Characterization of heparan sulfate proteoglycans synthesized by rat ovarian granulosa cells in culture

Rat ovarian granulosa cells were isolated from immature female rats after stimulation with pregnant mare's serum gonadotropin and maintained in culture. Proteoglycans were labeled using [35S]sulfate, [3H]serine, [3H]glucosamine, or [3H]mannose as precursors. A species of heparan sulfate proteoglycan was purified using DEAE-Sephacel chromatography under dissociative conditions in the presence of detergent. The heparan sulfate proteoglycan, which constituted approximately 15% of the 35S-labeled proteoglycans in the culture medium has a similar hydrodynamic size (Kd = 0.62 on Sepharose CL-2B) and buoyant density distribution in CsCl density gradients as the low buoyant density dermatan sulfate proteoglycan synthesized by the same granulosa cells and described in the accompanying report (Yanagishita, M., and Hascall, V. C. (1983) J. Biol. Chem. 258, 12847-12856). The heparan sulfate chains (average Mr = 28,000) have an average of 0.8-0.9 sulfate groups/repeating disaccharide, of which 50% are N-sulfate, 30% are alkaline-labile O-sulfate (presumably on the 6-position of glucosamine residues), and 20% are alkaline-resistant O-sulfate groups. Alkaline borohydride treatment released both N-linked oligosaccharide-peptides containing mannose, glucosamine, and sialic acid, and O-linked oligosaccharides. Trypsin digestion of the proteoglycan generated fragments which contain (a) glycosaminoglycan-peptides with an average of 2 heparan sulfate chains/peptide; (b) clusters of O-linked oligosaccharides on peptides; and (c) N-linked oligosaccharide-peptides, which are as small as single N-linked oligosaccharides. The compositions of the O-linked and N-linked oligosaccharides and the trypsin fragments of this heparan sulfate proteoglycan were very similar to those of the low buoyant density dermatan sulfate proteoglycan synthesized by the same cells.     

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.     

Schistosoma mansoni synthesizes glycoproteins containing terminal O-linked N-acetylglucosamine residues

In this report, we describe our studies on the structures of the O-linked oligosaccharides in glycoproteins synthesized by the human blood fluke Schistosoma mansoni. Adult male schistosomes were incubated with either [2-3H]mannose, [6-3H]glucosamine, or [6-3H]galactose to metabolically radiolabel newly synthesized glycoproteins. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorographic analyses indicated that many glycoproteins were labeled by each of the radioactive precursors. Glycopeptides were prepared from radiolabeled glycoproteins by pronase treatment and fractionated on columns of concanavalin A-Sepharose and pea lectin-agarose. The O-linked oligosaccharides were released from glycopeptides by treatment with mild base/borohydride. All O-linked material was found in glycopeptides not bound by either of the immobilized lectins. The structures of the released chains were then analyzed by a variety of techniques. Our results demonstrate that the schistosomes synthesize glycoproteins containing two major types of simple O-linked sugar chains. One type, which represents a minor fraction of the O-linked oligosaccharides, contains N-acetylgalactosamine linked to peptide. These O-linked chains occur as terminal O-linked N-acetylgalactosamine and the O-linked disaccharide, galactose----N-acetylgalactosamine. Sialic acid was not present in either of these O-linked chains or in any other glycopeptides derived from adult male schistosomes. However, the major type of O-linked chain in glycoproteins synthesized by adult schistosomes is an unusual terminal O-linked N-acetylglucosamine linked to peptide. This latter structure represents approximately 10% of the total radioactive N-acetylglucosamine recovered in all glycopeptides. Our results also suggest the possibility that the O-linked oligosaccharides are highly clustered on the glycopeptides.     

Tunicamycin inhibits proteoglycan synthesis in rat ovarian granulosa cells in culture

The effects of tunicamycin, an inhibitor of N-linked oligosaccharide biosynthesis, on the synthesis and turnover of proteoglycans were investigated in rat ovarian granulosa cell cultures. The synthesis of proteoglycans was inhibited (40% of the control at 1.6 micrograms/ml tunicamycin) disproportionately to that of general protein synthesis measured by [3H]serine incorporation (80% of control). Proteoglycans synthesized in the presence of tunicamycin lacked N-linked oligosaccharides but contained apparently normal O-linked oligosaccharides. The dermatan sulfate and heparan sulfate chains of the proteoglycans had the same hydrodynamic size as control when analyzed by Sepharose 6B chromatography. However, the disulfated disaccharide content of the dermatan sulfate chains was reduced by tunicamycin in a dose-dependent manner, implying that the N-linked oligosaccharides may be involved in the function of a sulfotransferase which is responsible for sulfation of the iduronic acid residues. When [35S]sulfate and [3H]glucosamine were used as labeling precursors, the ratio of 35S/3H in chondroitin 4-sulfate was reduced to approximately 50% of the control by tunicamycin, indicating that the drug reduced the supply of endogenous sugar to the UDP-N-acetylhexosamine pool. Neither transport of proteoglycans from Golgi to the cell surface nor their turnover from the cell surface (release into the medium, or internalization and subsequent intracellular degradation) was affected by the drug. Addition of mannose 6-phosphate to the culture medium did not alter the proteoglycan turnover. When granulosa cells were treated with cycloheximide, completion of proteoglycan diminished with a t1/2 of approximately 12 min, indicating the time required for depleting the core protein precursor pool. The glycosaminoglycan synthesizing capacity measured by the addition of p-nitrophenyl-beta-xyloside, however, lasted longer (t1/2 of approximately 40 min). Tunicamycin decreased the core protein precursor pool size in parallel to decreased proteoglycan synthesis, both of which were significantly greater than the inhibition of general protein synthesis. This suggests two possibilities: tunicamycin specifically inhibited the synthesis of proteoglycan core protein, or more likely a proportion of the synthesized core protein precursor (approximately 50%) did not become accessible for post-translational modifications, and was possibly routed for premature degradation.     

Beta-linked N-acetylgalactosamine residues present at the nonreducing termini of O-linked oligosaccharides of a cloned murine cytotoxic T lymphocyte line are absent in a Vicia villosa lectin-resistant mutant cell line

The O-linked oligosaccharides of the cloned, murine cytotoxic T cell line B6.1.SF.1 were compared with the corresponding oligosaccharides from a Vicia villosa lectin-resistant mutant of B6.1.SF.1 called VV6 (Conzelmann, A., Pink, R., Acuto, O., Mach, J.-P., Dolivo, S., and Nabholz, M. (1980) Eur. J. Immunol. 10, 860-868). The VV6 mutant cells are deficient in binding sites for this GalNAc-specific lectin. Cells were grown in the presence of [3H]glucosamine and [3H] galactose to label the glycoproteins, and the desialyzed, alkaline borohydride-released oligosaccharides were isolated and characterized. The VV6 cells contained a series of O-linked oligosaccharides ranging in size from a disaccharide to a pentasaccharide. These were composed of galactose, N-acetylglucosamine, and N-acetylhexosaminitol, the latter sugar being derived from the reducing terminus. The predominant oligosaccharide had the partial structure Gal beta GlcNAc beta-(Gal beta)N-acetylhexosaminitol. In contrast, the analogous oligosaccharides of the parental cells contained additional beta-linked GalNAc residues located at nonreducing termini. The smallest of these had the structure GalNAc beta 1,4Gal beta-N-acetylhexosaminitol. Neither cell line contained significant amounts of terminal GalNAc linked to Ser/Thr which is the main binding site for the V. villosa B4 lectin on Tn erythrocytes (Tollefsen, S. R., and Kornfeld, R. (1983) J. Biol. Chem. 258, 5172-5176). These findings suggest that the major binding sites for the V. villosa lectin on the parental cytotoxic T cell line consist of structures containing beta 1,4-linked GalNAc residues at the nonreducing ends of conventional O-linked structures. The VV6 cells lack these beta-linked GalNAc residues, and this may account for their deficiency of V. villosa lectin-binding sites. In the following paper (Conzelmann, A., and Kornfeld, S. (1984) J. Biol. Chem. 259, 12536-12542), we demonstrate that the VV6 cells are missing the N-acetylgalactosaminyltransferase that is responsible for the synthesis of these unusual oligosaccharides.     

Deaminated neuraminic acid-rich glycoprotein of rainbow trout egg vitelline envelope. Occurrence of a novel alpha-2,8-linked oligo(deaminated neuraminic acid) structure in O-linked glycan chains

Deaminated neuraminic acid-rich glycoprotein (KDN-gp), first found and isolated from the vitelline envelope of rainbow trout eggs (Inoue, S., Kanamori, A., Kitajima, K., and Inoue, Y. (1988) Biochem. Biophys. Res. Commun. 153, 172-176), has been found to contain a number of O-linked glycan. Oligosaccharides were released by alkaline borohydride treatment of KDN-gp. Following fractionation by DEAE-Sephadex A-25 and thin-layer chromatography, a series of acidic oligosaccharides were obtained and analyzed for their chemical structures. The structure is based on composition analysis, methylation analysis, alkali-catalyzed "peeling" reactions, periodate oxidation, 400-MHz one- and two-dimensional 1H NMR spectroscopy, and molecular secondary ion mass spectrometry. The O-linked oligosaccharides isolated from KDN-gp have been shown to contain a common core trisaccharide Gal beta 1-3GalNAc alpha 1-3GalNAc in which the terminal Gal residue is blocked by a single residue of deaminated neuraminic acid (KDN) and the proximal GalNAc residue is substituted by alpha-2,8-linked oligo(KDN) chains. Structures of KDN-oligosaccharide chains in the glycoprotein are novel and expressed by the following general formula, where n = 0-5, for which data are available. [formula: see text]     

Human transferrin receptor contains O-linked oligosaccharides

We have investigated the oligosaccharides in the human transferrin receptor from three different cell lines. During our studies on the structures of the N-linked oligosaccharides of the receptor, we discovered that the receptor contains O-linked oligosaccharides. This report describes the isolation and characterization of these O-linked oligosaccharides. Three different human cell lines--K562, A431, and BeWo--were grown in media containing either [2-3H] mannose or [6-3H]glucosamine. The newly synthesized and radiolabeled transferrin receptors were purified by immunoprecipitation from cell extracts and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The receptor was proteolytically digested or treated directly with mild base/borohydride. The released radiolabeled glycopeptides and oligosaccharides were separated by a variety of chromatographic techniques, and their structures were analyzed. The transferrin receptor from all three cell types contains O-linked oligosaccharides that are released from peptide by mild base/borohydride treatment. The receptor from K562 cells contains at least one O-linked oligosaccharide having two sialic acid residues and a core structure of the disaccharide galactose-N-acetyl-galactosamine. In contrast, the O-linked oligosaccharides in the transferring receptors from both A431 and BeWo cell lines are not as highly sialylated and were identified as both the neutral disaccharide galactose-N-acetylgalactosamine and the neutral monosaccharide N-acetylgalactosamine. In addition, the receptors from all three cell lines contain both complex-type and high mannose-type N-linked oligosaccharides. The complex-type chains in the receptor from A431 cells have properties of blood group A antigens, whereas oligosaccharides in receptors from both BeWo and K562 cells lack these properties. These results are interesting since both A431 and BeWo cells, but not K562 cells, are positive for blood group A antigens. Thus, our results demonstrate that the human transferrin receptor contains O-linked oligosaccharides and that there are differences in the structures of both the O-linked and complex-type N-linked oligosaccharides on the receptors synthesized by different cell types.     

Two linkage-region fragments isolated from skeletal keratan sulphate contain a sulphated N-acetylglucosamine residue.

Peptido-keratan sulphate fragments were isolated from the nucleus pulposus of bovine intervertebral discs (6-year-old animals) after chondroitin ABC lyase digestion followed by digestion of A1D1 proteoglycans by diphenylcarbamoyl chloride-treated trypsin and gel-permeation chromatography on Sepharose CL-6B. Treatment of these peptido-keratan sulphate fragments with alkaline NaB3H4 yielded keratan sulphate chains with [3H]galactosaminitol end-labels, and these chains were further purified by gel-permeation chromatography on Sephadex G-50 and ion-exchange chromatography on a Pharmacia Mono-Q column in order to exclude any contamination with O-linked oligosaccharides. The chains were then treated with keratanase, and the digest was chromatographed on a Bio-Gel P-4 column followed by anion-exchange chromatography on a Nucleosil 5 SB column. Two oligosaccharides, each representing 18% of the recovered radiolabel, were examined by 500 MHz 1H-n.m.r. spectroscopy, and shown to have the following structures: [formula: see text] The structure of oligosaccharide (I) confirms the N-acetylneuraminylgalactose substitution at position 3 of N-acetylgalactosamine in the keratan sulphate-protein linkage region found by Hopwood & Robinson [(1974) Biochem. J. 141, 57-69] but additionally shows the presence of a 6-sulphated N-acetylglucosamine. Electron micro-probe analysis specifically confirmed the presence of sulphur in this sample. This sulphate ester group differentiates the keratan sulphate linkage region from similar structures derived from O-linked oligosaccharides [Lohmander, De Luca, Nilsson, Hascall, Caputo, Kimura & Heinegård (1980) J. Biol. Chem. 255, 6084-6091].     

Age-related changes in hyaluronan, proteoglycan, collagen, and osteonectin synthesis by human bone cells.

Human bone cells grown in culture, representative of a preosteoblastic stage of maturation, produce an extracellular matrix composed of collagen, several noncollagenous glycoproteins, hyaluronan, and four distinct proteoglycans (PGs). The influence of donor age on the levels of expression of these molecules in vitro has not been well characterized. In this study, human bone cells derived from sources ranging from fetal to 60-year-old donors were grown in culture, radiolabeled for 24 h, and the amount of incorporation of [35S]sulfate into PGs, [3H]glucosamine into hyaluronan, [3H]leucine/proline into osteonectin, and [3H]proline into collagen was determined. Cell proliferation was most rapid in fetal-derived bone cells and decreased with increasing age. Total protein and PG synthesis also decreased with increasing age, falling to 1/3 and 1/4, respectively, of fetal levels after age 30. A large chondroitin sulfate PG (Mr approximately 600,000 Da) was the major fetal PG and its levels were highly correlated with cellular proliferation. [3H]Collagen and [35S]decorin levels increased with the increasing age of the donor, reached a maximum in puberty-derived cells, and decreased to 1/3 maximal levels after age 20. The heparan sulfate PG (Mr approximately 400,000 Da) exhibited steady-state levels regardless of donor age. [3H]Osteonectin and [35S]biglycan levels were high in fetal-derived cells and in cells derived from pubescent donors. The percentage of collagen and four proteoglycans associated with the cell layer pool changed with donor age. All fetal-derived PG core proteins possessed more N- and O-linked oligosaccharides than newborn or adult derived PGs.     

Structural variations of O-linked oligosaccharides present in leukosialin isolated from erythroid, myeloid, and T-lymphoid cell lines

Structures of O-linked oligosaccharides of leukosialin isolated from K562 erythroid, HL-60 promyelocytic, and HSB-2 T-lymphoid cell lines were examined. Leukosialin was isolated by specific immunoprecipitation from cells which were metabolically labeled with [3H]glucosamine, and glycopeptides were isolated after Pronase digestion. O-Linked oligosaccharides were released by alkaline borohydride treatment, and the structures of purified oligosaccharides were elucidated by specific exoglycosidase digestion, Smith degradation, and methylation anaylsis. Oligosaccharides from K562 cells were found to be GalNAcOH, Gal beta 1----3GalNAcOH, NeuNAc alpha 2----6GalNAcOH, NeuNAc alpha 2----3Gal beta 1----3GalNAcOH, Gal beta 1----3(NeuNAc alpha 2----6)GalNAcOH, and NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----6)GalNAcOH. On the other hand, oligosaccharides from HL-60 and HSB-2 cells were found to be NeuNAc alpha 2----3Gal beta 1----3GalNAcOH, NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6(Gal beta 1----3)GalNAcOH, Gal beta 1----4GlcNAc beta 1----6(NeuNAc alpha 2----3)Gal beta 1----3)GalNAcOH, and NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6(NeuNAc alpha 2----3Gal beta 1----3)GalNAcOH. These results clearly indicate that leukosialin can be differently glycosylated with O-linked chains, and each erythroid or myeloid (and T-lymphoid) cell line expresses a characteristic set of O-linked oligosaccharides which differ in core structures as well as in sialylation.     

Analysis of the proteoglycans synthesized by corneal explants from embryonic chicken. II. Structural characterization of the keratan sulfate and dermatan sulfate proteoglycans from corneal stroma

Radioisotopically labeled proteoglycans were isolated from a 4 M guanidine HCl, 2% Triton X-100 extract of corneal stroma from day 18 chicken embryos by anion-exchange chromatography. Two predominant proteoglycans in the sample were separated by octyl-Sepharose chromatography using a gradient elution of detergent in 4 M guanidine HCl. One proteoglycan had an overall mass of approximately 125 kDa, a single dermatan sulfate chain (approximately 85-90% chondroitin 4-sulfate, low iduronate content) of approximately 65 kDa, and a core protein after chondroitinase ABC digestion of approximately 45 kDa which also contained one to three N-linked oligosaccharides and one O-linked oligosaccharide. The other proteoglycan had an overall size of approximately 100 kDa, two to three keratan sulfate chains of approximately 15 kDa each, and a core protein following keratanase digestion of approximately 51 kDa which included two to three N-linked but no O-linked oligosaccharides. A larger size, a greater overall hydrophobicity (as measured by its interaction with octyl-Sepharose) and an absence of O-linked oligosaccharides argue that this core protein is a distinct gene product from the core protein of the dermatan sulfate proteoglycan.     

O-Linked glycome and proteome of high-molecular-mass proteins in human ovarian cancer ascites: Identification of sulfation, disialic acid and O-linked fucose

The O-linked glycosylation of the main acidic high-molecular-weight glycoprotein from ascites fluid from patients with ovarian cancer were analyzed. The O-linked oligosaccharides were shown to consist of mainly highly sialylated core 1 and 2 structures with a smaller amount of sulfated core 2 structures. These structures were shown to be able to be further extended into small keratan sulfate (KS)-type oligosaccharides with up to four N-acetyllactosamine units. Proteomic studies of the acidic fraction of ascites fluid from patients with ovarian cancer showed that this fraction was enriched in proteoglycans. Among them, lumican, agrin, versican and dystroglycans were potential candidates, with threonine- and serine-rich domains that could carry a significant amount of O-linked glycosylation, including also the O-linked KS. Glycomic analysis using liquid chromatography (LC)-tandem mass spectrometry (MS/MS) also showed that the disialic acid NeuAc-NeuAc- was frequently found as the terminating structure on the O-linked core 1 and 2 oligosaccharides from one ascites sample. Also, a small amount of the epidermal growth factor (EGF)-associated O-linked fucose structure Gal-GlcNAc-Fucitol was detected with and without sialic acid in the LC-MS/MS analysis. Candidate proteins containing O-linked fucose were suggested to be proteoglycan-type molecules containing the O-linked fucose EGF consensus domain.     

Heterogeneity of proteoglycans in monkey corneal stroma

The proteoglycans of the cynomolgus monkey corneal stroma were isolated and characterized by using a combination of physiochemical and biochemical methods. Proteoglycans were biosynthetically radiolabeled by incubating whole corneas in medium containing [³⁵S]sulfate and either [³H]serine or [³H]mannose as precursors. Macromolecules were extracted from the corneal stromas with 4 m guanidine-HCl. After dialysis into 8 m urea, proteoglycans in the extracts were initially purified by DEAE-cellulose chromatography. A portion of the proteoglycan fraction was digested with chondroitinase ABC, and the keratan sulfate proteoglycans were then isolated by rechromatography of the digest on DEAE-cellulose. Another portion of the proteoglycan fraction was digested with endo-β-galactosidase and the dermatan sulfate-proteoglycans were then isolated by chromatography of the digest on Sepharose CL-4B. Each proteoglycan population was further fractionated by chromatography on concanavalin A-Sepharose and by CsCl density gradient centrifugation. Four subpopulations for both the keratan sulfate proteoglycans and the dermatan sulfate proteoglycans were isolated. Based on differences in binding to concanavalin A-Sepharose, buoyant densities, and glycosaminoglycan content, subpopulations of each proteoglycan differ by the number and properties of both the glycosaminoglycan chains and the mannose-containing oligosaccharides attached to their protein core.     

Isolation and characterization of proteoglycans synthesized by adult human gingival fibroblasts in vitro

The proteoglycans synthesized by fibroblasts derived from healthy human gingivae were isolated and characterized. The largest medium proteoglycan was excluded from Sepharose CL-4B but not from Sepharose CL-2B; it was recovered in the most-dense density gradient fraction and identified as a chondroitin sulfate proteoglycan. The medium contained two smaller proteoglycans; one contained predominantly chondroitin sulfate proteoglycan, while the other was comprised predominantly of dermatan sulfate proteoglycan and was quantitatively the major species. The largest proteoglycan in the cell layer fraction, excluded from both Sepharose CL-2B and Sepharose CL-4B, was found in the least-dense density gradient fraction and contained heparan sulfate and chondroitin sulfate proteoglycan. It could be further dissociated by treatment with detergent, suggesting an intimate association with cell membranes. Two other proteoglycan populations of intermediate size were identified in the cell layer extracts which contained variable proportions of heparan sulfate, dermatan sulfate, or chondroitin sulfate proteoglycan. Some small molecular weight material indicative of free glycosaminoglycan chains was also associated with the cell layer fraction. Carbohydrate analysis of the proteoglycans demonstrated the glycosaminoglycan chains to have approximate average molecular weights of 25,000. In addition, N- and O-linked oligosaccharides which were associated with the proteoglycans appeared to be sulfated in varying degrees.     

Isolation and characterization of the heparan sulfate proteoglycans of brain. Use of affinity chromatography on lipoprotein lipase-agarose

Heparan sulfate proteoglycans were extracted from rat brain microsomal membranes or whole forebrain with deoxycholate and purified from accompanying chondroitin sulfate proteoglycans and membrane glycoproteins by ion-exchange chromatography, affinity chromatography on lipoprotein lipase-Sepharose, and gel filtration. The proteoglycan has a molecular size of approximately 220,000, containing glycosaminoglycan chains of Mr = 14,000-15,000. In [3H]glucosamine-labeled heparan sulfate proteoglycans, approximately 22% of the radioactivity is present in glycoprotein oligosaccharides, consisting predominantly of N-glycosidically linked tri- and tetraantennary complex oligosaccharides (60%, some of which are sulfated) and O-glycosidic oligosaccharides (33%). Small amounts of chondroitin sulfate (4-6% of the total glycosaminoglycans) copurified with the heparan sulfate proteoglycan through a variety of fractionation procedures. Incubation of [35S]sulfate-labeled microsomes with heparin or 2 M NaCl released approximately 21 and 13%, respectively, of the total heparan sulfate, as compared to the 8-9% released by buffered saline or chondroitin sulfate and the 82% which is extracted by 0.2% deoxycholate. It therefore appears that there are at least two distinct types of association of heparan sulfate proteoglycans with brain membranes.     

Chondroitin sulfate and heparan sulfate proteoglycans of PC12 pheochromocytoma cells

We have isolated and characterized the cell-associated and secreted proteoglycans synthesized by a clonal line of rat adrenal medullary PC12 pheochromocytoma cells, which have been extensively employed for the study of a wide variety of neurobiological processes. Chondroitin sulfate accounts for 70-80% of the [35S] sulfate-labeled proteoglycans present in PC12 cells and secreted into the medium. Two major chondroitin sulfate proteoglycans were detected with molecular sizes of 45,000-100,000 and 120,000-190,000, comprising 14- and 105-kDa core proteins and one or two chondroitin sulfate chains with an average molecular size of 34 kDa. In contrast to the chondroitin sulfate proteoglycans, one major heparan sulfate proteoglycan accounts for most of the remaining 20-30% of the [35S] sulfate-labeled proteoglycans present in the PC12 cells and medium. It has a molecular size of 95,000-170,000, comprising a 65-kDa core protein and two to six 16-kDa heparan sulfate chains. Both the chondroitin sulfate and heparan sulfate proteoglycans also contain O-glycosidically linked oligosaccharides (25-28% of the total oligosaccharides) and predominantly tri- and tetraantennary N-glycosidic oligosaccharides. Proteoglycans produced by the original clone of PC12 cells were compared with those of two other PC12 cell lines (B2 and F3) that differ from the original clone in morphology, adhesive properties, and response to nerve growth factor. Although the F3 cells (a mutant line derived from B2 and reported to lack a cell surface heparan sulfate proteoglycan) do not contain a large molecular size heparan sulfate proteoglycan species, there was no significant difference between the B2 and F3 cells in the percentage of total heparan sulfate released by mild trypsinization, and both the B2 and F3 cells synthesized cell-associated and secreted chondroitin sulfate and heparan sulfate proteoglycans having properties very similar to those of the original PC12 cell line but with a reversed ratio (35:65) of chondroitin sulfate to heparan sulfate.     

Metabolic pathways of heparan sulfate proteoglycans in a rat parathyroid cell line.

The distribution of heparan sulfate (HS) proteoglycans in clonal rat parathyroid cells is regulated by the extracellular Ca2+ concentration, which is a principal factor for parathyroid cell function (Takeuchi, Y., Sakaguchi, K., Yanagishita, M., Aurbach, G. D., and Hascall, V. C. (1990) J. Biol. Chem. 265, 13661-13668). Increasing the concentration of extracellular Ca2+ in the physiological range redistributes HS proteoglycans from the cell surface to an intracellular compartment. We have now examined effects of the extracellular Ca2+ concentration on the metabolism of the HS proteoglycans in detail using [35S]sulfate metabolic labeling-chase experiments. Two distinct metabolic pathways were demonstrated: (i) the intracellular generation of HS chains from HS proteoglycans in prelysosomal compartments followed by their release into the medium (pathway 1), and (ii) intracellular generation of HS oligosaccharides from HS chains in prelysosomal compartments, which are eventually degraded into free sulfate in lysosomes (pathway 2). The HS oligosaccharides were exclusively present within the cells, whereas HS chains were found primarily in the medium. The cells do not internalize either HS proteoglycans or HS chains from the medium. These observations indicate that these two degradation pathways are independent. In addition to these pathways, approximately 15% of the HS proteoglycans were released into the medium as a proteoglycan form. Treatment of cells with chloroquine, a lysosomotropic agent, did not affect generation of HS chains but inhibited conversion of HS chains to HS oligosaccharides or to free sulfate and resulted in the release of HS chains from the cells. The drug did not affect metabolic pathway 1. The extracellular Ca2+ concentration did not alter these intracellular degradation pathways for HS proteoglycans in the parathyroid cells. Thus, extracellular Ca2+ appears to regulate only the distribution of HS proteoglycans between the cell surface and intracellular compartments, and the process of cycling between these compartments when extracellular Ca2+ is low.     

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

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

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.     

Synthesis of sulfated glycoproteins by glial precursor cells

Populations of enriched glial precursor cells and astrocytes isolated from primary cultures of newborn rat brain were used to study the synthesis of sulfated glycoproteins. Both cell types incorporated [³H]glucosamine and [³⁵S]sulfate into carbohydrate side chains of proteoglycans and glycoproteins. The rate of incorporation of [³H]glucosamine into the oligosaccharides and the pattern of distribution of the label into high mannose and complex glycopeptides recovered from the glycoproteins appeared to be similar for the two glial cell types. However, clear differences were noted in the rate of oligosaccharide sulfation activities. Thus the cultures of precursor glia were about four times more active than cultures enriched in astroglia in their ability to incorporate [³⁵S]sulfate into glycoproteins.