Characterization of proteoglycans synthesized by a rat parathyroid cell line

The structure, biosynthesis, and distribution of cell-associated proteoglycans in a clonal line of parathyroid cells, which exhibit differentiated characteristics such as calcium-regulated hormone secretion and cell growth, were studied by metabolic labeling with [3H] glucosamine and [35S]sulfate as precursors. Proteoglycans were isolated by two consecutive ion exchange chromatography steps and then analyzed by gel filtration, polyacrylamide gel electrophoresis, and specific enzyme and chemical reactions. The cells synthesize almost exclusively (greater than 95%) heparan sulfate (HS) proteoglycans with a glycosaminoglycan synthesis rate of approximately 0.5 micrograms/10(6) cells/24 h. Two major HS proteoglycan species were identified. HS proteoglycan-I has a mass of approximately kDa with a single HS chain (approximately 12 kDa) and a core protein of approximately 150 kDa including oligosaccharides. HS proteoglycan-II has a mass of approximately 170 kDa with 3-4 HS chains (approximately 30 kDa) and a core protein of 70-80 kDa including oligosaccharides. In the medium with low ionized calcium (0.05 mM), HS proteoglycan-I is synthesized at approximately 1.6 times the rate and HS proteoglycan-II at a similar rate as for cells cultured in the medium with high ionized calcium (2.1 mM). The distribution of proteoglycans, examined by the accessibility of the molecules to trypsin, was dramatically influenced by environmental calcium concentration; at low calcium levels 70-80% of the HS proteoglycans are trypsin-accessible while only 20-30% are accessible at high calcium levels. This suggests that the proteoglycans are primarily on the cell surface in low calcium and in trypsin-inaccessible compartments in high calcium conditions.     

Metabolic labeling of glycosylphosphatidylinositol-anchor of heparan sulfate proteoglycans in rat ovarian granulosa cells.

The glycosylphosphatidylinositol (GPI)-anchor of the plasma membrane-associated heparan sulfate (HS) proteoglycan was metabolically radiolabeled with [3H]myristic acid, [3H]palmitic acid, [3H]inositol, [3H]ethanolamine, or [32P]phosphate in rat ovarian granulosa cell culture. Cell cultures labeled with [3H]myristic acid or [3H]palmitic acid were extracted with 4 M guanidine HCl buffer containing 2% Triton X-100 and the proteoglycans were purified by ion exchange chromatography after extensive delipidation. Specific incorporation of 3H into GPI-anchor was demonstrated by removing the label with a phosphatidylinositol-specific phospholipase C (PI-PLC). Incorporation of 3H activity into glycosaminoglycans and core glycoproteins was also demonstrated. However, the specific activity of 3H in these structures was approximately 2 orders of magnitude lower than that in the GPI-anchor, suggesting that 3H label was the result of the metabolic utilization of catabolic products of the 3H-labeled fatty acids. PI-PLC treatment of cell cultures metabolically labeled with [3H]inositol, [3H]ethanolamine, or [32P]phosphate specifically released radiolabeled cell surface-associated HS proteoglycans indicating the presence of GPI-anchor in these proteoglycans. GPI-anchored HS proteoglycans accounted for 20-30% of the total cell surface-associated HS proteoglycans and virtually all of them were removed by PI-PLC. These results further substantiate the presence of GPI-anchored heparan sulfate proteoglycan in ovarian granulosa cells and its cell surface localization.     

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.     

Heparan sulfate proteoglycan from the extracellular matrix of human lung fibroblasts. Isolation, purification, and core protein characterization

Confluent cultured human lung fibroblasts were labeled with 35SO4(2-). After 48 h of labeling, the pericellular matrix was prepared by Triton X-100 and deoxycholate extraction of the monolayers. Heparan sulfate proteoglycan (HSPG) accounted for nearly 80% of the total matrix [35S]proteoglycans. After solubilization in 6 M guanidinium HCl and cesium chloride density gradient centrifugation, the majority (78%) of these [35S] HSPG equilibrated at an average buoyant density of 1.35 g/ml. This major HSPG fraction was purified by ion-exchange chromatography on Mono Q and by gel filtration on Sepharose CL-4B, and further characterized by gel electrophoresis and immunoblotting. Intact [35S]HSPG eluted with Kav 0.1 from Sepharose CL-4B, whereas the protein-free [35S]heparan sulfate chains, obtained by alkaline borohydride treatment of the proteoglycan fractions, eluted with Kav 0.45 (Mr approximately 72,000). When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, core (protein) preparations, obtained by heparitinase digestion of 125I-labeled HSPG fractions, yielded one major labeled band with apparent molecular mass of approximately 300 kDa. Reduction with beta-mercaptoethanol slightly increased the apparent Mr of the labeled band, suggesting a single polypeptide structure and the presence of intrachain disulfide bonds. Immunoadsorption experiments and immunostaining of electrophoretically separated heparitinase-digested core proteins with monoclonal antibodies raised against matrix and cell surface-associated HSPG suggested that the major matrix-associated HSPG of cultured human lung fibroblasts is distinct from the HSPG that are anchored in the membranes of these cells. Binding studies suggested that this matrix HSPG interacts with several matrix components, both through its glycosaminoglycan chains and through its heparitinase-resistant core. Core (protein) interactions seem to be responsible for the association of the proteoglycan with the extracellular matrix.     

Glycosylphosphatidylinositol-anchored and core protein-intercalated heparan sulfate proteoglycans in rat ovarian granulosa cells have distinct secretory, endocytotic, and intracellular degradative pathways.

Rat ovarian granulosa cells synthesize two distinct species of plasma membrane-intercalated heparan sulfate (HS) proteoglycans; glycosylphosphatidylinositol (GPI)-anchored and core protein-intercalated HS proteoglycans. Both species of HS proteoglycans are primarily localized on the plasma membrane. Cell surface localization of GPI-anchored and protein-intercalated HS proteoglycans can be determined by their accessibility to exogenously added phosphatidylinositol-specific phospholipase C (PI-PLC) and trypsin, respectively. Kinetic parameters for the processes involving their transfer from the Golgi to the cell surface, endocytosis and secretion, and the modes of intracellular degradation were determined by metabolic labeling experiments using [35S]sulfate and various chase protocols in combination with the use of PI-PLC and trypsin in rat ovarian granulosa cells. The experiments demonstrated that (i) both HS proteoglycan species are transferred from the Golgi to the cell surface with an average transit time of approximately 12 min. (ii) GPI-anchored HS proteoglycans are endocytosed with a t1/2 approximately 3 h, without being shed into the medium, and they are rapidly degraded, t1/2 approximately 25 min, without generating recognizable degradation intermediates. (iii) Protein-intercalated HS proteoglycans are partly (approximately 30%) shed from the cell surface into the medium and the remaining approximately 70% are endocytosed with a t1/2 approximately 4 h. After endocytosis, they undergo a slow (t1/2 approximately 4 h) stepwise degradation generating distinct HS oligosaccharides as degradation intermediates. These results indicate that the GPI-anchored and the protein-intercalated HS proteoglycans have distinct secretory, endocytotic, and intracellular degradation pathways probably due to the differences in their anchor structures.     

Differentiation of 3T3-L1 preadipocytes with 3-isobutyl-1-methylxanthine and dexamethasone stimulates cell-associated and soluble chondroitin 4-sulfate proteoglycans.

The proteoglycans (cell-associated and culture media) in 3T3-L1 preadipocytes in culture were analyzed before and during differentiation into adipocytes. Cells were metabolically labeled with [35S]sulfate and [3H] glucosamine for 24 h and then extracted and analyzed. There was a 1.68 +/- 0.07-fold increase in the 35S in medium proteoglycan during differentiation, whereas cell-associated proteoglycan radioactivity showed no increase. Analyses of radiolabeled molecules using ion-exchange chromatography, gel filtration, and high performance liquid chromatography after enzymatic or alkaline digestion indicated that all of the 35S label was recovered as two major species of chondroitin 4-sulfate proteoglycans (CSPG-I and CSPG-II) and 7% as heparan sulfate proteoglycan. CSPG-I has a mass of approximately 970 kDa with multiple chondroitin sulfate chains (average of 50 kDa each) and a core protein of approximately 370 kDa including oligosaccharides. CSPG-II has a mass of 140 kDa with one or two chondroitin sulfate chains (average of 68 kDa each) and a core protein of 41 kDa including oligosaccharides. CSPG-I appears to be similar to versican, whereas CSPG-II is similar to decorin and/or biglycan, found in other fibroblastic cells. Cell differentiation was associated with a specific increase in CSPG-I (4.0 +/- 0.2-fold in media and 3.2 +/- 0.5-fold in the cell-associated form). This system should facilitate study of the functional roles of proteoglycans during growth and differentiation.     

Proteoglycans synthesized and secreted by pancreatic islet beta-cells bind amylin

Pancreatic islet amyloid deposits in type 2 diabetes are associated with decreased islet beta-cell function. They contain both amylin (islet amyloid polypeptide), the beta-cell-derived unique fibrillogenic component, and heparan sulfate proteoglycans (HSPGs). We hypothesized that beta-cell HSPGs contribute to islet amyloidogenesis. [35S]Sulfate-labeled proteoglycans from islet-derived beta-TC3 cell cultures eluted from diethylaminoethyl Sephacel at 0.35M NaCl. Chromatography on Sepharose CL-4B and SDS-PAGE analysis revealed distinct populations of proteoglycans. Medium HSPGs eluted at K(av) approximately 0.18 and 0.50 with glycosaminoglycan chains of approximately 28 and 19 kDa, respectively. A third population containing chondroitin/dermatan sulfate eluted at K(av) approximately 0.70 with glycosaminoglycan chains of approximately 10 kDa. A single size class of heparan and chondroitin/dermatan sulfate proteoglycans in the cell layer eluted at K(av) approximately 0.40 with glycosaminoglycan chains of approximately 19 kDa. Medium and cell layer proteoglycans bound exclusively to fibrillogenic amylin, as determined by gel mobility shift assays, indicating a possible role for beta-cell-derived proteoglycans in islet amyloid formation.     

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.     

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.     

Identification of heparan sulfate proteoglycan as a high affinity receptor for acidic fibroblast growth factor (aFGF) in a parathyroid cell line.

We have characterized two high affinity acidic fibroblast growth factor (aFGF) receptors in a rat parathyroid cell line (PT-r). Affinity labeling with 125I-aFGF showed that these two receptors, apparent molecular masses, 150 and 130 kDa, respectively, display higher affinity for aFGF than for bFGF. The 150-kDa receptor bears a heparan sulfate chain(s), demonstrated by a decrease in size of 15-20 kDa with heparitinase digestion after affinity labeling. Heparitinase digestion before affinity labeling markedly reduced the intensity of the 150 kDa species. Scatchard analysis showed two different high affinity binding sites (Kd of 3.9 pM with 180 sites/cell and Kd of 110 pM with 5800 sites/cell). The higher affinity site was completely eliminated by digestion with heparitinase before adding labeled aFGF; the lower affinity site was unaffected. In ion exchange chromatography after metabolic labeling of the cells with [3H]glucosamine and affinity labeling with 125I-aFGF, the larger receptor-ligand complex, 165 kDa, eluted with approximately 0.5 M NaCl, typical eluting conditions for heparan sulfate proteoglycans. Both of the receptor-ligand complexes were smaller on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than two major heparan sulfate proteoglycans, HSPG I and II, which we characterized in this cell line previously (Yanagishita, M., Brandi, M. L., and Sakaguchi, K. (1989) J. Biol. Chem. 264, 15714-15720). Both receptors have similar N-linked oligosaccharide and sialic acid contents, shown by analysis of affinity-labeled receptors upon digestion with glycopeptidase F and with neuraminidase. All together, these results suggest that PT-r cells bear two distinct high affinity receptors for aFGF, a 150-kDa receptor which is a heparan sulfate proteoglycan and another that is a glycoprotein. The heparan sulfate glycosaminoglycan moiety of the 150- kDa receptor is critical for high affinity binding of aFGF. These findings contrast with current concepts derived from other systems, suggesting that heparan sulfate glycosaminoglycans/proteoglycans function as a reservoir source for FGF or as a group of low affinity binding sites.     

Proteoglycan synthesis by cultured liver endothelium: the role of membrane-associated heparan sulfate in transferrin binding

Liver endothelium has been reported to possess membrane receptors for the iron-binding protein transferrin (Tf). Similarly, the core protein of proteoglycans (PG) associated with cell membrane in many cell systems can bind Tf. To find out if membrane-associated proteoglycans can explain Tf-binding ability of liver endothelium, we investigated the synthesis and distribution of proteoglycans by isolated, cultured liver capillary endothelium. Cells were isolated and cultured for 48 h in sulfate-free medium and pulse-labeled with 35SO4. The relative distribution of 35SO4-labeled macromolecules, determined in the extracellular (EC), membrane-associated (MA), and intracellular (IC) pools, was respectively 74, 15, and 10%. Membrane-associated proteoglycan (MA-PG) was further purified by ion exchange and gel chromatography. Glycosaminoglycan (GAG) chain characterization indicated about 78% chondroitin sulfate, 7% dermatan sulfate, and about 14% heparan sulfate (HS). Similar GAG chain characterization was made for PG in the EC and IC pools. Transferrin-binding ability of MA-PG was studied by affinity column chromatography, using CNBr-activated sepharose bound to transferrin. About 15% of the labeled MA-PG was specifically bound to Tf-affinity column and could be eluted by excess soluble Tf. This proportion was similar to the proportion of HS in the total membrane-associated pool. Moreover, the eluted labeled material was susceptible to pretreatment with heparitinase, confirming its HS nature. We conclude that the transport capillary endothelium of the liver can synthesize HS proteoglycans which are membrane-associated and this MA-HS pool can bind transferrin. The finding may provide a molecular basis for transferrin binding to liver endothelium and may explain the subsequent transendothelial transport of iron-transferrin complexes into the liver.     

Proteoglycan biosynthesis in murine monocytic leukemic (M1) cells before and after differentiation

Murine monocytic leukemic (M1) cells were cultured in the presence of [3H]glucosamine and [35S]sulfate. Labeled proteoglycans were purified by anion exchange chromatography and characterized by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with chemical and enzymatic degradation. M1 cells synthesize a single predominant species of proteoglycan which distributes almost equally between the cell and medium after 17 h labeling. The cell-associated proteoglycan has an overall size of about 135 kDa and contains three to five chondroitin sulfate chains (28-31 kDa each) attached to a chondroitinase-generated core protein of 28 kDa. The synthesis and subsequent secretion of this proteoglycan was enhanced 4-5-fold in cells induced to differentiate into macrophages. This was not a phenomenon of arrest in the G0/G1 stage of the cell cycle, since density inhibited undifferentiated cells arrested at this stage did not increase proteoglycan synthesis. The chondroitin sulfate chains contained exclusively chondroitin 4- and 6-sulfate; however, the ratio of these two disaccharides differed between the medium- and cell-associated proteoglycans, and changed during progression of the cells into a fully differentiated phenotype. Pulse-chase kinetics indicate the presence of two distinct pools of proteoglycan; one that is secreted very rapidly from the cell after a approximately 1-h lag, and a second pool that is turned over in the cell with a half-time of approximately 3.5 h. Subtle differences in the glycosylation patterns of the medium- and cell-associated species are consistent with synthesis of two pools. Papain digestion suggests that the chondroitin sulfate chains are clustered on a small protease resistant peptide. The data suggest that this proteoglycan is similar to the serglycin proteoglycan family.     

Proteoglycans synthesized by cultured mouse osteoblasts

Proteoglycan synthesis in nonmineralizing osteoblast cultures was investigated. Cultures were labeled with [35S]sulfate or [3H]serine, and proteoglycans were extracted from medium and cell layer with 4 M guanidine HCl. Labeled material was subjected to Sepharose CL-4B and DEAE-Sephacel chromatography and polyacrylamide gel electrophoresis. The size and composition of the glycosaminoglycan chains and the protein core size were determined. Two proteoglycan populations were isolated by Sepharose CL-4B chromatography: a minor excluded species with chondroitin sulfate chains of apparent Mr 25,000 and a smaller population (Kav = 0.43) accounting for 80% of the total labeled material. This small population resolved into two species by polyacrylamide gel electrophoresis. Both species contain dermatan sulfate chains of apparent Mr 40,000 and a core protein with Mr 45,000 on sodium dodecyl sulfate gels. With the exception of their glycosaminoglycan composition these species appear similar to those extracted from bone. In addition, high molecular weight hyaluronic acid and glycosaminoglycan peptides were found in cell extracts.     

Secreted and cellular proteochondroitin sulfates of a human B lymphoblastoid cell line contain different protein cores.

Proteoglycans of the human B lymphoblastoid cell line LICR-LON-HMy2 were metabolically labeled with [35S]sulfate. High-density fractions of 35S-labeled material separated by CsCl gradient ultracentrifugation were further purified by anion exchange chromatography and gel filtration. Two proteoglycans, isolated from cell lysates and culture supernatants, were characterized by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in combination with enzymatic degradation. Treatment with chondroitinase AC completely degraded the glycosaminoglycan moiety of the proteoglycans. Three to 4 chondroitin sulfate chains (average molecular mass = 26 kDa) were estimated for each of the two proteoglycans. Differences between the proteochondroitin sulfates (CSPG) were observed in the content of N-linked oligosaccharides. After chondroitinase AC treatment the resulting band in SDS-PAGE of the secreted CSPG was sensitive to treatment with endoglycosidase F (Endo F) which further reduced the molecular mass from 30 to 21.5 kDa, whereas the band of the cellular CSPG after chondroitinase AC treatment (molecular mass = 30 kDa) remained resistant to Endo F treatment. The composition of amino acids was different in the protein cores, suggesting differences in the primary structure. Both CSPG contained a high percentage of glycine and serine. For both CSPG a molecular mass of approximately 135 kDa was deduced from the hydrodynamic sizes of the glycosaminoglycan chains obtained after alkaline/borohydride treatment and the migration of the protein/oligosaccharide complexes in SDS-PAGE. 75% of all [35S]sulfate-labeled molecules were found in the culture supernatant and 25% in the cellular fraction. 35S-Labeled material in the culture supernatant consisted exclusively of intact CSPG, whereas 35S-Labeled molecules in the cellular preparation consisted largely of free chondroitin sulfate chains. Only 8.3% of the cellular material, isolated from the microsomal fraction, was intact CSPG. In pulse-chase experiments maximal secretion of CSPG was found after 4 h, comprising approximately 40% of totally synthesized CSPG. From these experiments we tentatively conclude that a small proportion of CSPG synthesized by LICR-LON-HMy2 cells is membrane-associated, a larger portion is secreted, and another portion is intracellularly degraded.     

Presence of the HNK-1 epitope on poly(N-acetyllactosaminyl) oligosaccharides and identification of multiple core proteins in the chondroitin sulfate proteoglycans of brain

The chondroitin sulfate proteoglycans of brain contain several core proteins bearing HNK-1 antibody epitopes. Endo-beta-galactosidase treatment resulted in the almost complete disappearance of HNK-1 staining of proteoglycan immunoblots, indicating that a significant portion of the 3-sulfated sugar residues recognized by this antibody are present on poly(N-acetyllactosaminyl) oligosaccharides. However, after treatment with chondroitinase ABC followed by endo-beta-galactosidase, several proteoglycan species showed HNK-1 reactivity, presumably due to the presence of this epitope on other oligosaccharides which are both resistant to endo-beta-galactosidase and inaccessible to the antibody in the native proteoglycan. Immunostaining of the endo-beta-galactosidase degradation products after separation by thin-layer chromatography demonstrated that HNK-1 reactivity was confined to a minor population of large oligosaccharides. Only a relatively small portion of the native chondroitin sulfate proteoglycans of brain enter a 6-12% SDS-polyacrylamide gel. However, after treatment of the proteoglycans with chondroitinase ABC (or chondroitinase and endo-beta-galactosidase) in the presence of protease inhibitors, seven bands with molecular sizes ranging from 80 to 200 kDa appear in Coomassie Blue stained gels, and two additional bands with molecular sizes of 67 and 350-400 kDa are apparent in fluorographs of sodium [35S]sulfate labeled proteoglycans. Most of these components probably represent individual proteoglycan species rather than different degrees of nonchondroitin sulfate/keratan sulfate glycosylation of a single protein core, since [35S]methionine-labeled proteins of comparable molecular size were synthesized by an in vitro translation system. These findings suggest that chondroitin sulfate proteoglycans which differ in molecular size and composition may be specific to particular cell types in brain.     

Effect of insulin on the altered production of proteoglycans in rib cartilage of experimentally diabetic rats

Costal cartilage from experimentally diabetic rats, labeled in vivo or in vitro with [35S]sulfate, was shown to incorporate less label into proteoglycans than cartilage from nondiabetic rats. Analyses of guanidine HCl cartilage extracts by gel chromatography on Sepharose CL-2B showed two major peaks at Kav approximately 0.4 and 0.8 (peaks I and II, respectively). Cartilage extracts from the diabetic rats contained predominantly peak II proteoglycans, while 60 and 55%, respectively, of the total 35S-labeled proteoglycans extracted from control cartilage labeled in vivo and in vitro with [35S]sulfate were present in peak I. After insulin treatment of the diabetic rats, the relative amount of peak I 35S-labeled proteoglycans synthesized in vivo was increased to 70%. The overall in vivo incorporation of [35S]sulfate into proteoglycans was also stimulated in diabetic rats treated with insulin to levels above those found for control rats. Thus, diabetes-induced changes in the biosynthesis of rat costal cartilage proteoglycans may be alleviated by normalization of the diabetic state by insulin treatment. However, addition of insulin (10(-5)-10(-9) M) to the culture medium did not affect the amount of 35S-labeled proteoglycans synthesized in vitro or the relative amounts of peak I proteoglycans produced by control or diabetic cartilage, suggesting that insulin does not have a direct effect on proteoglycan production. Moreover, no decrease in the amount of 35S-labeled proteoglycans produced was found when glucose at high concentrations was present in the culture medium. However, the presence of rat serum resulted in an increase in the amount of 35S-labeled proteoglycans produced by both control and diabetic cartilage, demonstrating that the cartilage explants were metabolically responsive to stimulatory factors.     

Structural characterization of heparan sulfate proteoglycan subclasses isolated from bovine aortic endothelial cell cultures

Labeled heparan sulfate proteoglycans (HSPG) were isolated from wounded and confluent cultures of bovine aortic endothelial cells by nondegradative extraction with 4 M guanidine hydrochloride and detergent. HSPG were separated from more highly charged chondroitin or dermatan sulfate proteoglycans by ion-exchange chromatography, and subclasses of different hydrodynamic size were isolated by gel filtration. Three major subclasses of HSPG were characterized structurally with respect to the presence and relative size of protein core, the presence and amount of nonsulfated oligosaccharide, and size and structure of heparan sulfate (HS) chains. The largest (600-800-kDa) HSPG subclass (I), isolated from cell layers and media of confluent cultures, bears 38-kDa HS chains on an apparently heterogeneous class of relatively large glycoprotein cores. HSPG II (150-200 kDa), isolated from cell layer or media, has 22-kDa HS chains and smaller core glycoproteins (less than 50 kDa). HSPG III, the subclass of smallest hydrodynamic size, has 13-kDa HS chains and a glycopeptide core of less than 15 kDa. All subclasses bear varying proportions of non-sulfated oligosaccharides of similar sizes. Comparisons of HS chain structure indicated that the different subclasses have similar proportions (49-55%) of N-sulfate, with both O-sulfate and highly N-sulfated blocks of disaccharide distributed similarly along HS chains. In addition, HS chains from subclasses II and III contain sequences that are insensitive to periodate oxidation or heparitinase digestion, suggesting that they contain increased proportions of iduronate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transforming growth factor-beta induces selective increase of proteoglycan production and changes in the copolymeric structure of dermatan sulphate in human skin fibroblasts.

Human embryonic skin fibroblasts were pretreated with transforming growth factor-beta (TGF-beta) for 6 h and then labeled with [35S]sulphate and [3H]leucine for 24 h. Radiolabeled proteoglycans from the culture medium and the cell layer were isolated and separated by isopycnic density-gradient centrifugation, followed by gel, ion-exchange and hydrophobic-interaction chromatography. The major proteoglycan species were examined by polyacrylamide gel electrophoresis in sodium dodecyl sulphate before and after enzymatic degradation of the polysaccharide chains. The results showed that TGF-beta increased the production of several different 35S-labelled proteoglycans. A large chondroitin/dermatan sulphate proteoglycan (with core proteins of approximately 400-500 kDa) increased 5-7-fold and a small dermatan sulphate proteoglycan (PG-S1, also termed biglycan, with a core protein of 43 kDa) increased 3-4-fold both in the medium and in the cell layer. Only a small effect was observed on another dermatan sulphate proteoglycan, PG-S2 (also named decorin). These observations are generally in agreement with results of other studies using similar cell types. In addition, we have found that the major heparan sulphate proteoglycan of the cell layer (protein core approximately 350 kDa) was increased by TGF-beta treatment, whereas all the other smaller heparan sulphate proteoglycans with protein cores from 250 kDa to 30 kDa appeared unaffected. To investigate whether TGF-beta also influences the glycosaminoglycan (GAG) chain-synthesizing machinery, we also characterized GAGs derived from proteoglycans synthesized by TGF-beta-treated cells. There was generally no increase in the size of the GAG chains. However, the dermatan sulphate chains on biglycan and decorin from TGF-beta treated cultures contained a larger proportion of D-glucuronosyl residues than those derived from untreated cultures. No effect was noted on the 4- and 6-sulphation of the GAG chains. By the use of p-nitrophenyl beta-D-xyloside (an initiator of GAG synthesis) it could be demonstrated that chain synthesis was also enhanced in TGF-beta-treated cells (approximately twofold). Furthermore, the dermatan sulphate chains synthesized on the xyloside in TGF-beta-treated fibroblasts contained a larger proportion of D-glucuronosyl residues than those of the control. These novel findings indicate that TGF-beta affects proteoglycan synthesis both quantitatively and qualitatively and that it can also change the copolymeric structure of the GAG by affecting the GAG-synthesizing machinery. Altered proteoglycan structure and production may have profound effects on the properties of extracellular matrices, which can affect cell growth and migration as well as organisation of matrix fibres.     

Energy of the low-lying excited levels for some reduced [4Fe-4S] ferredoxins, from the relaxation broadening of the E.P.R. signals

Two different forms of cell-associated [³⁵S]-heparan sulfate proteoglycans were identified in prelabeled cultured cells, including glial cells, endothelial cells and fibroblasts. One of them migrated characteristically in the excluded volume fraction in Sepharose CL-2B chromatography and flotated in CsCl density gradient centrifugation. Further, it showed affinity for a hydrophobic gel, Octyl-Sepharose. The molecular size was markedly reduced and the density elevated by treatment with detergent or lipid solvents. These findings indicate an admixture of lipid in this proteoglycan and suggest a location for the molecule in the plasma membrane. This proteoglycan was found in all cell species examined. - The other type of heparan sulfate proteoglycan had a larger molecular size than most previously described heparan sulfate proteoglycans and had a buoyant density around 1.32 g/ml, probably due to an unusually high ratio of protein to carbohydrate. This heparan sulfate proteoglycan was found only in extracts of cells capable of forming a fibrillar extracellular matrix, but not in extracts of cells devoid of matrix. It was retained in cell-free preparations of extracellular matrix, indicating that it may be a specific product of this compartment.     

Identification of a heparin-releasable lipoprotein lipase binding protein from endothelial cells.

Triglycerides in circulating plasma lipoproteins are hydrolyzed by lipoprotein lipase (LPL) which is thought to bind to proteoglycans on the luminal endothelial cell surface. Previous studies from this laboratory using LPL-Sepharose affinity chromatography identified a 220-kDa LPL binding proteoglycan. Using ligand blotting with 125I-LPL, we now report a 116-kDa LPL binding protein in plasma membrane preparations of endothelial cells. 125I-LPL binding to this protein was abolished by addition of unlabeled LPL. When the cell surface of endothelial cells was labeled with biotin, a 116-kDa protein was biotinylated. Furthermore, the biotinylated 116-kDa protein bound to LPL-Sepharose and eluted with 0.4 M NaCl suggesting that the 116-kDa LPL binding protein is present on the cell surface. When detergent extracts of endothelial cells were applied to LPL-Sepharose in the presence of 0.15 M NaCl, the 116-kDa, but not the 220-kDa, protein still bound to LPL-Sepharose. The 116-kDa protein was not labeled with 35SO4 and eluted from DEAE-cellulose prior to proteoglycans, suggesting that it is not a proteoglycan. However, a 116-kDa endothelial cell surface protein was metabolically labeled with [35S]methionine. This protein was dissociated from the cell surface by incubating cells with heparin (50 units/ml)-containing buffer. After heparin treatment of endothelial cells, LPL binding to and internalization by the cells decreased greater than 70% compared to control cells. These results suggest that endothelial cells synthesize a heparin-releasable, high affinity 116-kDa LPL binding protein. We postulate that this protein is associated with proteoglycans on luminal endothelial surfaces and mediates LPL binding, internalization, and recycling. We name this protein hrp (heparin-releasable protein)-116.