Partial characterization of proteoglycans synthesized by human glomerular epithelial cells in culture

Confluent adult and fetal human glomerular epithelial cells were incubated for 24 h in the presence of [3H]-amino acids and [35S]sulfate. Two heparan-35SO4 proteoglycans were released into the culture medium. These 35S-labeled proteoglycans eluted as a single peak from anion exchange chromatographic columns, but were separable by gel filtration on Sepharose CL-6B columns. The larger heparan-35SO4 proteoglycan eluted with the column void volume and at a Kav of 0.26 from Sepharose CL-4B columns. The most abundant medium heparan-35SO4 proteoglycan was a high buoyant density proteoglycan similar in hydrodynamic size (Sepharose CL-6B Kav 0.23) to those previously described in glomerular basement membranes and isolated glomeruli. Heparan-35SO4 chains from both proteoglycans were 36 kDa. A smaller proportion of Sepharose CL-6B excluded dermatan-35SO4 proteoglycan was also synthesized by these cells. The predominant protein cores of both medium heparan-35SO4 proteoglycans were approximately 230 and 180 kDa. A hybrid chondroitin/dermatan-heparan-35SO4 proteoglycan with an 80-kDa protein core copurified with the smaller medium heparan-35SO4 proteoglycan. This 35S-labeled proteoglycan appeared as a diffuse, chondroitinase ABC sensitive 155-kDa fluorographic band in sodium dodecyl sulfate-polyacrylamide gels after the Sepharose CL-6B Kav 0.23 35S-labeled proteoglycan fraction was digested with heparitinase. The heparitinase generated heparan sulfate proteoglycan protein cores and the 155-kDa hybrid proteoglycan fragment had molecular weights similar to those previously identified in rat glomerular basement membrane and glomeruli using antibodies against a basement membrane tumor proteoglycan precursor (Klein et al. J. Cell Biol. 106, 963-970, 1988). Thus, human glomerular epithelial cells in culture are capable of synthesizing, processing, and releasing heparan sulfate proteoglycans which are similar to those synthesized in vivo and found in the glomerular basement membrane. These proteoglycans may belong to a family of related basement membrane proteoglycans.     

Human glomerular epithelial cell proteoglycans

Proteoglycans synthesized by cultures of human glomerular epithelial cells have been isolated and characterized. Three types of heparan sulfate were detected. Heparan sulfate proteoglycan I (HSPG-I; Kav 6B 0.04) was found in the cell layer and medium and accounted for 12% of the total proteoglycans synthesized. HSPG-II (Kav 6B 0.25) accounted for 18% of the proteoglycans and was located in the medium and cell layer. A third population (9% of the proteoglycan population), heparan sulfate glycosaminoglycan (HS-GAG; Kav 6B 0.4-0.8), had properties consistent with single glycosaminoglycan chains or their fragments and was found only in the cell layer. HSPG-I and HSPG-II from the cell layer had hydrophobic properties; they were released from the cell layer by mild trypsin treatment. HS-GAG lacked these properties, consisted of low-molecular-mass heparan sulfate oligosaccharides, and were intracellular. HSPG-I and -II released to the medium lacked hydrophobic properties. The cells also produced three distinct types of chondroitin sulfates. The major species, chondroitin sulfate proteoglycan I (CSPG-I) eluted in the excluded volume of a Sepharose CL-6B column, accounted for 30% of the proteoglycans detected, and was found in both the cell layer and medium. Cell layer CSPG-I bound to octyl-Sepharose. It was released from the cell layer by mild trypsin treatment. CSPG-II (Kav 6B 0.1-0.23) accounted for 10% of the total 35S-labeled macromolecules and was found predominantly in the culture medium. A small amount of CS-GAG (Kav 6B 0.25-0.6) is present in the cell extract and like HS-GAG is intracellular. Pulse-chase experiments indicated that HSPG-I and -II and CSPG-I and -II are lost from the cell layer either by direct release into the medium or by internalization where they are metabolized to single glycosaminoglycan chains and subsequently to inorganic sulfate.     

Proteoglycan synthesis by primary chick skeletal muscle during in vitro myogenesis

The proteoglycans synthesized by primary chick skeletal muscle during in vitro myogenesis were compared with those of muscle-specific fibroblasts. Cultures of skeletal muscle cells and muscle fibroblasts were separately labeled using [35S] sulfate as a precursor. The proteoglycans of the cell layer and medium were separately extracted and isolated by ion-exchange chromatography on DEAE-Sephacel followed by gel filtration chromatography on Sepharose CL-2B. Two cell layer-associated proteoglycans synthesized both by skeletal muscle cells and muscle fibroblasts were identified. The first, a high molecular weight proteoglycan, eluted from Sepharose CL-2B with a Kav of 0.07 and contained exclusively chondroitin sulfate chains with an average molecular weight greater than 50,000. The second, a relatively smaller proteoglycan, eluted from Sepharose CL-2B with a Kav of 0.61 and contained primarily heparan sulfate chains with an average molecular weight of 16,000. Two labeled proteoglycans were also found in the medium of both skeletal muscle and muscle fibroblasts. A high molecular weight proteoglycan was found with virtually identical properties to that of the high molecular weight chondroitin sulfate proteoglycan of the cell layer. A second, smaller proteoglycan had a similar monomer size (Kav of 0.63) to the cell layer heparan sulfate proteoglycan, but differed from it in that this molecule contained primarily chondroitin sulfate chains with an average molecular weight of 32,000. Studies on the distribution of these proteoglycans in muscle cells during in vitro myogenesis demonstrated that a parallel increase in the relative amounts of the smaller proteoglycans occurred in both the cell layer and medium compared to the large chondroitin sulfate proteoglycan in each compartment. In contrast, muscle-derived fibroblasts displayed a constant ratio of the small proteoglycans of the cell layer and medium fractions, compared to the larger chondroitin sulfate proteoglycan of the respective fraction as a function of cell density. Our results support the concept that proteoglycan synthesis is under developmental regulation during skeletal myogenesis.     

Biosynthesis of heparan sulfate proteoglycans of developing chick breast skeletal muscle in vitro

Previous studies have reported an increase in heparan sulfate glycosaminoglycan (HSGAG) during skeletal muscle differentiation in culture. We have investigated this phenomenon further in relation to the heparan sulfate proteoglycans (HSPG) produced by myogenic cultures. Pulse-chase analysis indicated an approx. 3-fold increase in heparan sulfate synthesis in myotube cultures over that in proliferating or aligning myoblast cultures. Muscle fibroblast culture heparan sulfate synthesis was higher than that of myoblasts but was lower than myotubes. The turnover rates appeared to be the same for all stages of development, with a t1/2 of approx. 5 h. Enrichment for heparan sulfate by Sepharose CL-4B and DEAE-Sephacel chromatography indicated an increase in the hydrodynamic size of the proteoglycan produced by myotubes over that from myoblasts, with a shift in Kav from 0.14-0.19 to 0.07. Fibroblasts synthesized the smallest proteoglycan, with a Kav of 0.22. All of the proteoglycans contained similar sized glycosaminoglycan chains with an estimated molecular weight of 30,000-40,000. Localization of the heparan sulfate proteoglycan in myotube cultures by trypsin sensitivity indicated much of the intact proteoglycan to be closely associated with the cell surface, while internalized material appeared in a degraded form.     

Changes in heparan sulfate structure during transition from the proliferating to the non-dividing state of cultured arterial smooth muscle cells

Cultured arterial smooth muscle cells synthesize a cell-associated heparan sulfate proteoglycan which consists of a 92 kDa core protein with 3 to 4 heparan sulfate side chains covalently attached. Biosynthesis of the cell-associated heparan sulfate proteoglycan was compared in proliferating and in non-dividing vascular smooth muscle cells which are preincubated in the presence of [35]sulfate or a combination of [35S]methionine and [3H]glucosamine. The Mr of the core protein was identical in either growth state, but changes in the structure of the heparan sulfate side chains were observed. Non-dividing (postconfluent) arterial smooth muscle cells form longer heparan sulfate chains with a higher proportion of hydrophobic (N-acetyl) groups than proliferating (preconfluent) cells as judged from gel filtration experiments, hydrophobic interaction chromatography and heparitinase degradation. An enzyme preparation from proliferating cells catalyzes deacetylation and N-sulfation of heparan sulfate at a 5-fold higher activity than from non-dividing cells. Cell density-dependent structural differences of heparan sulfate are related to the finding that heparan sulfate isolated from non-dividing cells has a 10-fold higher antiproliferative potency than heparan sulfate from proliferating (preconfluent) cells.     

Detergent-solubilized proteoglycans in rat testicular Sertoli cells

Rat Sertoli cells were cultured for 48 h in the presence of [35S]sulfate and extracted with 4 M guanidine chloride. In this extract, a Sepharose CL-2B Kav 0.10 proteoheparan appeared lipid associated, since after addition of detergent it emerged at Kav = 0.65 on Sepharose CL-2B. Treatment of cells with 0.2% Triton X-100 released 35S-labeled material which was purified by ion-exchange chromatography and hydrophobic interaction chromatography on octyl-Sepharose. Proteoglycan with affinity for octyl-Sepharose (Kav = 0.30 and 0.12 on Sepharose CL-4B and CL-6B, respectively) mostly carried heparan sulfate chains with Kav = 0.38 and minor proportion of heparan chains with Kav = 0.77 on Sepharose CL-6B. An association with lipids was confirmed by intercalation into liposomes of this proteoheparan which might be anchored in the plasma membrane, via an hydrophobic segment and/or covalently linked to an inositol-containing phospholipid. Non-hydrophobic material consisted of: (i) proteoheparan slightly smaller in size than lipophilic proteoheparan and possibly deriving from this one and (ii) two heparan sulfate glycosaminoglycan populations (Kav = 0.38 and 0.86 on Sepharose CL-6B) corresponding to single glycosaminoglycan chains and their degradation products.     

Identification of chondroitin sulfate E in human lung mast cells

Human lung mast cells (HLMC) enriched up to 99% purity by counter current elutriation and density gradient centrifugation were labeled with 35S-sulfate to determine cell-associated proteoglycans. The 35S-labeled proteoglycans were extracted by the addition of detergent and 4 M guanidine-HCl, and separated from unincorporated precursor by Sephadex G-50 chromatography. 35S-Proteoglycans chromatographed over Sepharose 4B with a Kav of 0.48. 35S-Glycosaminoglycans separated from the parent 35S-proteoglycans by beta-elimination and chromatographed over Sepharose 4B with a Kav of 0.63. Characterization of 35S-proteoglycans by chondroitin ABC lyase treatment revealed approximately 36% of the proteoglycan to be composed of chondroitin sulfates. Analysis by HPLC of component disaccharides liberated by chondroitin ABC lyase using an amino-cyano-substituted silica column indicated that the chondroitin sulfates consisted of the monosulfated A disaccharide (GlcUA----GaINAc4SO4) (75%) and the over-sulfated E disaccharide (GlcUA----GaINAc4,6-diSO4) (25%). Nitrous acid/heparinase-susceptible heparin proteoglycans accounted for approximately 62% of the total 35S-proteoglycans present in the HLMC. Proteoglycans remaining after exposure of the original proteoglycan extract to either heparinase or chondroitin ABC lyase were of similar size, suggesting that the majority of heparin and chondroitin sulfate glycosaminoglycans were on separate protein cores. Proteoglycans extracted from HLMC were protease insensitive. Hence, in addition to heparin proteoglycans, HLMC synthesize a hitherto unrecognized quantity of chondroitin sulfate E proteoglycans.     

A heparan sulfate-degrading endoglycosidase from rat liver tissue

Incubation of a rat liver lysosomal fraction with [³⁵S]heparan sulfate resulted in degradation of the polymer to oligosaccharides, demonstrating the presence of a heparan sulfate-degrading endoglycosidase. Judging from the size of the oligosaccharides, representing degradation end-products, only a limited number of the glycosidic linkages in the heparan sulfate molecule would seem to be susceptible to the heparitinase.The pH-dependence of the enzyme (active at pH 5.6; inactive at pH 3.8) was found to differ from that of liver hyaluronidase (active at pH 3.8; inactive at pH 5.6), suggesting that the heparitinase is a previously unknown enzyme.     

Characterization of extracellular matrix-associated glycosaminoglycans produced by untransformed and transformed bovine corneal endothelial cells in culture

A cloned bovine corneal endothelial cell line was transformed in vitro by simian virus 40, and the subendothelial extracellular matrix-associated sulfated glycosaminoglycans synthesized by the cells were isolated and compared with their untransformed counterpart. The transformed endothelial cells grew at faster rates to higher stationary cell densities in the absence of fibroblast growth factor than did the untransformed cells. On a per-cell basis, the transformed cells produced slightly lower amounts of sulfated glycosaminoglycans. The rate of production of sulfated glycosaminoglycans in extracellular matrix increased during seven days of culture. At confluency the extracellular matrix-associated sulfated glycosaminoglycans synthesized by the untransformed endothelial cells consisted of about 80% heparan sulfate and about 20% chondroitin sulfate. Extracellular matrix-associated sulfated glycosaminoglycans of transformed endothelial cells were composed of about 70% heparan sulfate and about 30% chondroitin sulfate plus dermatan sulfate. High-speed gel permeation chromatography profiles on Fractogel TSK HW-55(S) of matrix-associated heparan sulfate from untransformed and transformed endothelial cells were very similar, and gave single peaks (Kav = 0.19). Apparent Mr estimated from the eluting position of the peaks were approximately 47000. Heparan sulfate from both untransformed and transformed endothelial cells was degraded by incubation with a metastatic B16 melanoma cell lysate containing heparanase (heparan-sulfate-specific endo-beta-glucuronidase). The eluting position of the heparan sulfate degradation products on gel permeation column were similar (Kav = 0.43). Size analysis and anion-exchange chromatography of the degradation products after nitrous acid deamination at low pH indicated that the degree of N-sulfation of heparan sulfate was similar in untransformed and transformed endothelial cells. The results indicated that transformation of endothelial cells only slightly changes the molecular nature of subendothelial matrix-associated sulfated glycosaminoglycans.     

Stimulation of human arterial smooth muscle cell chondroitin sulfate proteoglycan synthesis by transforming growth factor-beta

Summary Human platelet-derived transforming growth factor-beta (TGF-beta) is a cell-type specific promotor of proteoglycan synthesis in human adult arterial cells. Cultured human adult arterial smooth muscle cells synthesized chondroitin sulfate, dermatan sulfate, and heparan sulfate proteoglycans, and the percent composition of these three proteoglycan subclasses varied to some extent from cell strain to cell strain. However, TGF-beta consistently stimulated the synthesis of chondroitin sulfate proteoglycan. Both chondroitin 4- and chondroitin 6-sulfate were stimulated by TGF-beta to the same extent. TGF-beta had no stimulatory effect on either class of [³⁵S]sulfate-labeled proteoglycans which appeared in an approximately 1:1 and 2:1 ratio of heparan sulfate to dermatan sulfate of the medium and cell layers, respectively, of arterial endothelial cells. Human adult arterial endothelial cells synthesized little or no chondroitin sulfate proteoglycan. Pulse-chase labeling revealed that the appearance of smooth muscle cell proteoglycans into the medium over a 36-h period equaled the disappearance of labeled proteoglycans from the cell layer, independent of TGF-beta. Inhibitors of RNA synthesis blocked TGF-beta-stimulated proteoglycan synthesis in the smooth muscle cells. The incorporation of [³⁵S]methionine into chondroitin sulfate proteoglycan core proteins was stimulated by TGF-beta. Taken together, the results presented indicate that TGF-beta stimulates chondroitin sulfate proteoglycan synthesis in human adult arterial smooth muscle cells by promoting the core protein synthesis. Supported in part by grants from the Public Health Service, U.S. Department of Health and Human Services, Washington, DC (CA 37589 and HL 33842), RJR Nabisco, Inc., and Chang Gung Biomedical Research Foundation (CMRP 291).     

Heterogeneity of heparan sulfate proteoglycans synthesized by PYS-2 cells

Antibodies to the basement membrane proteoglycan produced by the EHS tumor were used to immunoprecipitate [35S]sulfate-labeled protoglycans produced by PYS-2 cells. The immunoprecipitated proteoglycans were subsequently fractionated by CsCl density gradient centrifugation and Sepharose CL-4B chromatography. The culture medium contained a low-density proteoglycan eluting from Sepharose CL-4B at Kav = 0.18, containing heparan sulfate side chains of Mr = 35-40,000. The medium also contained a high-density proteoglycan eluting from Sepharose CL-4B at Kav = 0.23, containing heparan sulfate side chains of Mr = 30,000. The corresponding proteoglycans of the cell layer were all smaller than those in the medium. Since the antibodies used to precipitate those proteoglycans were directed against the protein core, this suggests that these proteoglycans share common antigenic features, and may be derived from a common precursor which undergoes modification by the removal of protein segments and a portion of each heparan sulfate chain.     

Dog mastocytoma proteoglycans: occurrence of heparin and oversulfated chondroitin sulfates, containing trisulfated disaccharides, in three cell lines

The cell-associated proteoglycans synthesized by three dog mastocytoma cell lines were isolated and their structural features compared. The lines were propagated as subcutaneous tumors in athymic mice for over 25 generations. In primary cell culture, all three lines incorporated [35S]sulfate into high molecular weight proteoglycans which were heterogeneous in size and glycosaminoglycan content. Two lines, BR and G, synthesized both a heparin proteoglycan (HPG) and a chondroitin sulfate proteoglycan (ChSPG) in different proportions. The third line, C2, synthesized predominantly a ChSPG with little or no detectable heparin. Gel filtration of the 35S-labeled HPG and ChSPG from the BR line on Sepharose CL-4B in dissociative conditions (4 M guanidine, Triton X-100) yielded a major polydisperse peak (Kav = 0.22) accounting for 70% of 35S activity. Under aggregating conditions (0.1 M sodium acetate) on Sepharose CL-4B, the BR proteoglycans eluted in the excluded volume. Proteoglycans from lines G and C2 also eluted in the void volume under nondissociative conditions, however the C2 line yielded additional fractions of smaller hydrodynamic size (Kav = 0.81) suggesting the presence of intracellular proteoglycan cleavage products or incompletely processed proteoglycans. As assessed by dissociative chromatography on Sepharose CL-4B, proteoglycans from the BR line were resistant to proteinase cleavage under conditions which degraded a rat chondrosarcoma proteoglycan. For all lines, glycosaminoglycans released by pronase/alkaline-borohydride had molecular weights ranging from 20,000 to 50,000 on gel filtration. For line BR, 75% of 35S-labeled glycosaminoglycans were degraded to oligosaccharides by nitrous acid, and the remaining 25% were degraded by chondroitinase ABC. Corresponding percentages for line G were 89% and 11%, and for line C2, 2% and 98%. Paper chromatography of the chondroitinase digestion products from lines BR and C2 showed products corresponding to unsaturated standards delta Di-diSB and delta Di-diSE, derived from the disaccharides IdoUA-2-SO4----GalNAc-4-SO4 and GlcUA----GalNAc-4,6-diSO4 respectively, in addition to smaller amounts of monosulfated disaccharides. Glycans from lines C2 and BR contained small quantities of a trisulfated disaccharide which was degraded to delta Di-diSB upon incubation with chondro-6-sulfatase. The results demonstrate the simultaneous presence of heparin and polysulfated chondroitin sulfate in dog mast cells of clonal origin.     

Composition of proteoglycans synthesized by rabbit aortic explants in culture and the effect of experimental atherosclerosis

The synthesis of proteoglycans by aorta explants from rabbits with diet-induced atherosclerosis and controls was studied by 35S-incorporation. Proteoglycans were isolated under dissociative conditions from incubation medium and from arterial explants. Additionally, the tissue proteoglycans that were not extracted by 4 M guanidine-HCl were solubilized by digestion of the tissue by elastase in the presence of proteinase inhibitors. The residual tissue was hydrolyzed by papain and glycosaminoglycans were isolated. The atherosclerotic aorta tissue incorporated twice the amount of 35S into proteoglycans than observed for controls; in both groups about 70% of the label incorporated into the tissue was noted in the proteoglycans extracted by guanidine-HC;, while about 30% of the total 35S-labeled proteoglycans synthesized by the explants were found in the media. Atherosclerotic tissue incorporated 35S predominantly into chondroitin sulfate proteoglycans when compared to control tissue. The chondroitinase ABC-digestable proteoglycans that were extracted by guanidine-HCl from atherosclerotic tissues were of larger molecular size than those from control tissue, but the core proteins from these preparations were similar. The heparan sulfate proteoglycan that was obtained by dissociative extraction from atherosclerotic tissue had greater amounts of N-acetyl and lesser amounts of N-sulfate ester groups than the preparation from control tissue. Digestion of the tissue by elastase yielded heparan sulfate proteoglycan as the major constituent in both groups, although atherosclerotic tissue contained relatively small amounts of this proteoglycan. The residual tissue from both groups contained chondroitin sulfate and heparan sulfate as the major glycosaminoglycans with the latter showing a decrease with atherosclerosis. Atherosclerotic tissue secreted into the medium about two-fold more 35S-labeled proteoglycans with larger molecular size than control tissue; proteoglycans of the heparan sulfate and chondroitin sulfate types were the major constituents in the culture medium of both tissues. Thus, proteoglycans undergo both quantitative and qualitative changes in atherosclerosis, reflecting the enhanced smooth muscle cell activity. These changes are potentially important in modulating lipoprotein binding and hemostatic properties, as well as fibrillogenesis of the arterial wall.     

Metabolism of sulfated glycosaminoglycans in rat hepatocytes. Synthesis of heparan sulfate and distribution into cellular and extracellular pools

Primary cultures of rat hepatocytes grown in a serum-free medium supplemented with [35S]sulfate synthesize 35S-labelled glycosaminoglycans at an almost constant rate for 58 h. Approx. 57% of the newly synthesized 35S-labelled glycosaminoglycans remain within the hepatocytes, approx. 30% become associated with the cell surface and only 13% are secreted into the medium. The amount of cell-surface-associated 35S-labelled glycosaminoglycans became constant within 36 h, whereas no equilibrium was reached in the intra- and extracellular pool. During a 24 h chase more than 50% of the intracellular and cell-surface-associated 35S-labelled glycosaminoglycans disappears, more than 80% of this material is degraded and radioactivity is recovered as inorganic sulfate. A minor part is released into the medium in a macromolecular form. Heparan sulfate accounts for more than 95% of the 35S-labelled glycosaminoglycans in each of the three pools. It is distinguished from heparan sulfates from other sources by the presence of unsubstituted glucosamine residues. In all three pools, heparan sulfate chains of mean molecular weights between 24 000 and 30 000 are part of an alkali labile proteoglycan. Intra- and extracellularly, however, part of the heparan sulfate appears to have little, if any, protein attached. Hepatocytes contain heparan sulfate-degrading endoglycosidase activity, which may contribute to the variation of molecular weights observed for the heparan sulfate.     

Synthesis by Schwann cells of basal lamina and membrane-associated heparan sulfate proteoglycans

Primary cultures that contain only Schwann cells and sensory nerve cells synthesize basal lamina. The assembly of this basal lamina appears to be essential for normal Schwann cell development. In this study, we demonstrate that Schwann cells synthesize two major heparan sulfate-containing proteoglycans. Both proteoglycans band in dissociative CsCl gradients at densities less than 1.4 g/ml, and therefore, presumably, have relatively low carbohydrate-to-protein ratios. The larger of these proteoglycans elutes from Sepharose CL-4B in 4 M guanidine hydrochloride (GuHCl) at a Kav of 0.21 and contains heparan sulfate and chondroitin sulfate chains of Mr 21,000 in a ratio of approximately 3:1. This proteoglycan is extracted from cultures by 4 M GuHCl but not Triton X-100 and accumulates only when Schwann cells are actively synthesizing basal lamina. The smaller proteoglycan elutes from Sepharose CL-4B at a Kav of 0.44 and contains heparan sulfate and chondroitin sulfate chains of Mr 18,000 in a ratio of approximately 4:1. This proteoglycan is extracted by 4 M GuHCl or by Triton X-100. The accumulation of this proteoglycan is independent of basal lamina production.     

Three distinct molecular species of proteoglycan synthesized by the rat limb bud at the prechondrogenic stage

To characterize proteoglycans in the prechondrogenic limb bud, proteoglycans were extracted with 4 M guanidine HCl containing a detergent and protease inhibitors from Day 13 fetal rat limb buds which had been labeled with [35S]sulfate for 3 h in vitro. About 90% of 35S-labeled proteoglycans was solubilized under the conditions used. The proteoglycan preparation was separated by DEAE-Sephacel column chromatography into three peaks; peak I eluted at 0.45 M NaCl concentration, peak II at 0.52 M, and peak III at 1.4 M. Peaks I and III were identified as proteoglycans bearing heparan sulfate side chains. The heparan sulfate proteoglycan in peak III was larger in hydrodynamic size than the proteoglycan in peak I. The heparan sulfate side chains of peak III proteoglycan were smaller in the size and more abundant in N-sulfated glucosamine than those of peak I proteoglycan. Peak II contained a chondroitin sulfate proteoglycan with a core protein of a doublet of Mr 550,000 and 500,000. The chondroitin sulfate proteoglycan was easily solubilized with a physiological salt solution and the heparan sulfate proteoglycan in peak I was partially solubilized with the physiological salt solution. The remainder of the proteoglycan in peak I and the heparan sulfate proteoglycan in peak III could be solubilized effectively only with a solution containing a detergent, such as nonanoyl-N-methylglucamide. This observation indicates the difference in the localization among these three proteoglycans in the developing rat limb bud.     

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.     

Identification of the precursor protein to basement membrane heparan sulfate proteoglycans

The precursor protein of a basement membrane specific heparan sulfate proteoglycan has been identified as a 400,000 Mr polypeptide. Antibodies against large and small forms of this proteoglycan, isolated from a basement membrane (Engelbreth-Holm-Swarm, EHS) tumor, immunoprecipitated the same 400,000 protein from pulse-labeled EHS cells. The proteoglycan precursor protein was not recognized by antibodies against other basement membrane components or by antibodies to the cartilage proteoglycan. Furthermore, heparan sulfate proteoglycan purified from the EHS tumor blocked the immunoprecipitation of the precursor protein. Pulse-chase studies with [35S]methionine showed the precursor protein was converted to a proteoglycan. Pulse-chase studies with 35SO4 showed the large, low density proteoglycan appeared first and was degraded to a smaller, high density proteoglycan. We propose that the precursor protein is used after very little or no modification in the assembly of a large, low density heparan sulfate proteoglycan and that a portion of the population of these macromolecules are subsequently degraded to a smaller form.     

Turnover of heparan sulfate proteoglycan in human colon carcinoma cells. A quantitative biochemical and autoradiographic study

The metabolism of heparan sulfate proteoglycan, a major product of human colon carcinoma cells, was investigated in a series of pulse-chase experiments using a combination of quantitative biochemistry and electron microscope autoradiography. This was possible primarily because these cells incorporate [35S]sulfate exclusively into heparan sulfate proteoglycan, thus allowing the possibility of correlating the two sets of information. The results showed a progressive movement of the newly synthesized proteoglycan from the Golgi to the cell surface, where it became closely associated with the plasma membrane and was labeled ultrastructurally by both ruthenium red and radiosulfate. Subsequently, about 55% was released into the medium (t1/2 approximately 2.5 h) where it resided as intact macromolecule and was neither endocytosed nor degraded further. The remaining 45% was internalized and converted into smaller species through a series of degradative steps. Initially (Step 1) there was proteolytic cleavage of the protein core and partial endoglycosidic cleavage of the heparan sulfate chains (t1/2 approximately 6 h), with generation of larger glycosaminoglycan-peptide intermediates with chains of Mr approximately 10,000, about one-third their original size. These components were subsequently converted (Step 2) to yet smaller, limiting fragments of Mr approximately 5,000, which were finally depolymerized (Step 3) with quantitative release of free sulfate. The intracellular degradation of the proteoglycan, particularly Steps 2 and 3, was markedly inhibited by choloroquine, implicating the involvement of acidic compartments in the catabolism of these macromolecules. This was corroborated by the autoradiographic studies which showed the close association of 35S-labeled products with secondary lysosomes. However, the initial degradation of the proteoglycan might have occurred in a prelysosomal compartment since Step 1 was not totally blocked by chloroquine. The combined results indicate that the intracellular degradation of heparan sulfate follows structural as well as functional compartmentalization and provide a model that may be shared by other cell systems.