Characterization of proteoglycans synthesized by rat thyroid cells in culture and their response to thyroid-stimulating hormone

A Fisher rat thyroid cell line was maintained in culture and the cells were labeled with [3H]glucosamine, [35S]sulfate, and [35S]cysteine to examine the synthesis of proteoglycans. 3H and 35S radioactivity from these precursors were incorporated into both chondroitin sulfate (CS) and heparan sulfate (HS) proteoglycans. CS proteoglycans were almost exclusively secreted into the medium while HS proteoglycans remained mainly associated with the cell layer. Single chain glycosaminoglycans released by papain digestion or alkaline borohydride treatment of either the CS or HS proteoglycans had average molecular weights of approximately 30,000 on Sepharose CL-6B chromatography. Both CS and HS proteoglycans were relatively small and contained only one or two glycosaminoglycans chains. 3H and 35S incorporation into both CS and HS proteoglycans were increased by thyroid-stimulating hormone (TSH) in a dose-dependent manner, which is in part explained by an adenylate cyclase-dependent mechanism as indicated by a similar effect in response to dibutyryl cAMP. TSH enhanced the incorporation of 35S into CS from [35S]cysteine about 1.5-fold and that from [35S]sulfate about 2-fold. This result demonstrated that the increased 35S incorporation from the [35S]sulfate precursor reflects an actual increase in sulfate incorporation and is not simply a result from an apparent increase in specific activity of the phosphoadenosine phosphosulfate donor. Analysis of disaccharides from chondroitinase digests revealed that the proportion of non-sulfated, 4-sulfated, and 6-sulfated disaccharides was not altered appreciably by TSH. These results, together with the disproportionate increase in 3H incorporation into CS from [3H]glucosamine, indicated that TSH increased the specific activity of the 3H label as well. Chase experiments revealed that CS proteoglycans were rapidly (t1/2 = 15 min) secreted into the medium and that the degradation of cell-associated proteoglycans was enhanced by TSH.     

High-glucose-induced structural changes in the heparan sulfate proteoglycan, perlecan, of cultured human aortic endothelial cells

Hyperglycemia is an independent risk factor for diabetes-associated cardiovascular disease. One potential mechanism involves hyperglycemia-induced changes in arterial wall extracellular matrix components leading to increased atherosclerosis susceptibility. A decrease in heparan sulfate (HS) glycosaminoglycans (GAG) has been reported in diabetic arteries. The present studies examined the effects of high glucose on in vitro production of proteoglycans (PG) by aortic endothelial cells. Exposure of cells to high glucose (30 vs. 5 mM glucose) resulted in decreased [(35)S] sodium sulfate incorporation specifically into secreted HSPG. Differences were not due to hyperosmolar effects and no changes were observed in CS/DSPG. Enzymatic procedures, immunoprecipitation and Western analyses demonstrated that high glucose induced changes specifically in the HSPG, perlecan. In double-label experiments, lower sulfate incorporation in high-glucose-treated cells was accompanied by lower [(3)H] glucosamine incorporation into GAG but not lower [(3)H] serine incorporation into PG core proteins. Size exclusion chromatography demonstrated that GAG size was unchanged and GAG sulfation was not reduced. These results indicate that the level of regulation of perlecan by high glucose is posttranslational, involving a modification in molecular structure, possibly a decrease in the number of HS GAG chains on the core protein.     

Influence of collagen gel substrata on certain biochemical activities of hepatocytes in primary culture

In order to study the influence of cell shape as modulated by the extracellular matrix on the cellular activity, hepatocytes isolated from liver were maintained on collagen I coated plastic substrata and collage I gel substrata and certain hepatocyte specific functions were investigated. The incorporation of³[H]-leucine into total proteins and albumin secreted by cells maintained on collagen gel was found to be significantly higher compared to those maintained on a collagen coated plastic substrata, indicating that hepatocytes on collagen gel have an enhanced albumin synthesizing capacity. Increased incorporation of³⁵[S]-sulphate into total proteoglycans (PG) and a relatively higher fraction of the³⁵[S]-PG in the extracellular space showed an increased rate of synthesis and secretion of sulphated PGs by cells maintained on collagen gels. But in contrast to the above results, the incorporation of³[H]-leucine into cytokeratins C₈, C₁₈ and actin were significantly low in cells maintained on collagen gel. The tyrosine amino transferase activity exhibited by hepatocytes preincubated with dexamethasone on collagen gel was also significantly low. The different forms of collagen substrata appeared to have no effect on the amino acid transport by hepatocytes, further suggesting that the various hepatocyte specific functions are not uniformly altered when hepatocytes are maintained on three-dimensional collagen gel substrata. These results indicate that the shape of the cell as determined by the nature of the matrix substratum influences the synthetic activity of secretory proteins and those remaining intracellularly, differently.     

Changes in Glycosaminoglycans During the Neuritogenesis in PC12 Pheochromocytoma Cells Induced by Nerve Growth Factor

Abstract: Previously, we had suggested that heparan sulfate (HS) makes some contribution to a flat-shaped morphology of PC12D cells. Therefore, we carried out quantitative and qualitative analyses of glycosaminoglycans (GAGs), the polysaccharide moiety of proteoglycans, during neuritogenesis in PC12 cells that is induced by nerve growth factor (NGF). (a) In PC12 cells, NGF induced a flat-shaped morphology with a few short processes after 3 days of culture, and then it elicited short and long neurites after 6 (in ～30% of cells) and 9 (in 60–70%) days of culture, respectively, (b) HS and chondroitin sulfate (CS) were detected in the cell layer at all times. Only CS was found in the medium at 3 and 6 days, whereas a low level of HS, in addition to CS, was detectable on day 9. (c) In the NGF-treated cultures, the amounts of cell-associated HS per cell were two to three times as high as those in the respective nontreated cultures at all times, whereas the amount based on phospholipid was about twofold higher after 3 days of culture. (d) The levels of HS labeled with [³⁵S]sulfate during the last 48 h of the culture were 1.5-to twofold higher in the NGF-treated cultures than in the respective controls at any time. (e) The amount of cell-associated CS per cell (or per unit of phospholipid), but not of labeled CS per cell, was transiently enhanced at 3 days in culture with or without NGF. At all times, NGF treatment caused an increase in the levels of total and [³⁵S]sulfate-labeled CS associated with the cells and released into the medium, (f) NGF enhanced the amount of N-sulfation of glucosamine residues of HS at all times, but it did not change the ratio of 4-sulfate units to 6-sulfate units in CS. (g) At 3 days in culture, the uptake of [³⁵S]sulfate by PC12 cells was lower in the NGF-treated culture than in the nontreated control. (h) In chase experiments, the percentage of unrecovered CS was about twofold higher in the NGF-treated culture than in the non-treated control. These results suggest that the enhanced synthetic activity and the accumulation of GAGs as well as the structural change of HS induced by NGF occur preceding the neurite elongation from PC12 cells. Also, it is suggested that the increase in content of HS is closely correlated with the morphological change from round to flat in PC12 cells.     

Isolation of heparan sulfate proteoglycan from beneath the monolayers of rat hepatocytes and its binding to type IV collagen

Primary cultures of rat hepatocytes maintained as monolayer in a serum-free medium synthesise and secrete sulphated proteoglycans. Nearly 5% of the total 35(S)-sulphated material was obtained in a soluble form from beneath the cell layer. A shift in gel filtration pattern on beta-elimination with alkali suggested that it is a sulphated proteoglycan. On ion exchange chromatography over Dowex AG 1 x 2, the major fraction was eluted with 1.25 M NaCl. Further, nearly 80% of the 35(S)-labeled material was susceptible to nitrous acid degradation and more than 90% of the material was resistant to chondroitinase ABC digestion suggesting that it is predominantly a heparan sulphate proteoglycan (HSPG). Since HSPG is a major component of basement membrane, its binding with collagen was studied by a solid phase binding assay. About 75% of the 35(S) HSPG bound to wells coated with type IV collagen whereas only about 20% bound to type I collagen at physiological pH. Binding to collagen IV was reduced by about 50% when free GAG chains were used indicating that the protein core is also involved in interaction with the collagen. These results indicate the possible role of this basal extracellular heparan sulphate proteoglycan in the basal lamina formation.     

Proteoglycan biosynthesis by chick embryo retina glial-like cells

In this report we present biochemical evidence that purified cultures of chick embryo retina glial-like cells actively synthesize heparan sulfate (HS) and chondroitin sulfate/dermatan sulfate (CS/DS) proteoglycans as well as hyaluronic acid. Glial-like cell cultures were metabolically labeled with [3H]glucosamine and 35SO4, and the medium, cell layer, and substratum-bound fractions were analyzed separately. Proteoglycans were characterized according to charge, apparent molecular size, and glycosaminoglycan (GAG) composition and were found to be differentially distributed among the cellular compartments. HS was the predominant GAG overall and was the major species found in the cell layer and substratum-bound fractions. CS/DS was also present in each fraction and comprised the largest proportion of GAGs in the medium. The major GAG-containing material resolved into three different size classes. The first, found in the cell layer and substratum-bound fractions, contained both CS/DS and HS and was of large size. A second, intermediately sized class with a higher CS/DS:HS ratio was found in the medium. The smallest class was found in the cell layer fraction and comprised HS, most likely present as free GAG chains. In addition, each fraction contained hyaluronic acid. Characteristics of these macromolecules differ from those produced by purified cultures of chick embryo retina neurons and photoreceptors in terms of size, compartmental distribution, and presence of hyaluronic acid.     

Localization of newly synthesized precursors of basal lamina in the regenerating planarian as revealed by autoradiography

Autoradiography has been carried out to investigate the site of synthesis of the basal lamina in the regenerating planarian, Dugesia japonica. Since the basic collagenous structures of the basal lamina arose from RR-positive amorphous precursor, [³H]proline, [³H]glucose and [³⁵S]sodium sulphate were used as radioactive precursors of collagen, unsulphated and sulphated GAG respectively. Cytoplasm of the most regenerating epidermal cells was heavily labeled with [³H]proline during epithelization. A quantitative uptake analysis of [³H]proline indicates a progressive decline in the amount of labeled precursor in the epidermis with a corresponding increase in deposition of the labeled collagen at the presumptive basal lamina. Several myoblasts at the subepidermal region were highly labeled with both [³H]glucose and [³⁵S]sodium sulphate. Silver grains of these labeled precursors were also present in the presumptive portion of basal lamina. These observations suggest that the regenerating epidermal cell is the only site of synthesis of the basal lamina collagen while the myoblast exclusively secretes extracellular GAG. Some of the GAG may be closely associated with the amorphous zone.     

Organ culture of human articular cartilage: Studies on sulphated glycosaminoglycan synthesis

Summary An organ culture system is described for adult human articular cartilage obtained from joints afterfemoral head replacement operations. Cartilage slices maintain maximal viability for 2 days in culture as assessed by uptake of [³H]uridine and [³H]leucine into whole tissue, and³⁵SO₄ into sulphated glycosaminoglycans (GAGs). Since GAGs are the components of cartilage matrix, the depletion of which is associated with osteoarthrosis, a method for measuring sulphated GAG synthesis in culture has been investigated.     

Extracellular matrix components influence DNA synthesis of rat hepatocytes in primary culture

The effects of several extracellular matrix components (EMCs)--fibronectin (Fn), laminin (Ln), type I (C-I) and type IV (C-IV) collagen--on DNA synthesis in rat hepatocytes in primary culture were examined by both quantitative scintillation spectrometry and autoradiography of [3H]thymidine incorporation. Hepatocytes cultured on Fn showed the most active DNA synthesis initiated by epidermal growth factor (EGF) with decreasing levels of [3H]thymidine uptake exhibited in the cells cultured on C-IV, C-I, and Ln, respectively. The decreasing level of DNA synthesis in hepatocytes cultured on Fn, C-IV, C-I, and Ln respectively was not influenced by cell density. The number of EGF receptors of hepatocytes was also not influenced by EMCs. These data suggest that EMCs modify hepatocyte DNA synthesis by means of post-EGF-receptor mechanisms which are regulated by both growth factors and cell density.     

Changes in the sulfation extent of membrane-associated proteoglycans produced by sertoli cells in culture

Sertoli cells in culture synthesize two different membrane-associated proteoglycans (MA-PG): a proteoglycan containing heparan sulfate (HS) and chondroitin sulfate (CS) glycosaminoglycan (GAG) chains and a CS-PG containing only CS-GAG chains. The structure of these molecules is regulated by the presence of fetal calf serum (FCS) in the culture medium. Changes in the concentration of FCS resulted in changes in the total ³⁵SO₄ incorporation into MA-PG and a shift in the elution profile of each component subjected to ion-exchange chromatography. Thus, without FCS, the incorporation was low, while in 1% and 10% FCS, the uptake of the precursor was 1.7 and 4.5 times higher, respectively. MA-PG synthesized by Sertoli cells cultured in 10% FCS eluted from DEAE-Sephacel columns at higher salt concentration than the MA-PG synthesized by cells cultured in 0% or 1% FCS. Double-labeled experiments showed that the ³⁵SO_4/3H-glucosamine ratio incorporated into MA-PG produced by Sertoli cells, increased from 17.6 to 23.6 and 50.9 in cells cultured at 0, 1, and 10% FCS, respectively. However, the presence of FCS affected neither the hydrodynamic size nor the chemical nature of GAG chains of MA-PG. These results show that changes in the FCS concentration promote changes in the sulfation extent of MA-PG molecules produced by Sertoli cells.     

Sulfated proteoglycans synthesized by Neuro 2a neuroblastoma cells: Comparison between cells with and without ganglioside-induced neurites

Mouse neuroblastoma Neuro 2a cells are known to extend neurite-like processes in response to gangliosides added to the culture medium. We compared the structural features of proteoglycans (PG) synthesized by conventional Neuro 2a cells with those of neurite-bearing cells. Two different proteoglycans labeled with [³⁵S]sulfate, namely, chondroitin sulfate proteoglycan (CS-PG) and heparan sulfate proteoglycan (HS-PG), were found both in the cell layer and in the culture medium of the conventional cells. CS-PG isolated from the cell layer had a K_av value of 0.38 on Sepharose CL-6B, and had CS side chains with Mr of 27,000. HS-PG in the cell layer was slightly larger (K_av of 0.33) in terms of hydrodynamic size than CS-PG, and the apparent Mr of the heparan sulfate side chains was 10,000. The structural parameters of CS-PG and HS-PG isolated from the medium were almost identical to those of the PGs in the cell layer. In addition to these PGs, single-chain HS, with an average Mr of 2,500, was observed only in the cell layer and this component was the major sulfated component in the cell layers of both control and ganglioside treated cells. The neurite-bearing cells also synthesized both CS-PG and HS-PG which were very similar in hydrodynamic size to those synthesized by the conventional cells, but the size of HS side chains was greater. Radioactivity, as³⁵S, of each sulfated component from the gangliosideteated culture seemed to be slightly less than that of the corresponding component from the control culture. These findings indicate that the marked morphological change in Neuro 2a cells, induced by gangliosides is not accompanied by major changes in the synthesis of PGs.     

Effects of monensin on the synthesis, transport, and intracellular degradation of proteoglycans in rat ovarian granulosa cells in culture

Rat ovarian granulosa cells, isolated from immature female rats 48 h after stimulation with 5 IU of pregnant mare's serum gonadotropin, were maintained in culture. The effects of monensin, a monovalent cationic ionophore, on various aspects of proteoglycan metabolism were studied by metabolically labeling cultures with [35S]sulfate, [3H]glucosamine, or [3H]glucose. Monensin inhibited post-translational modification of both heparan sulfate (HS) proteoglycans and dermatan sulfate (DS) proteoglycans, resulting in decreased synthesis of completed proteoglycans [( 35S]sulfate incorporation decreased to 10% of control by 30 microM monensin, with an ED50 approximately 1 microM). Proteoglycans synthesized in the presence of monensin showed undersulfation of both DS and HS glycosaminoglycans and altered N-linked and O-linked oligosaccharides, suggesting that the processing of all sugar moieties is closely associated. Monensin caused a decrease in the endogenous sugar supply to the UDP-N-acetylhexosamine pool as indicated by an increased 3H incorporation into DS chains [( 3H]glucosamine as precursor) in spite of the decrease in glycosaminoglycan synthesis. Monensin reduced and delayed transport of both secretory and membrane-associated proteoglycans from the Golgi complex to the cell surface. It took 2-4 min for newly labeled proteoglycans to reach the main transport process inhibited by monensin. Monensin at 30 microM did not prevent internalization of cell surface 35S-labeled proteoglycans but almost completely inhibited their intracellular degradation to free [35S]sulfate (ED50 approximately 1 microM), resulting in intracellular accumulation of both DS and HS proteoglycans. Pulse-chase experiments demonstrated that one of the intracellular degradation pathways involving proteolysis of both DS and HS proteoglycans and limited endoglycosidic cleavage of HS continued to operate in the presence of monensin. These results suggest that the intracellular degradation of proteoglycans involve both acidic and nonacidic compartments with monensin inhibiting those processes that normally occur in such acidic compartments as endosomes or lysosomes by raising their pH.     

Growth modulation and proteoglycan turnover in cultured mesangial cells

Proliferation of mesangial cells is a common feature of renal disease, and conditioned media from glomerular epithelial and endothelial cells have been found to contain heparin-like molecules that suppress proliferation of rat mesangial cells (RMC). We have partially characterized the glycosaminoglycans that are labeled with ³⁵SO₄^2? by RMC in culture at early passage and examined their ability to inhibit mitogenic stimulation of the cells. Four chondroitin/dermatan sulfate proteoglycans (CS/DSPG) were identified, the largest and smallest of which (K_d of 0.04 and 0.26 on Superose 6) were retained in the cell layer while the other two (K_d = 0.17 and 0.22) were secreted into the medium. Heparan sulfate proteoglycans (HSPG) with K_d values of 0.09, 0.13, and 0.39 were minor components of the cell layer, while a single heparan sulfate (K_d = 0.17) was recovered from the medium. After 16 h of labeling in serum-free medium, about 60% of macromolecular ³⁵S was cell-associated and 40% was in the medium. Cell-associated label consisted of 7% CS/DSPG, 9% HSPG, and 84% free glycosaminoglycan chains (mostly CS/DS), whereas the medium contained 52% CS/DSPG, 17% HSPG, and approximately equal amounts of free HS and CS/DS chains. Bovine lung heparin (1 μg/ml) decreased by 45% the incorporation of [³H]-thymidine into DNA after release of serum-starved RMC from growth arrest. Heparin acted prior to the G₁/S interface; arrest of the cells in early S phase with aphidicolin abrogated the heparin response. The endogenous HSPGs had a slight antimitogenic effect on the RMC, but heparan sulfate chains from both the medium and cell layer had a potent effect. On an equivalent mass basis, only the free glycosaminoglycan chains were more potent than heparin in this regard, decreasing thymidine incorporation by over 90% when present at 1 μg/ml. These results demonstrate that heparan sulfate glycosaminoglycans derived from mesangial proteoglycans are potential negative autocrine growth regulators. Proteoglycan metabolism releases these soluble heparan sulfate chains, determining the level of this activity. © 1994 wiley-Liss, Inc.     

Cell surface glycosaminoglycans (GAGs) of cultured chick fibroblasts : Modifications in relation to the stage of embryo development

We have investigated the changes in glycosaminoglycan (GAG) composition between cultured fibroblasts derived from 8- and 16-day chick embryos. GAG composition has been studied after [3H]glucosamine and [35S]sulfate labeling. Both the 8- and 16-day embryo fibroblasts were found to contain hyaluronic acid (HA), dermatan sulfate (DS), heparan sulfate (HS) and chondroitin sulfates (CS), the latter being the major component in 8- and 16-day cells. These four GAGs were quantified after their separation using cellulose acetate electrophoresis. The amounts of HA and CS were respectively shown to increase 2-fold and 4-fold between the 8th and 16th day of development, whereas the amounts of HS and DS resp. diminished 2.5-fold and 1.2-fold. These results show that the relative proportions of the different GAGs alter during embryo development. The fibroblasts from 8-day-old embryos detached more rapidly from the culture dishes than the cells from 16-day-old embryos when treated with trypsin. However, this difference was not directly related to the different GAG content.     

IGF-1 stimulates synthesis of undersulfated proteoglycans and of hyaluronic acid by peritubular cells from immature rat testis

The exposure of confluent peritubular (PT) cells from immature rat testis to insulin-like growth factor-1 (IGF-1) induced a time and dose-dependent increase of [³⁵S]-sulfate and [³H]-d-glucosamine incorporations in newly synthesized proteoglycans (PG). This increased content of PG was the result of an enhancement of PG synthesis rather than a decreased rate of degradation. IGF-1 had no effect on the molecular weight of synthesized PG nor on the nature and distribution of the constitutive glycosaminoglycan chains, both in medium and in cell layer. The stimulation of PG synthesis by IGF-1 appeared to be due, at least partially, to an increase of glycosylation processes. IGF-1 effect was mediated by the classical tyrosine kinase signalling process, since IGF-1 action on PG synthesis was abolished by genistein and tyrphostin A9, two well known tyrosine kinase inhibitors. The increase of PG synthesis was accompanied with an undersulfation of constitutive glycosaminoglycan (GAG) chains (chondroitin sulfate and heparan sulfate chains) since the [³⁵S]/[³H] ratio was reduced by about 20–25% in presence of IGF-1. Although the mechanism of hyaluronic acid synthesis was completely different from those of other GAG, IGF-1 also dramatically enhanced its production by PT cells.     

Distinctive fibroblastic subpopulations in skin and oral mucosa demonstrated by differences in glycosaminoglycan content

Summary The glycosaminoglycan (GAG) content of rabbit skin, oral mucosa, and cultured [³H]-glucosamine-labeled dermal and submucosal fibroblasts was compared. Skin contained predominantly dermatan sulfate (DS) and a small amount of hyaluronic acid (HA), whereas mucosa contained primarily keratan sulfate (KS) and smaller quantities of HA and DS. Culture medium from dermal and submucosal fibroblasts contained GAGs co-electrophoresing with DS, HA, and chondroitin sulfate (CS), although the relative proportions of these GAG differed. CS isolated from dermal and mucosal fibroblast culture medium co-electrophoresed with chondroitin 4-sulfate (C4-S) on cellulose acetate, whereas dermal medium CS was resistant to digestion by chondroitinase ABC, and mucosal medium CS was chondroitinase ABC-susceptible. The pericellular matrix of dermal fibroblasts contained primarily DS and C4-S/C6-S, as confirmed by chondroitinase ABC digestion; the corresponding fraction of mucosal fibroblasts contained HS and a GAG co-electrophoresing with a C6-S standard, yet resistant to digestion by chondroitinase ABC. Thus the GAG content of dermal and mucosal fibroblasts differed both qualitatively in terms of the type of GAG secreted into the culture medium and pericellular matrix, and quantitatively, in terms of the relative proportions of these GAGs in both fractions. These differences support the concept of distinctive fibroblastic subpopulations in skin and mucosal tissue, inasmuch as the cells were subjected to identical culturing conditions. This work was supported by research grant 15878 (C.N.B.) from the Shriners Hospitals for Crippled Children and DE 07803 (C.N.B.) from the National Institute of Dental Research, National Institutes of Health, Bethesda, MD.     

Characteristics of human chondrocyte cultures in completely defined medium

Summary Chondrocytes derived from normal human adult articular cartilage were established and maintained for over 5 months in a completely defined medium without the addition of serum or any other growth factors. At the end of 5 months, these cells were still metabolically active. The cells incorporated [³H]thymidine into DNA, incorporated [³⁵S]sulfate into proteoglycans, and exhibited lysosomal enzyme activities. The³⁵S-labeled proteoglycans isolated from the culture medium had elution profiles on high pressure liquid chromatography (HPCL) similar to those observed from proteoglycans from other mammalian sources. This self-contained growth competence may reflect a need produced by the unusual avascular and alymphatic character of articular cartilage. This research was supported, in part, by Grant AM22057 from the National Institutes of Health, Bethesda, MD.     

Effect of temperature on endocytosis and degradation of sulphated proteoglycans by cultured skin fibroblasts

Temperature up to 16‡C reduced endocytosis of [³⁵S]-proteoglycans by human skin fibroblasts to less than 15% of that at 37‡C. At temperatures between 20–26‡C endocytosis was more than 50%. At temperatures below 26‡C, the relative rate of degradation of endocytosed [³⁵S]-proteoglycans was several fold less than the rate of endocytosis. Codistribution of endocytosed [³⁵S]-proteoglycans and the lysosomal marker enzyme Β-hexosaminidase upon subcellular fractionation indicated that endocytotic vesicles containing [³⁵S]-proteoglycans had fused with lysosomes at 37‡C and at 16‡C. The prolonged halflives of endocytosed [³⁵S]-proteoglycans at 16–26‡C could not be explained merely by a temperature dependent reduction of catalytic activity of lysosomal enzymes participating in the degradation of sulphated proteoglycans.     

Partial characterization of heparan and dermatan sulfate proteoglycans synthesized by normal rat glomeruli

Rat glomerular heparan sulfate (HS) and dermatan sulfate (DS) proteoglycan synthesis was studied in vitro and in vivo. Incorporation of [35S]sulfate into macromolecules was linear over 16 h in vitro, and DS was the predominant glycosaminoglycan (GAG), while HS dominated in vivo incubations. Proteoglycans were found in the bottom 2/5 (high density) CsCl gradient fractions and eluted as two overlapping peaks from DEAE-Sephacel columns. The proportion of low density 35S-glycoproteins and 35S-proteoglycans increased with time. Two high buoyant density HS proteoglycans were extracted from glomeruli and eluted in DEAE peak I. The first, HS-tIA, had an Mr of 130 X 10(3) with Mr 12.5 X 10(3) GAG chains. This proteoglycan was released from the tissue by trypsin and was partially displaced by heparin treatment. In addition, it was rapidly released into the medium of label-chase experiments after which it migrated slightly more rapidly than HS-tIA in gels, with HS chains similar in length to its tissue counterpart. The second, HS-tIB, had an Mr of 8.6 X 10(3) with little or no attached protein. This proteoglycan was characterized as intracellular as it resisted release by trypsin treatment or heparin extraction in medium and was not detected in the medium of label-chase experiments. Two tissue DS proteoglycans were characterized. The first, DS-tIA, co-purified with HS-tIA and was the predominant proteoglycan synthesized during 4-h in vitro incubations. Like HS-tIA, it was rapidly released into medium and displaced from cell surfaces or tissue "receptors" by heparin or trypsin treatments. A second, Sepharose CL-6B-excluded DS proteoglycan from DEAE peak II, DS-tII, accumulated in tissue over 16 h in vitro. This proteoglycan was self-associating and contained clusters of iduronic acid residues along its Mr 26 X 10(3) DS chains. It resisted extraction from the tissue with heparin, trypsin, and detergent. No DS-tII was detected in the incubation medium. Instead, medium proteoglycans eluted as single Sepharose CL-6B-included peaks. DS chains from medium proteoglycans were shorter (Mr 18 X 10(3)) and had more regularly spaced iduronic acid residues than GAGs from DS-tII. The length and sulfation patterns of DS-mII GAG were similar to GAG from DS-tIA. Thus, glomeruli rapidly synthesized and released Sepharose CL-6B-included heparin-displaceable DS and HS proteoglycans while retaining a Sepharose CL-6B-excluded self-associating DS proteoglycan and an intracellular HS.     

Extracellular calcium regulates distribution and transport of heparan sulfate proteoglycans in a rat parathyroid cell line

The regulation of the cellular distribution of proteoglycans in a clonal rat parathyroid cell line by extracellular Ca2+ concentrations ([Ca2+]e) was studied. Proteoglycans synthesized by the cells metabolically labeled with [35S]sulfate have been shown to be almost exclusively heparan sulfate (HS) proteoglycans (Yanagishita, M., Brandi, M.L., and Sakaguchi, K. (1989) J. Biol. Chem. 264, 15714-15720), which are generally associated with the plasma membrane. The proportion of HS proteoglycans on the cell surface was approximately 20% in 2.1 mM (high) [Ca2+]e, whereas it increased to 50-60% in 0.05 mM (low) [Ca2+]e. Cell-associated HS proteoglycans redistribute in response to changing [Ca2+]e with a t 1/2 less than 4 min; HS proteoglycans appear on the cell surface as [Ca2+]e decreases and disappear from the cell surface as [Ca2+]e increases. Further, HS proteoglycans on the cell surface recycle to and from an intracellular compartment approximately 10 times before their degradation in low [Ca2+]e but do not recycle in high [Ca2+]e. The distribution of newly synthesized HS proteoglycans is regulated by [Ca2+]e but is independent of [Ca2+]e during biosynthesis. In low [Ca2+]e, at least 50% of the HS proteoglycans pulse-labeled for 10 min are transported from the Golgi complex to the cell surface or to the recycling compartment with a t 1/2 of approximately 20 min. Another approximately 10% appear on the cell surface in either low or high [Ca2+]e in a compartment with a long half-life. Addition of Mg2+ or Ba2+ to the low [Ca2+]e cultures had little effect on the distribution of HS proteoglycans. These observations suggest that [Ca2+]e specifically regulates the distribution and recycling of cell-associated HS proteoglycans in the parathyroid cells.