A unique heparan sulfate in the nuclei of hepatocytes: structural changes with the growth state of the cells

Growing and confluent cultures of a rat hepatocyte cell line were labeled with 35SO4(2-) and the heparan sulfate in the culture medium, the pericellular matrix, the nucleus, the nuclear outer membrane, and the remaining cytoplasmic pool was purified by DEAE-cellulose chromatography. The heparan sulfate in all pools from the confluent cells was bound more strongly on the DEAE-cellulose column than the corresponding pools from the growing cells. Gel filtration of each pool before and after beta-elimination showed that the heparan sulfate from the nuclear and nuclear membrane pools was composed of primarily free chains, whereas the heparan sulfate in all of the other pools was a mixture of proteoglycans and free chains. The heparan sulfate in each pool was cleaved with nitrous acid to obtain mixtures of di- and tetrasaccharides. Analysis of these mixtures showed that the structural features of the heparan sulfates in each pool were different and were altered significantly when the growing cells became confluent. The nuclear-plus-nuclear membrane pools represented 6.5% and 5.4% of the total cell-associated heparan sulfate in the growing cells and the confluent cells, respectively. The structural features of the heparan sulfate in the two nuclear pools were very similar to each other, but were markedly different from those of the heparan sulfate from the other pools or from any previously described heparan sulfate or heparin. The most unusual aspect of these structures was the high content of beta-D-glucuronosyl(2-SO4)----D-glucosamine-N,O-(SO4)2 disaccharide units in these sequences. The mode of biosynthesis and delivery of these unusual sequences to the nucleus and the potential significance of these observations are discussed.     

Secretion of chondroitin SO4 by monolayer cultures of chick embryo chondrocytes

Monolayer cultures of chick embryo tibial chondrocytes incorporate 35SO42- into chondroitin SO4 which is rapidly secreted from the cells into two extracellular pools. Part of the extracellular chondroitin SO4 is recovered in a soluble form in the culture medium, and the remainder is associated with the cell matrix from which it is released by isotonic trypsinization. At 38 degrees C labeled chondroitin SO4 appears in the cell matrix fraction within 5 min after addition of 35SO42- and in the culture medium fraction 15 min after 35SO42- is added. The intracellular pool of labeled chondroitin SO4 reaches a steady state level of 150 to 200 pmol of bound SO4 per 10(6) cells in 60 min, while the cell matrix and medium fractions increase at rates of 3 and 1 nmol of bound SO4 per h per 10(6) cells, respectively. After 4 h of labeling, less than 20% of the newly synthesized cell-associated chondroitin SO4 is in the intracellular fraction. By labeling cells for 15 min at 25 degrees C 80% of the cell-associated chondroitin 35SO4 is obtained in the intracellular fraction. This material is chased without lag into both the cell matrix fraction and the medium fraction. A mixture of NaF and NaCN, both at 30 mM, lowers the cellular ATP level to 15% of normal and blocks secretion of the intracellular chondroitin SO4 into both extracellular fractions. Colchicine at 10(-6) M gives a partial inhibition of both synthesis and secretion of chondroitinSO4. Sucrose density gradient sedimentation analysis of the intracellular chondroitin SO4 and the two extracellular fractions shows that all three fractions contain both a heavy and light proteoglycan fraction. The intracellular light proteoglycan fraction is secreted preferentially into the culture medium where it represents 30% of the total culture medium pool. The ratio of 6-sulfated GalNAc to 4-sulfated GalNAc in the heavy proteochondroitin SO4 fraction is approximately twice that found for the light fraction.     

Transport of heparan sulfate into the nuclei of hepatocytes

Monolayer cultures of a rat hepatocyte cell line shown previously to accumulate a nuclear pool of free heparan sulfate chains that are enriched in sulfated glucuronic acid (GlcA) residues (Fedarko, N.S., and Conrad, H.E., (1986) J. Cell Biol. 587-599) were incubated with 35SO4(2-), and the rate of appearance of heparan [35S]sulfate in the nuclei was measured. Heparan [35S]sulfate began to accumulate in the nuclei 2 h after the administration of 35SO4(2-) to the cells and reached a steady state level after 20 h. Heparan [35S]sulfate was lost from the nuclei of prelabeled cells with a t1/2 of 8 h. Chloroquine did not inhibit the transport of heparan sulfate into the nucleus, but increased the t1/2 for the exit of heparan sulfate from the nucleus to 20 h and led to a doubling of the steady state level of nuclear heparan sulfate. Heparan [35S]sulfate which was obtained from the medium or from the cell matrix of a labeled culture and which contained only low levels of GlcA-2-SO4 residues was incubated with cultures of unlabeled cells, and the uptake of the exogenous heparan [35S]sulfate was studied. At 37 degrees C the cells took up proteoheparan [35S]sulfate and transported about 10% of the internalized heparan [35S]sulfate into the nucleus, where it appeared as free chains. The heparan [35S]sulfate isolated from the nucleus was enriched in GlcA-2-SO4 residues, whereas the heparan [35S]sulfate remaining in the rest of the intracellular pool showed a corresponding depletion in GlcA-2-SO4 residues. At 16 degrees C, where endocytosed materials do not enter the lysosomes, the cells also transported exogenous proteoheparan [35S]sulfate to the nucleus with similar processing. Thus, the metabolism of exogenous heparan sulfate by hepatocytes follows the same pathway observed in continuously labeled cells and does not involve lysosomal processing of the internalized heparan sulfate.     

Structure and synthesis of intracellular proteoglycan in HL-60 human leukemic promyelocytes

The structure, biosynthesis, and metabolism of proteoglycans in the HL-60 human promyelocytes were studied by metabolic labeling in culture with [35S]sulfate, [3H]glucosamine, [3H]serine, and [3H]leucine. These cells synthesize a single predominant species of intracellular proteoglycan with an approximate molecular weight of 100,000. The cells contain about 1 microgram of proteoglycan/million cells. The proteoglycan is turned over within the cells in two apparent pools with half-lives of about 0.6 and 27 h, respectively. The fast pool represents secretion into medium in an apparently intact form, whereas the slow pool represents intracellular degradation to free chondroitin sulfate chains and smaller fragments. The proteoglycan contains a protein core with an apparent Mr on gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of about 20,000-30,000. To the core protein are attached an average of six or seven chondroitin sulfate chains, each with an Mr of about 10,000. The chondroitin sulfate chains contain approximately 85% 4-sulfated and approximately 15% nonsulfated disaccharides. The chondroitin sulfate attachment region of the core protein is essentially resistant to trypsin and elastase, whereas the remainder of the protein core is readily degraded by proteases. The size of the chondroitin sulfate attachment region peptide generated by trypsin was estimated to be approximately 5 kDa. Based on the molecular size, distribution of amino acids, protease susceptibility, and the extent of O-glycosylation, we propose that the intracellular proteoglycan characterized in this study is the translation product of a proteoglycan gene reported to be present in these cells (Stevens, R.L., Avraham, S., Gartner, M.C., Bruns, G.A., Austen, K.E., and Weis, J.H. (1988) J. Biol. Chem. 263, 7287-7291).     

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.     

Catabolism of newly synthesized decorin by explant cultures of bovine ligament.

The catabolism of newly synthesized decorin by explant cultures of bovine collateral ligament was investigated. The tissue was placed in explant culture for 6 days then incubated with radiolabeled sulfate for 6 h and replaced in culture for 5 days to allow for the loss of the radiolabeled large proteoglycan. The metabolic fate of the remaining radiolabeled decorin present in the matrix of the tissue over the next 9-day period was determined. It was shown that this pool of decorin was lost from ligament explant cultures either directly into the culture medium or taken up and degraded within the cells of the tissue. The intracellular degradation of the radiolabeled pool of decorin by ligament explant cultures was shown to result in the generation of [35S]sulfate. This process required metabolically active cells and involved the lysosomal system since sulfate generation was inhibited when cultures were maintained at 4 degrees C or in the presence of either 10 mM ammonium chloride or 0. 05 mM chloroquine. The inhibition of intracellular processing of decorin resulted in an increase in the rate of loss of this proteoglycan into the medium of the cultures. The inhibition of intracellular degradation of decorin was reversible on incubation of the explant cultures at 37 degrees C or removal of ammonium chloride from the culture medium. After removal of the ammonium chloride from the culture medium the rate of intracellular catabolism was greater than that observed in cultures maintained in medium alone, which suggested that there was an intracellular accumulation of native and/or partially degraded material within the cells.     

Temporal regulation of hyaluronan and proteoglycan metabolism by human bone cells in vitro

Osteoblasts elaborate a dynamic extracellular matrix that is constructed and mineralized as bone is formed. This matrix is primarily composed of collagen, along with noncollagenous proteins which include glycoproteins and proteoglycans. After various times in culture, human bone cells were labeled with [35S]sulfate, [3H] leucine/proline, or [3H]glucosamine and the metabolism of hyaluronan and four distinct species of proteoglycans (PGs) was assayed in the medium, cell layer, and intracellular pools. These cells produce hyaluronan (Mr approximately 1,400,000; a chondroitin sulfate PG (CSPG), Mr approximately 600,000; a heparan sulfate PG (HSPG), Mr approximately 400,000; and two dermatan sulfate PGs with Mr approximately 270,000 (biglycan, PG I) and Mr approximately 135,000 (decorin, PG II) that distribute between the medium and cell layer. Two days following subculture, 12 h [35S]sulfate steady-state labeling yielded a composition of 24, 27, 31, and 18% for total CSPG, HSPG, biglycan, and decorin, respectively. While HSPG and decorin levels and distribution between medium and cell layer remained relatively constant during steady-state labeling at different times in culture, CSPG and biglycan levels increased dramatically at late stages of growth, and their distribution changed throughout culture. These results were independent of cell density, media depletion, and labeling pool effects. In contrast, hyaluronan synthesis was uncoupled from PG synthesis and apparently density-dependent. Pulse chase labeling at different stages of culture showed that the CSPG and decorin behaved as secretory PGs. Both HSPG and biglycan underwent catabolism, with HSPG possessing a t1/2 of 8 h and biglycan a t1/2 of 4 h. While the rate of HSPG turnover did not appreciably change between early and late culture, that of biglycan decreased. The mRNA for decorin was constant, while that of biglycan changed during culture. These results suggest that each PG possesses a distinct pattern of cellular and temporal distribution that may reflect specific stages in matrix formation and maturation.     

Proteoglycan and glycosaminoglycan synthesis by cultured rat mesangial cells

The synthesis of metabolically labeled proteoglycans and glycosaminoglycans from medium, cell layer and substrate attached material by rat glomerular mesangial cells in culture was characterized. The cellular localization of the labeled proteoglycans and glycosaminoglycans was determined by treating the cells with Flavobacterial heparinase. Of the total sulfated glycosaminoglycans, 33% were heparan sulfate; 55% of the cell layer material was heparan sulfate; 80% of sulfated proteins in the medium were chondroitin sulfate/dermatan sulfate. Putative glycosaminoglycan free chains of heparan sulfate and chondroitin sulfate were found in both the medium and cell layer; 95% of total proteoglycans and most (90%) of the putative heparan sulfate free chains were removed from the cell layer by the heparinase, whereas only 50% of the chondroitin sulfate and 25% of dermatan sulfate were removed. Large amounts of hyaluronic acid labeled with 3H glucosamine were found in the cell layer. In summary, approximately 60% of total sulfated glycoproteins was in the form of putative glycosaminoglycan free chains. Thus rat mesangial cells may synthesize large amounts of putative glycosaminoglycan free chains, which may have biological functions in the glomerulus independent of proteoglycans.     

Altered synthesis of heparan sulfate proteoglycans at low sulfate concentration

The structural alterations in heparan sulfate produced by sulfate deprivation were studied in cell cultures of the Engelbreth-Holm-Swarm tumor. Tumor cells were labeled in vitro with [3H]glucosamine and/or [35S]sulfate in media containing either 300 microM MgSO4 or no added carrier sulfate, and the newly synthesized proteoglycans isolated by chromatography on DEAE-Sephacel. The proteoglycans isolated from low sulfate cultures showed a reduced affinity for the column eluting at lower salt concentrations compared with the proteoglycans isolated from cultures containing sulfate, suggesting that the former were undersulfated. Analysis of the isolated heparan sulfate side chains indicated that two pools of heparan sulfate were present which differed in their degree of sulfation. Both pools were synthesized by both high sulfate and low sulfate cultures, but the highly sulfated pool was the predominant form produced in sulfate containing cultures, while the undersulfated pool was the predominant form synthesized in low sulfate cultures. The more sulfated pool contained more N-sulfate than the less sulfated pool. Few if any free amino groups were detected in either pool, suggesting that the initial deacetylation step in the biosynthesis of heparan sulfate is tightly coupled to the N-sulfation step in the cells.     

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

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

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.     

Biosynthesis of heparan sulfate proteoglycan by human colon carcinoma cells and its localization at the cell surface

After 24 h of continuous labeling with radioactive precursors, a high molecular weight heparan sulfate proteoglycan (HS-PG) was isolated from both the medium and cell layer of human colon carcinoma cells (WiDr) in culture. The medium HS-PG eluted from a diethylaminoethyl anion exchange column with 0.45-0.50 M NaCl, had an average density of 1.46-1.49 g/ml on dissociative CsCl density-gradient ultracentrifugation, and eluted from Sepharose CL-2B with a Kav = 0.57. This proteoglycan had an estimated Mr of congruent to 8.5 X 10(5), with glycosaminoglycan chains of Mr = 3 X 10(4) which were all susceptible to HNO2 deaminative cleavage. Deglycosylation of the HS-PG with polyhydrogen fluoride resulted in a 3H-core protein with Mr congruent to 2.4 X 10(5). The cell layer contained a population of HS-PG with characteristics almost identical to that released into the medium but with a larger Mr = 9.5 X 10(5). Furthermore, an intracellular pool contained smaller heparan sulfate chains (Mr congruent to 1 X 10(4)) which were mostly devoid of protein core. In pulse chase experiments, only the large cell-associated HS-PG was released (approximately 58%) into the medium as intact proteoglycan and/or internalized and degraded (approximately 42%), with a t1/2 = 6 h. However, the small intracellular component was never released into the medium and was degraded at a much slower rate. When the cells were subjected to mild proteolytic treatment, only the large cell-associated HS-PG, but none of the small component, was displaced. Addition of exogenous heparin did not displace any HS-PG into the medium. Both light and electron microscopic immunocytochemistry revealed that the cell surface reacted with antibody against an HS-PG isolated from a basement membrane-producing tumor. Electron microscopic histochemistry using ruthenium red and/or cuprolinic blue revealed numerous 10-50-nm diam granules and 70-220-nm-long electron-dense filaments, respectively, on the surface of the tumor cells. The results indicate that colon carcinoma cells synthesize HS-PGs with distinct structural and metabolic characteristics: a large secretory pool with high turnover, which appears to be synthesized as an integral membrane component and localized primarily at the cell surface, and a small nonsecretory pool with low turnover localized predominantly within the cell interior. This culture system offers an opportunity to investigate in detail the mechanisms involved in the regulation of proteoglycan metabolism, and in the establishment of the neoplastic phenotype.     

Inhibition of intracellular degradation of proteoglycans by leupeptin in rat ovarian granulosa cells

Previous work (Yanagishita, M., and Hascall, V. C. (1984) J. Biol. Chem. 259, 10270-10283) has indicated that heparan sulfate (HS) proteoglycans in rat ovarian granulosa cells are degraded by two kinetically distinct pathways. Pathway 1 degrades proteoglycans rapidly with a t 1/2 approximately 25 min without generating appreciable degradative intermediates. Pathway 2 degrades proteoglycans more slowly with a t 1/2 approximately 4 h, generating distinct degradative intermediates: single HS chains of Mr = approximately 10,000 and approximately 5,000. Effects of leupeptin, an inhibitor of thiol proteases, on the intracellular degradation of proteoglycans in the rat ovarian granulosa cell culture were examined using various chase protocols after labeling cells with [35S]sulfate. The presence of leupeptin at 100 micrograms/ml in the culture medium inhibited the intracellular degradation of proteoglycans by approximately 80% during a 7-h chase period after a 20-h labeling. Leupeptin affected neither the cellular content nor the in vitro activities of beta-hexosaminidase and arylsulfatase. Structural analyses of heparan sulfate species in leupeptin-treated cells demonstrated that the drug inhibited the degradation of HS proteoglycans at two distinct points. First, degradation of the core protein was partially inhibited and delayed before the start of glycosaminoglycan degradation. This resulted in the accumulation of degradative intermediates with partially degraded core proteins bearing intact glycosaminoglycan chains. This establishes the initial sequence for HS proteoglycan degradation, with proteolysis preceding endoglycosidase digestion, and suggests that these two degradation steps may occur in physically separate compartments. Second, the final depolymerization of HS fragments through pathway 2 was totally inhibited, resulting in the continuous accumulation of Mr = 5,000 HS chains. This is not due to the direct inhibition of the lysosomal exoglycosidase and sulfatase enzymes responsible for the complete depolymerization of HS chains, since pathway 1, while slowed, continued to completely depolymerize the HS chains in the presence of leupeptin. The results suggest that the intracellular compartment which completely degrades heparan sulfate chains is separate from those containing partially, endoglycosidically processed heparan sulfate chains and that leupeptin interfered with the translocation of glycosaminoglycans to the final degradation site.     

Biochemical and ultrastructural studies of proteoheparan sulfates synthesized by PYS-2, a basement membrane-producing cell line

The mouse teratocarcinoma-derived cell line, PYS-2, has been shown to produce laminin, a basement membrane-specific glycoprotein. In these studies we demonstrate that PYS-2 cells synthesize and secrete into the culture medium a proteoglycan which contains only heparan sulfate as its sulfated polysaccharide side chains, as well as type IV procollagen and laminin. The apparent molecular weights of the proteoglycan and its heparan sulfate side chain were estimated to be 400,000 and 25,000, respectively, by gel chromatography. A proteoheparan sulfate with properties closely similar, if not identical, to those of the proteoglycan in the medium, together with two heparan sulfate single chains of different molecular size, were extracted from the cell layer with 2% SDS in the presence of protease inhibitors. Ultrastructurally, a fine fibrillar intercellular matrix was recognized which contained discrete 100-200 A diameter ruthenium red-positive granules interspersed throughout the filamentous meshwork. The PYS-2 cultures were shown by immunofluorescence to react with antibodies against the heparan sulfate-containing proteoglycan isolated from the mouse EHS sarcoma (Hassell, J. R., P. G. Robey, H. J. Barrach, J. Wilczek, S. I. Rennard, and G. R. Martin. 1980. Proc. Natl. Acad. Sci. U. S. A. 77:4494-4498). Immunoelectron microscopic examination, using the same antibodies, revealed that the proteoheparan sulfate was located not only at the edges but also within the interstices of the matrix. These findings indicate that PYS-2 cells synthesize and secrete a proteoglycan with properties similar to those of basement membrane proteoglycan. These cells may therefore serve as a useful model system for the study of the biosynthesis and structure of basement membranes.     

Metabolism of sulfated glycosaminoglycans in cultivated bovine arterial cells. I. Characterization of different pools of sulfated glycosaminoglycans.

"Fibroblast-like" cells from the intimal layer of bovine aorta were grown in culture. The formation, composition, molecular weight and turnover rate of different pools of glycosaminoglycans were investigated in cultures incubated in the presence [35S]sulfate or [14C]glucosamine. The newly synthesized glycosaminoglycans are distributed into an extracellular pool (37 - 58%), a cell-membrane associated or pericellular pool (23 - 33%), and an intracellular pool (19 - 30%), each pool exhibiting a characteristic distribution pattern of chondroitin sulfate, dermatan sulfate, heparan sulfate and hyaluronate. The distribution pattern of the extracellular glycosaminoglycans resembles closely that found in bovine aorta. A small subfraction of the pericellular pool - tentatively named "undercellular" pool--has been characterized by its high heparan sulfate content. The intracellular and pericellular [35S]glycosaminoglycan pools reach a constant radioactivity after 8-12 h and 24 h, respectively, whereas the extracellular [35S]glycosaminoglycans are secreted into the medium at a linear rate over a period of at least 6 days. The intracellular glycosaminoglycans are mainly in the process of degradation, as indicated by their low molecular weight and by their half-life of 7 h, but intracellular dermatan sulfate is degraded more rapidly (half-life 4-5 h) than intracellular chondroitin sulfate and heparan sulfate (half-life 7-8 h). Glycosaminoglycans leave the pericellular pool with a half-life of 12-14 h by 2 different routes: about 60% disappear as macromolecules into the culture medium, and the remainder is pinocytosed and degraded to a large extent. Extracellular and at least a part of the pericellular glycosaminoglycans are proteoglycans. Even under dissociative conditions (4M guanidinium chloride) their hydrodynamic volume is sufficient for partial exclusion from Sepharose 4B gel. The existence of topographically distinct glycosaminoglycan pools with varying metabolic characteristics and differing accessibility for degradation requiresa reconsideration and a more reserved interpretation of results concerning the turnover rates of glycosaminoglycans as determined in arterial tissue.     

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.     

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.     

Heparan sulfate at the surface of HeLa cells.

HeLa cells, labeled with Na235SO4, release into the culture medium 35SO4 bound to plasma membrane vesicles next to 35SO4-glycoproteins and free 35SO4. Plasma membrane vesicles, experimentally produced by treatment with formaldehyde, contain 35SO4 and their surface can be stained with high iron diamine. Scanning of chromatograms of the trypsinate from labeled cells demonstrates radioactivity on the spot of heparan sulfate. It is concluded that HeLa cells synthesize heparan sulfate, which is incorporated at the plasma membrane and released by shedding of small vesicles.     

Evidence of a small hydrophobic domain in the core protein of the heparan sulfate proteoglycan from human colon carcinoma cells

We demonstrate that the cell surface heparan sulfate proteoglycan of human colon carcinoma cells has an affinity for a hydrophobic matrix. This property is mediated by sequences in the core protein, since papain-or alkaline borohydride-released heparan sulfate chains do not bind to the matrix. Trypsin releases a [3H]leucine-rich, unsulfated, hydrophobic peptide, with Mr approximately 5000. This domain is present in neither the proteoglycan released into the medium nor in the intracellular degradation products. It is proposed that this peptide may represent the portion of the core protein intercalated into the plasma membrane.