growth-inhibited cells, heparan sulfate chains primed by the active xyloside were degraded to products that contained anhydromannose and appeared in the nuclei. Hence the degradation products were generated by nitric oxide-dependent cleavage. Accordingly, nitric oxide depletion reduced nuclear localization of the degradation products and counteracted the growth-inhibitory effect of the xyloside. We propose that 2-(6-hydroxynaphthyl)-beta-D-xylopyranoside entered cells and primed synthesis of heparan sulfate chains that were subsequently degraded by nitric oxide into products that accumulated in the nucleus. In vivo experiments demonstrated that the xyloside administered subcutaneously, perorally, or intraperitoneally was adsorbed and made available to tumor cells located subcutaneously. Treatment with the xyloside reduced the average tumor load by 70-97% in SCID mice. The present xyloside may serve as a lead compound for the development of novel antitumor strategies.     

Effect of beta-D-xyloside on the glomerular proteoglycans. I. Biochemical studies

The effect of p-nitrophenyl-beta-D-xylopyranoside on glomerular extracellular matrices (glomerular basement membrane and mesangial matrix) proteoglycans was studied. The proteoglycans of rat kidneys were labeled with [35S]sulfate in the presence or absence of beta- xyloside (2.5 mM) by using an isolated organ perfusion system. The proteoglycans from the glomeruli and perfusion medium were isolated and characterized by Sepharose CL-6B chromatography and by their behavior in CsCl density gradients. With xyloside treatment there was a twofold decrease in 35S-labeled macromolecules in the tissues but a twofold increase in those recovered in the medium as compared with the control. The labeled proteoglycans extracted from control kidneys eluted as a single peak with Kav = 0.25 (Mr = approximately 130,000), and approximately 95% of the radioactivity was associated with heparan sulfate proteoglycan (HS-PG), the remainder with chondroitin (or dermatan) sulfate proteoglycan (CS-PG). In the xyloside-treated kidneys, the proteoglycans extracted from the tissue eluted as two peaks, Kav = 0.25 (Mr = approximately 130,000) and 0.41 (Mr = approximately 46,000), which contained approximately 40 and approximately 60% of the total radioactivity, respectively. The first peak contained mostly the HS-PG (approximately 90%) while the second peak had a mixture of HS-PG (approximately 70%) and CS-PG (approximately 30%). In controls, approximately 90% of the radioactivity, mostly HS-PG, was confined to high density fractions of a CsCl density gradient. In contrast, in xyloside experiments, both HS- PG and CS-PG were distributed in variable proportions throughout the gradient. The incorporated 35S activity in the medium of xyloside- treated kidneys was twice that of the controls and had three to four times the amount of free chondroitin (or dermatan) sulfate glycosaminoglycan chains. The data suggest that beta-xyloside inhibits the addition of de novo synthesized glycosaminoglycan chains onto the core protein of proteoglycans and at the same time stimulates the synthesis of chondroitin or dermatan sulfate chains which are mainly discharged into the perfusion medium.     

Effect of 4-methylumbelliferyl-β-d-xyloside on collagen synthesis in chick limb bud mesenchymal cell cultures

Previous studies showed that cultures of chick limb bud mesenchymal cells plated at high density, to maximize chondrogenic expression, had a much reduced extracellular matrix around chondrocytes when exposed to 4-methyl-, umbelliferyl-β-d-xyloside. The majority of newly synthesized chondroitin sulfate chains were found in the culture medium presumably bound to the xyloside as opposed to their normal deposition on the core protein of proteoglycan. The question remained open as to whether the development of an abnormal matrix affected the synthesis of extracellular deposition of other cartilage-specific macromolecules. We have analyzed, both morphologically and biochemically, the synthesis and deposition of Type I and Type II collagen by β-d-xyloside-treated cultures of limb mesenchymal cells. While the rate of collagen synthesis per plate and its extracellular accumulation after 8 days in culture were reduced to some extent, the ratios of Type II to Type I collagen and the morphological distribution of these macromolecules were not affected by exposure to β-d-xyloside. We conclude that the expression of the cartilage-specific Type II collagen during chondrogenic differentiation is, although reduced, qualitatively not dependent on the amount of extracellular chondroitin sulfate chains attached to matrix-associated proteoglycan core protein. However, prolonged exposure of limb bud cells to xylosides leads to the formation of a chondroitin sulfate- and collagen-deficient matrix which, in turn, reduces the capacity of limb bud cells to synthesize Types I and II collagen.     

Primary mesenchyme cell migration requires a chondroitin sulfate/dermatan sulfate proteoglycan

Primary mesenchyme cell migration in the sea urchin embryo is inhibited by sulfate deprivation and exposure to exogenous beta-D-xylosides, two treatments known to disrupt proteoglycan synthesis. We show that in the developing sea urchin, exogenous xyloside affects the synthesis by the primary mesenchyme cells of a very large, cell surface chondroitin sulfate/dermatan sulfate proteoglycan. This proteoglycan is present in a partially purified fraction that restores migratory ability to defective cells in vitro. The integrity of this chondroitin sulfate/dermatan sulfate proteoglycan appears essential for primary mesenchyme cell migration since treatment of actively migrating cells with chondroitinase ABC reversibly inhibited their migration in vitro.     

p-Nitrophenyl-β-d-xyloside modulates proteoglycan synthesis and secretory differentiation in mouse mammary epithelial cell cultures

Summary Primary cultures of mouse mammary epithelial cells synthesize significant quantities of chondroitin and heparan sulfate proteoglycans (16). Long term treatment of such cultures with p-nitrophenyl-β-D-xylopyranoside leads to a 10–20 fold increase in the synthesis and secretion of free chondroitin sulfate glycosaminoglycan (GAG) chains and assembly of a cell-associated matrix that is relatively enriched in heparan sulfate proteoglycan. This modulation of cell-synthesized proteoglycans leads to significant changes in cell morphology and cellular differentiation. Notably cells cultured on plastic culture dishes change from being flattened to cuboidal. The synthesis of the milk proteins α1, α2, and β-casein is also increases as is the formation of fat droplets and fat droplet membrane components. Promotion of differentiation increases with increasing xyloside concentration in the range 0–1.5 mM, but there may be a block in secretion at higher xyloside concentrations. While the detailed mechanisms remain to be elucidated, we conclude that the composition of proteoglycans incorporated into the matrix (and possibly the glycosaminoglycans secreted into the medium), may play a significant role in maintaining the phenotypic characteristics of terminally differentiated mammary epithelial cells. This research was supported by the Office of Health and Environmental Research, Office of Energy Research, U.S. Dept. of Energy under contract No. DEAC-03-76SF00098 and by National Institutes of Health Grant CA44398-01 (G. Parry) Editor's Statement Exogenous elements of extracellular matrix affect expression of cultured mammary cell function. This work reports manipulation of cell-derived endogenous matrix elements and shows correlative changes in cell functions.     

Maturation-related differences in the structure and composition of proteoglycans synthesized by chondrocytes from bovine articular cartilage

Calf (2-3-month-old) and steer (approximately 18-month-old) bovine articular chondrocytes were isolated and cultured as high density monolayers. The proteoglycans synthesized on day 5 during a 15-h period of labeling with [35S]sulfate or [3H]glucosamine were isolated and characterized. The majority (greater than 70%) of the newly synthesized proteoglycans were found in the medium. When viewed in the electron microscope, medium-derived proteoglycans of high buoyant density were longer in calf than in steer. The medium and extracts of the cell layer were pooled and the radiolabeled proteoglycans were fractionated by isopycnic density gradient centrifugation performed under dissociative conditions. The low buoyant density fraction contained, in both calf and steer, small-sized nonaggregating proteoglycans containing chondroitin sulfate. The high buoyant density fraction contained greater than 90% of the newly synthesized proteoglycans. The majority were able to interact with hyaluronic acid to form aggregates. Calf high buoyant density fraction proteoglycans were larger, had longer chondroitin sulfate chains and lower ratios of keratan sulfate chains/chondroitin sulfate chains than steer high buoyant density fraction proteoglycans. These maturation-related differences are typical of those present in the proteoglycans of the calf and steer cartilage matrix from which the chondrocytes were isolated. Experiments with beta-D-xylosides showed that steer cultures had the capacity to synthesize twice as many chondroitin sulfate chains/cell as calf cultures. At each xyloside concentration used, chondroitin sulfate chains were longer in calf than steer. At both ages, chain size decreased with increase in rate of synthesis; the relationship between chain size and rate of synthesis was, however, quite different at the two ages. The results of these studies suggest that articular chondrocytes have an inherent program that determines the quality of proteoglycans synthesized at different ages.     

Xyloside inhibits synthesis of the class II-associated chondroitin sulfate proteoglycan and antigen presentation events

An alternative form of the human invariant chain exists as a chondroitin sulfate proteoglycan (CSPG) with invariant chain as the core protein. The selective inhibitor of proteoglycan synthesis, p-nitrophenyl beta-D-xyloside was used to study the role of this CSPG in class II biology. At xyloside concentrations of 2.5 and 5.0 mM, CSPG synthesis was completely inhibited with marginal inhibition of protein synthesis. The inhibitory effect on CSPG synthesis was completely reversible. The number of class II molecules on the cell surface was not affected by xyloside, but biosynthesis and appearance of newly synthesized class II molecules at the cell surface were both decreased by xyloside. Recognition of influenza virus-infected cells by class II-restricted, virus-specific cytotoxic T lymphocytes was not diminished by the presence of xyloside in the effector phase of the cytotoxicity assay. However, sensitization of target cells was markedly inhibited when target cells were exposed to virus in the presence of xyloside. These results are consistent with the hypothesis that the CSPG form of invariant chain has a role in antigen processing.     

Inhibition of cell migration in sea urchin embryos by beta-D-xyloside

This investigation examines the effect of exogenous xylosides on primary mesenchyme cell behavior in Strongylocentrotus purpuratus embryos. In confirmation of studies in some other species the addition of 2 mM p-nitrophenyl-beta-D-xylopyranoside blocks the migration but not the initial ingression of primary mesenchyme cells. The blastocoel matrix of treated embryos appears deficient in a 15- to 30-nm-diameter granular component that is observed extensively on the basal lamina and on filopodia of migrating primary mesenchyme cells in untreated embryos. Other blastocoel components appear unaffected by ultrastructural criteria. The incorporation of 35SO4(2-) per embryo into ethanol precipitates of isolated blastocoel matrices was reduced significantly after xyloside treatment but the distribution of 35SO4(2-) after polyacrylamide gel electrophoresis or the glycosaminoglycan composition was unaffected. Chromatography on Sepharose CL-2B demonstrates a reduction in size of sulfated components of the blastocoel. While over 60% of the 35S-labeled material from the blastocoel of normal mesenchyme blastulae is voided from a Sepharose CL-2B column run in a dissociative solvent, only 10% from xyloside treated embryos is voided. Instead, there is a large included peak with Kav of 0.33. This material is acid soluble but cetylpyridinium chloride precipitable. It apparently consists largely of free glycosaminoglycan chains. Based on analysis of chondroitinase ABC digestion products this material consists of 41% chondroitin-6-sulfate and 58% dermatan sulfate. These results are consistent with a role in cell migration for intact chondroitin sulfate/dermatan sulfate proteoglycans in the sea urchin blastocoel matrix.     

The effect of cycloheximide on synthesis of proteoglycans by cultured chondrocytes from the Swarm rat chondrosarcoma

Proteoglycan synthesis by cultured chondrocytes from the Swarm rat chondrosarcoma was examined after treatment with 0.1 mg/ml of cycloheximide which inhibited [3H]serine incorporation into total protein by greater than 90%. Incorporation of [35S]sulfate into proteoglycans decreased with nearly first order kinetics (t 1/2 = 96 +/- 6 min) with an accompanying increase in the size of the proteoglycan molecules, primary due to an increase in chondroitin sulfate chain sizes. After 5 h of cycloheximide treatment, when [35S]sulfate incorporation was inhibited by about 90%, addition of 1 mM beta-D-xyloside restored 76% of the incorporation into chondroitin sulfate observed in cultures treated only with xyloside. This suggests that the biochemical pathways for the affected by cycloheximide treatment. Cultures were prelabeled for 15 min with either [3H]serine or [35S]-methionine, and then cycloheximide was added to block further protein synthesis. Both precursors appeared in completed proteoglycan molecules with nearly first order kinetics with t 1/2 values of 92 +/- 8 and 101 +/- 11 min for [3H]serine and [35S]methionine, respectively, values in close agreement with the t 1/2 from the [35S]sulfate data. These results suggest that after cycloheximide treatment, the rate of [35S]sulfate incorporation into proteoglycan, after a correction for increases in chondroitin sulfate chain size, was directly proportional to the size of the intracellular pool of core protein. From the steady state rate of proteoglycan synthesis (estimated to be about 80 ng/min/10(6) cells in separate experiments) and a corrected t 1/2 value of 60 min, the amount of precursor core protein can be calculated to be about 500 ng/10(6) cells in these experiments.     

Role of proteoglycans in renal development

The role of proteoglycans (PGs) in morphogenesis was investigated. Fetal kidneys were obtained from 13-day-old mouse embryos and maintained for 7 days in culture. The biosynthesis of PGs was perturbed by addition of p-nitrophenyl-beta-D-xylopyranoside in the culture medium. The kidneys were processed for morphological and biochemical studies. The morphological studies included staining of tissues with anti-basement membrane antibodies and ruthenium red. [35S]sulfate was used as the precursor product for biosynthetic and autoradiographic studies. The kidneys treated with xyloside had loose mesenchyme, inhibition of ureteric bud branching, diminution in the population of developing nephron elements, decreased immunofluorescence with anti-proteoglycan antibodies and staining with ruthenium red, and a reduced [35S]sulfate incorporation into poorly organized extracellular matrices. The biochemical studies included characterization of PGs/glycosaminoglycans (GAGs) by Sepharose CL-4B, -6B, and DEAE-Sephacel chromatographies and cellulose acetate electrophoresis. Under the influence of xyloside, the total radioactivities decreased 2 to 4-fold in tissues and increased 18 to 42-fold in media fractions. A reduction in the size of macromolecular form of PGs, i.e., from MW approximately 2.5 X 10(6) to approximately 2.5 X 10(4), was noted. The PGs/GAGs synthesized were mainly made up of heparan sulfate and small amounts of chondroitin sulfate. They eluted at a lower salt concentration as compared to the controls. A similar diminution in the size of media PGs, i.e., from MW approximately 1.8 X 10(5) to approximately 2.8 X 10(4), was observed. Additional studies with [3H]xyloside indicated that the chains initiated on xyloside residues were similar in size and composition to GAG-chains. These findings indicate that a perturbance in the biosynthesis of PGs/GAGs leads to abnormalities in renal organogenesis.     

beta-D-xylosides and their analogues as artificial initiators of glycosaminoglycan chain synthesis. Aglycone-related variation in their effectiveness in vitro and in ovo.

A series of aryl and alkyl O-beta-D-xylosides and their analogues with S, NH or CH2 in the glycosidic linkage were prepared and examined for their ability to act as artificial chain initiators of chondroitin (dermatan) sulphate synthesis in embryonic chick cartilage, foetal rat skin and 6-week-old-rat aorta under conditions where normal protein-core synthesis was inhibited by cycloheximide. For all these tissues in culture, phenyl O-beta-D-xyloside and phenyl beta-D-thioxyloside were clearly more effective than the corresponding N-xyloside and homo-C-xyloside. Introduction of a carboxy group to the para position of their aglycone yielded derivatives with far lower initiator activity. In a concentration range lower than 0.1 mM, the effectiveness of alkyl beta-D-thioxyloside was greatly influenced by the carbon number (n) of the alkyl group and was at a maximum at n = 7 or 8 for the cartilage, at n = 5 for the skin and at n = 4 for the aorta. In the beta-xyloside-treated cartilages, the average length of newly formed chondroitin sulphate chains reflected the chain-initiator activity of added xyloside, i.e. the higher the initiator activity, the shorter the average chain length. In the skin and aorta, none of the drugs could relieve the inhibition of heparan sulphate synthesis caused by cycloheximide. Fertilized hens' eggs were each injected on day 9 with 9.2 mumol of beta-xyloside and the skeletal systems of embryos were examined a week later. The embryos treated with beta-xylosides of relatively high initiator activity showed a 30-40% decrease in the overall growth rate of skeletons, whereas those treated with beta-xylosides of low initiator activity showed little or no decrease in the growth rate. The results are consistent with the notion that the observed change in skeletal morphology results mainly, if not completely, from beta-xyloside-induced synthesis of core-protein-free chondroitin sulphate, and further suggest that a procedure employing a series of beta-xyloside homologues with various initiator activities will furnish an easily applied criterion on which to test the specificity of xyloside action on biological processes.     

Effect of beta-xylosides on synthesis of cartilage-specific proteoglycan in chondrocyte cultures.

Monolayer cultures of embryonic chick chondrocytes were incubated with ³⁵SO₄ in the presence and absence of 1.0 mM p-nitrophenyl-β-D-xylose for 2 days. The relative amounts of chondroitin sulfate proteoglycan and free chondroitin sulfate chains were measured following gel filtration on Sephadex G-200. Synthesis of β-xyloside-initiated polysaccharide chains was accompanied by an apparent decrease in chondroitin sulfate proteoglycan production by the treated cultures. The amount of core protein was determined from equivalent numbers of β-xyloside-treated and untreated cells by a radioimmune assay. Similar amounts of core protein were found in both types of cultures, indicating that decreased synthesis of cartilage-specific core protein is not responsible for the observed decrease in overall chondroitin sulfate proteoglycan production.     

Ovarian carcinoma cells synthesize both chondroitin sulfate and heparan sulfate cell surface proteoglycans that mediate cell adhesion to interstitial matrix

Metastatic ovarian carcinoma metastasizes by intra-peritoneal, non-hematogenous dissemination. The adhesion of the ovarian carcinoma cells to extracellular matrix components, such as types I and III collagen and cellular fibronectin, is essential for intra-peritoneal dissemination. The purpose of this study was to determine whether cell surface proteoglycans (a class of matrix receptors) are produced by ovarian carcinoma cells, and whether these proteoglycans have a role in the adhesion of ovarian carcinoma cells to types I and III collagen and fibronectin. Proteoglycans were metabolically labeled for biochemical studies. Both phosphatidylinositol-anchored and integral membrane-type cell surface proteoglycans were found to be present on the SK-OV-3 and NIH:OVCAR-3 cell lines. Three proteoglycan populations of differing hydrodynamic size were detected in both SK-OV-3 and NIH:OVCAR-3 cells. Digestions with heparitinase and chondroitinase ABC showed that cell surface proteoglycans of SK-OV-3 cells had higher proportion of chondroitin sulfate proteoglycans (75:25 of chondroitin sulfate:heparan sulfate ratio), while NIH:OVCAR-3 cells had higher proportion of heparan sulfate proteoglycans (10:90 of chondroitin sulfate:heparan sulfate ratio). RT-PCR indicated the synthesis of a unique assortment of syndecans, glypicans, and CD44 by the two cell lines. In adhesion assays performed on matrix-coated titer plates both cell lines adhered to types I and III collagen and cellular fibronectin, and cell adhesion was inhibited by preincubation of the matrix with heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, or chondroitin glycosaminoglycans. Treatment of the cells with heparitinase, chondroitinase ABC, or methylumbelliferyl xyloside also interfered with adhesion confirming the role of both heparan sulfate and chondroitin sulfate cell surface proteoglycans as matrix receptors on ovarian carcinoma cells.     

Effects of primer-concentration on uronosyl-epimerization and sulfation patterns in p-hydroxyphenyl-O-β-D-xylopyranoside-primed galactosaminoglycans produced by skin fibroblasts

By supplying skin fibroblasts with different concentrations of the galactosaminoglycan chain-primer p-hydroxyphenyl-O-β-D-xylopyranoside we have produced and recovered glycan-chains that were subsequently radio-iodinated in the hydroxyphenyl group and subjected to sequence analysis by using graded enzymic treatment followed by a combination of gel chromatography and electrophoresis. Fragments extending from the tagged reducing end to the cleavage-point were identified and quantified. Degradation by chondroitin B lyase of chains primed at 0.1 or 0.5 mM xyloside gave profiles indicating a periodic and wave-like distribution of iduronate-containing repeats, with high incidence around positions 2, 5 and onwards, whereas in chains produced at 1.0 mM xyloside the incidence of iduronate was similar in positions 1–4 and then declined. Degradation by chondroitin AC lyase indicated a high incidence of glucuronate in or near the linkage-region. There was a relatively uniform degree of sulfation in chains primed at low xyloside concentration, whereas chains primed at 1.0 mM xyloside gave very heterogeneous charge-patterns in all segments of the chain, including the linkage-region, giving the impression that adequate sulfation, probably at C-4 and at the first opportunity, is necessary to obtain an ordered and periodic epimerization pattern. Abbreviations:CS, chondroitin sulfate; DS, dermatan sulfate; GAG, glycosaminoglycan; Gal, D-galactose, GaINAc, N-acetyl-D-galactosamine; GlcUA, D-glucuronic acid; GlyUA, glycuronic acid; ΔGlyUA, 4,5-unsaturated glycuronic acid; IdoUA, L-iduronic acid; Xyl-Phe-OH, p-hydroxyphenyl-O-β-D-xylopyranoside; Xyl, D-xylose This revised version was published online in November 2006 with corrections to the Cover Date.