Kinetics of proteoheparan sulfate synthesis, secretion, endocytosis, and catabolism by a hepatocyte cell line

The metabolism of heparan sulfate proteoglycan was studied in monolayer cultures of a rat hepatocyte cell line. Late log cells were labeled with 35SO4(2-) or [3H] glucosamine, and labeled heparan sulfate, measured as nitrous acid-susceptible product, was assayed in the culture medium, the pericellular matrix, and the intracellular pools. Heparan sulfate in the culture medium and the intracellular pools increased linearly with time, while that in the matrix reached a steady-state level after a 10-h labeling period. When pulse-labeled cells were incubated in unlabeled medium, a small fraction of the intracellular pool was released rapidly into the culture medium while the matrix heparan sulfate was taken up by the cells, and the resulting intracellular pool was rapidly catabolized. The structures of the heparan sulfate chains in the three pools were very similar. Both the culture medium pool and the cell-associated fraction of heparan sulfate contained proteoheparan sulfate plus a polydisperse mixture of heparan chains which were attached to little, if any, protein. Pulse-chase data suggested that the free heparan sulfate chains were formed as a result of catabolism of the proteoglycan. When NH4Cl, added to inhibit lysosomal function, was present during either a labeling period or a chase period, the total catabolism of the heparan sulfate chains to monosaccharides plus free SO2-4 was blocked, but the conversion of the proteoglycan to free heparan sulfate chains continued at a reduced rate.     

Control of cell division in hepatoma cells by exogenous heparan sulfate proteoglycan

The effects of cell surface heparan sulfate proteoglycan (HSPG) prepared from log and confluent monolayers of a rat hepatoma cell line on hepatoma cell growth were studied. When HSPG isolated from confluent cells was added exogenously to log phase cells, it was internalized and free heparan sulfate (HS) chains appeared transiently in the nucleus. Concurrently, the growth of the treated cells was inhibited, but the cells resumed logarithmic growth as the level of nuclear HS fell, and the cells grew to confluence and became contact inhibited. When HSPG prepared from log-phase hepatoma cells was added exogenously to log phase cells, it was internalized but very little of the internalized HS appeared in the nucleus, and there was no change in the rate of cell growth. However, when the rate of cell growth was reduced by culture of the cells in serum- and insulin-deficient medium, HSPG prepared from log-phase cells stimulated the growth rate of these slow-growing cells. The cell cycle dependency of HSPG uptake and growth inhibition was studied in cultures synchronized by a thymidine/aphidicolin double block. When [35SO4]HSPG from confluent cells was added to synchronized cells just as they were released from the second block, a portion of the [35SO4]HSPG was internalized and [35SO4]HS appeared in the nucleus. However, at mitosis the [35SO4]HS disappeared almost completely from all of the cellular pools, and after mitosis, more of the [35SO4]HSPG was taken up and [35SO4]HS reappeared in the nucleus and remained in the nucleus until the cells divided again. When cultures were released from the aphidicolin block, both control and HSPG-treated cells progressed through the S, the G2, and the M phases of the cell cycle. However, the length of the G1 phase of the cycle was increased in the HSPG-treated cells. The treated cultures then progressed through the second S, G2, and M phases. Thus, the inhibition of cell division occurred in the G1 phase of the cell cycle, prior to the G1/S boundary. Addition of the HSPG to the synchronized cultures just after the first mitosis resulted in an immediate arrest of the cell cycle in G1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biosynthesis of glycosaminoglycans in the human colonic tumor cell line Caco-2: structural changes occurring with the morphological differentiation of the cells

The human colon cancer cell line Caco-2 cultured in vitro displayed morphological differentiation which was shown to be a growth-related event. We have investigated this phenomenon further in relation to the cell surface glycosaminoglycans produced by growing (5-day, i.e., prior to differentiation) and confluent (9-day, i.e., after morphological and functional differentiation) cultures. Neosynthesized [35S]glycosaminoglycans were purified on DEAE-cellulose; at confluency, they were bound more strongly to the column than the corresponding fractions from the growing cells. Analysis of Kav values of heparan sulfate and chondroitin sulfates from growing and confluent cells indicated an increase in chain length of both glycosaminoglycans in morphologically differentiated cells. Heparan sulfate was the main 35S-labeled glycosaminoglycan of the cell surface of both 5-day and 9-day cultures. Paper chromatography of the unsaturated disaccharides obtained by chondroitinase digestion showed that chondroitin sulfate chains were primarily 6-sulfated in the 2 studied extracts. Heparan sulfate chains were isolated as chondroitinase-resistant material and treated with nitrous acid. Analysis of N- and O-sulfate group-related radioactivity showed an increase in the amount of 35S-label in the form of N-sulfate groups and an increase in the O-35S-sulfation pattern in heparan sulfate from morphologically differentiated cells. Thus, the structural features of both chondroitin sulfates and heparan sulfate were significantly different when the growing cells became morphologically differentiated.     

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.     

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.     

Correlations between heparan sulfate metabolism and hepatoma growth

A rat hepatoma cell line (Gershenson et al., Science, 170:859-861, 1970) contains a dynamic steady-state pool of free heparan sulfate (HS) chains in the nucleus that increases in amount when growing cells reach confluence (Fedarko and Conrad, J. Cell Biol., 102:587-599, 1986). In logarithmically growing cells labeled with 35SO4(2-) steady-state levels of [35SO4]HS in the nucleus are altered by a variety of culture conditions. Rapidly dividing cells (doubling time = 18-22 h) growing under optimized conditions had steady-state levels of nuclear HS within the range of 40-50 pmol 35SO4 in nuclear HS/10(6) cells. The steady-state levels of nuclear HS were lowered by several changes in culture conditions, including 1) additions of 1 mM p-nitrophenyl-beta-D-xyloside, 0.25-0.5 mM (+)-catechin, 0.5 ng/ml transforming growth factor beta, 20 ng/ml phorbol-12-myristate-13-acetate, 1 mM dibutyryl cAMP, or 1 mM inositol-2-PO4; 2) decreased levels of D-glucose; or 3) deletions of serum, insulin, or inositol. In all cases lowering of the nuclear HS level was accompanied by an increase in the cell doubling times, suggesting a correlation in which nuclear HS levels must be optimized for maximal growth rates. When cells cultured under optimal growth conditions reached confluence, the level of nuclear HS increased threefold and the cells stopped dividing. The same culture conditions that lowered the steady-state levels of HS in the logarithmically growing cells prevented this rise in the nuclear HS as the cells reached confluence and resulted in loss of contact inhibition and overgrowth of the confluent cultures. These observations suggest a second correlation in which elevated nuclear HS levels are found when cell growth is inhibited at confluence; prevention of this rise results in continued growth. Consistent with this correlation between elevated nuclear HS and reduced growth rates, it was observed that addition of either 0.5 microgram/ml hydrocortisone or 0.05 microgram/ml retinoic acid to the culture medium of logarithmically growing cultures resulted in increases in steady-state levels of nuclear HS that were accompanied by increased cell doubling times. The two agents that increased the levels of nuclear HS in logarithmically growing cultures had little effect on levels of nuclear HS in confluent cells or on contact inhibition.(ABSTRACT TRUNCATED AT 400 WORDS)     

Disulfide-bonded aggregates of heparan sulfate proteoglycans

Heparan sulfate proteoglycans have been isolated from Swiss mouse 3T3 cells by using two nondegradative techniques: extraction with 4 M guanidine or 2.5% 1-butanol. These proteoglycans were separated from copurifying chondroitin sulfate proteoglycans by using ion-exchange chromatography on DEAE-cellulose in the presence of 2 M urea. The purified heparan sulfate proteoglycans are substantially smaller, ca. Mr 20 000, than those isolated from these same cells with trypsin, ca. Mr 720 000 [Johnston, L.S., Keller, K. L., & Keller, J. M. (1979) Biochim. Biophys. Acta 583, 81-94]. However, all of the heparan sulfate proteoglycans extracted by these three methods contain similar glycosaminoglycan chains (Mr 7500) and are derived from the same pool of cell surface associated molecules. The trypsin-released heparan sulfate proteoglycan (ca. Mr 720 000) can be significantly reduced in size (ca. Mr 33 000) under strong denaturing conditions in the presence of the disulfide reducing agent dithiothreitol, which suggests that this form of the molecule is a disulfide-bonded aggregate. The heparan sulfate proteoglycan isolated from the medium also undergoes a significant size reduction in the presence of dithiothreitol, indicating that a similar aggregate is formed as part of the normal release of heparan sulfate proteoglycans into the medium. These results suggest that well-shielded disulfide bonds between individual heparan sulfate proteoglycan monomers may account for the large variation in sizes which has been reported for heparan sulfate proteoglycans isolated from a variety of cells and tissues with a variety of extraction procedures.     

Composition and distribution of glycosaminoglycans in cultures of human normal and malignant glial cells.

The glycosaminoglycans of human cultured normal glial and malignant glioma cells were studied. [35S]Sulphate or [3H]glucosamine added to the culture medium was incorporated into glycosaminoglycans; labelled glycosaminoglycans were isolated by DEAE-cellulose chromatography or gel chromatography. A simple procedure was developed for measurement of individual sulphated glycosaminoglycans in cell-culture fluids. In normal cultures the glycosaminoglycans of the pericellular pool (trypsin-susceptible material), the membrane fraction (trypsin-susceptible material of EDTA-detached cells) and the substrate-attached material consisted mainly of heparan sulphate. The intra- and extra-cellular pools showed a predominance of dermatan sulphate. The net production of hyaluronic acid was low. The accumulation of 35S-labelled glycosaminoglycans in the extracellular pool was essentially linear with time up to 72h. The malignant glioma cells differed in most aspects tested. The total production of glycosaminoglycans was much greater owing to a high production of hyaluronic acid and hyaluronic acid was the major cell-surface-associated glycosaminoglycan in these cultures. Among the sulphated glycosaminoglycans chondroitin sulphate, rather than heparan sulphate, was the predominant species of the pericellular pool. This was also true for the membrane fraction and substrate-attached material. Furthermore, the accumulation of extracellular 35S-labelled glycosaminoglycans was initially delayed for several hours and did not become linear with time until after 24 h of incubation. The glioma cells produced little dermatan sulphate and the dermatan sulphate chains differed from those of normal cultures with respect to the distribution of iduronic acid residues. The observed differences between normal glial and malignant glioma cells were not dependent on cell density; rather they were due to the malignant transformation itself.     

Transformed mouse mammary epithelial cells synthesize undersulfated basement membrane proteoglycan

Proteoglycans deposited in the basal lamina of [14C] glucosamine-labeled normal and [3H]glucosamine-labeled transformed mouse mammary epithelial cells grown on type I-collagen gels, were extracted in 4 M guanidinium chloride and cofractionated over Sepharose CL 4B. The heparan sulfate chains carried by these proteoglycans were isolated by treatment with alkaline borohydride, protease K, chondroitinase ABC, and cetylpyridinium chloride precipitation. Heparan sulfate isolated from transformed cell cultures consistently eluted from DEAE-cellulose at lower salt concentrations and was of smaller apparent Mr when chromatographed over Sepharose CL 6B, than heparan sulfate of normal cell cultures. Experiments using doubly labeled cultures ([3H]glucosamine and [35S]sulfate) demonstrated an approximately 30% reduction in the sulfate/hexosamine ratio in heparan sulfate derived from transformed cultures. Both N- and O-sulfate were decreased. The decreased Mr and decreased sulfation of heparan sulfate upon transformation appear sufficient to explain the altered heparan sulfate/chondroitin sulfate ratios previously observed in these cells. These changes may have implications for the molecular interactions in which these proteoglycans are normally engaged during basal lamina assembly, and cause the poor basal lamina formation displayed by these transformed cells.     

Multiple heparan sulfate proteoglycans synthesized by a basement membrane producing murine embryonal carcinoma cell line

The murine embryonal carcinoma derived cell line M1536-B3 secretes the basement membrane components laminin and entactin and, when grown in bacteriological dishes, produces and adheres to sacs of basement membrane components. Heparan sulfate proteoglycans have been isolated from these sacs, the cells, and the medium. At least three different heparan sulfate proteoglycans are produced by these cells as determined by proteoglycan size, glycosaminoglycan chain length, and charge density. The positions of the N- and O-sulfate groups in the glycosaminoglycan chains from each proteoglycan appear to be essentially the same despite differences in the size and culture compartment locations of the heparan sulfate proteoglycan. Additionally, small quantities of chondroitin sulfate proteoglycans are found in each fraction and copurify with each heparan sulfate proteoglycan. Because this cell line appears to synthesize at least three different heparan sulfate proteoglycans which are targeted to different final locations (basement membrane, cell surface, and medium), this will be a useful system in which to study the factors which determine final heparan sulfate proteoglycan structures and culture compartment targeting and the possible effects of the protein core(s) on heparan sulfate carbohydrate chain synthesis and secretion.     

Biosynthesis of proteoglycans by isolated rabbit glomeruli

Isolated rabbit glomeruli were incubated in vitro with 35SO4 in order to analyze the proteoglycans synthesized. Proteoglycans extracted with 4 M guanidine HCl from whole isolated glomeruli and from purified glomerular basement membrane (GBM) were analyzed by gel filtration chromatography. Two types of sulfated proteoglycans were found to be synthesized by rabbit glomeruli and these contained either heparan sulfate or chondroitin/dermatan sulfate glycosaminoglycan chains. These glycosaminoglycans were characterized by their sensitivity to selective degradation by nitrous acid or chondroitinase ABC, respectively. The major proteoglycan extracted from the whole glomeruli was a chondroitin/dermatan sulfate species (75%), while purified GBM contained mostly heparan sulfate (70%). The glycosaminoglycan chains were estimated to be about 12,000 molecular weight which is consistent with previous estimates for similar molecules extracted from the rat GBM.     

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.     

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.     

The effect of precursor structures on the action of glucosaminyl 3-O-sulfotransferase-1 and the biosynthesis of anticoagulant heparan sulfate

To understand how 2-O-sulfation of uronic acid residues influences the biosynthesis of anticoagulant heparan sulfate, the cDNA encoding glucosaminyl 3-O-sulfotransferase-1 (3-OST-1) was introduced into wild-type Chinese hamster ovary cells and mutant pgsF-17 cells, which are defective in 2-O-sulfation. 3-OST-1-transduced cells gained the ability to bind to antithrombin. Structural analysis of the heparan sulfate chains showed that 3-OST-1 generates sequences containing GlcUA-GlcN(SO(3))3(SO(3)) and GlcUA-GlcN(SO(3))3(SO(3))6(SO(3)) in both wild-type and mutant cells. In addition, IdoUA-GlcN(SO(3))3(SO(3)) and IdoUA-GlcN(SO(3))3(SO(3))6(SO(3)) accumulate in the mutant chain. These disaccharides were also observed by tagging [6-(3)H]GlcN-labeled pgsF-17 heparan sulfate in vitro with [(35)S]PAPs and purified 3-OST-1. Heparan sulfate derived from the transduced mutant also had approximately 2-fold higher affinity for antithrombin than heparan sulfate derived from the transduced wild-type cells, and it inactivated factor Xa more efficiently. This study demonstrates for the first time that (i) 3-O-sulfation by 3-OST-1 can occur independently of the 2-O-sulfation of uronic acids, (ii) 2-O-sulfation usually occurs before 3-O-sulfation, (iii) 2-O-sulfation blocks the action of 3-OST-1 at glucosamine residues located to the reducing side of IdoUA units, and (iv) that alternative antithrombin-binding structures can be made in the absence of 2-O-sulfation.     

Heparan sulfate-chondroitin sulfate hybrid proteoglycan of the cell surface and basement membrane of mouse mammary epithelial cells

Chondroitin sulfate represents approximately 15% of the 35SO4-labeled glycosaminoglycans carried by the proteoglycans of the cell surface and of the basolateral secretions of normal mouse mammary epithelial cells in culture. Evidence is provided that these chondroitin sulfate-carrying proteoglycans are hybrid proteoglycans, carrying both chondroitin sulfate and heparan sulfate chains. Complete N-desulfation but limited O-desulfation, by treatment with dimethyl sulfoxide, of the proteoglycans decreased the anionic charge of the chondroitin sulfate-carrying proteoglycans to a greater extent than it decreased the charge of their constituent chondroitin sulfate chains. Partial depolymerization of the heparan sulfate residues of the proteoglycans with nitrous acid or with heparin lyase also reduced the effective molecular radius of the chondroitin sulfate-carrying proteoglycans. The effect of heparin lyase on the chondroitin sulfate-carrying proteoglycans was prevented by treating the proteoglycan fractions with dimethyl sulfoxide, while the effect of nitrous acid on the dimethyl sulfoxide-treated proteoglycans was prevented by acetylation. This occurrence of heparan sulfate-chondroitin sulfate hybrid proteoglycans suggests that the substitution of core proteins by heparan sulfate or chondroitin sulfate chains may not solely be determined by the specific routing of these proteins through distinct chondroitin sulfate and heparan sulfate synthesizing mechanisms. Moreover, regional and temporal changes in pericellular glycosaminoglycan compositions might be due to variable postsynthetic modification of a single gene product.     

Characterization of heparan sulfate proteoglycan from calf lens capsule and proteoglycans synthesized by cultured lens epithelial cells. Comparison with other basement membrane proteoglycans

After extraction with 4 M guanidinium chloride and purification by DEAE-cellulose chromatography, the heparan sulfate proteoglycan (HSPG) of calf anterior lens capsule was found to consist of two immunologically related components (Mr = 340,000 and 250,000) which upon deglycosylation with trifluoromethanesulfonic acid yielded core proteins with Mr values of 170,000 and 145,000. The heparan sulfate chains were uniform in size (Mr = 14,000) and manifested a clustering of sulfate groups in a peripheral domain. From the decrease in Mr observed after heparitinase digestion, it could be estimated that 6 and 11 glycosaminoglycan chains were present in the Mr = 250,000 and 340,000 components respectively. The occurrence of N-linked oligosaccharides was evident from the size difference of the heparitinase- and trifluoromethane-sulfonic acid-treated proteoglycans (approximately 20 kDa), as well as from the presence of a substantial number of mannose residues; furthermore, interaction of the capsule proteoglycan with Bandeiraea simplicifolia I suggested that these carbohydrate units contains terminal alpha-D-Gal groups. Cultured lens epithelial cells deposited a single [35S]sulfate-labeled proteoglycan into their matrix (Mr = 400,000) which was immunologically related to the lens capsule proteoglycan and contained only heparan sulfate chains. In addition to this component, the medium from these cells contained an immunologically unrelated HSPG (Mr = 150,000) as well as a chondroitin sulfate proteoglycan (Mr = 240,000). Examination of bovine glomeruli indicated that, in addition to the previously described 200-kDa HSPG, an immunologically related 350-kDa component was also present. This size heterogeneity, which is comparable to that seen in the lens capsule, is most readily attributable to proteolytic processing of a precursor molecule. Studies with polyclonal antibodies demonstrated only limited cross-reactivities between the Engelbreth-Holms-Swarm proteoglycan and the components from lens capsule and glomerular basement membrane; since even the latter two differed somewhat in their antigenic sites, it would appear that cell- and species-dictated genetic differences as well as post-translational events contribute to the diversity observed in basement membrane HSPGs.     

Identification of the precursor protein for the heparan sulfate proteoglycan of human colon carcinoma cells and its post-translational modifications

Human colon carcinoma cells synthesize a high-molecular-weight heparan sulfate proteoglycan which is localized at the cell surface. In this study we have performed a series of immunoprecipitation and pulse-chase experiments associated with various pharmacological agents that interfere with the synthesis and post-translational modification of the proteoglycan. We demonstrate that colon carcinoma cells synthesize the heparan sulfate proteoglycan from a 400-kDa precursor protein that is immunologically related to the Engelbreth-Holm-Swarm (EHS) tumor cell proteoglycan. The cells contain a large pool of precursor protein with a half-life of about 75 min. Most of the precursor protein receives heparan sulfate side chains and is then transported to the cell surface and released into the medium. A portion of the precursor pool, however, does not receive heparan sulfate chains but is secreted into the medium. The glycosylation and subsequent secretion of the 400-kDa precursor protein was inhibited by NH4Cl and even more by monensin, indicating that the transit of precursor from the rough endoplasmic reticulum to the cell surface occurred through the Golgi complex and acidic compartments. The existence of a sizable pool of precursor protein was confirmed by additional experiments using cycloheximide and xyloside. These experiments showed that the half-life of the precursor protein was also 75 min and that stimulation of heparan sulfate synthesis by xyloside was greatly enhanced (about 12-fold) after new protein core synthesis was blocked by cycloheximide. Although the structural models proposed for the EHS and colon carcinoma heparan sulfate proteoglycans differ, the observation that they are derived from a precursor protein with dimensional and immunological similarities suggests that they may be genetically related.     

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.     

Tumor attenuation by 2(6-hydroxynaphthyl)-beta-D-xylopyranoside requires priming of heparan sulfate and nuclear targeting of the products

We have previously reported that the heparan sulfate-priming glycoside 2-(6-hydroxynaphthyl)-beta-D-xylopyranoside selectively inhibits growth of transformed or tumor-derived cells. To investigate the specificity of this xyloside various analogs were synthesized and tested in vitro. Selective growth inhibition was dependent on the presence of a free 6-hydroxyl in the aglycon. Because cells deficient in heparan sulfate synthesis were insensitive to the xyloside, we conclude that priming of heparan sulfate synthesis was required for growth inhibition. In growth-inhibited cells, heparan sulfate chains primed by the active xyloside were degraded to products that contained anhydromannose and appeared in the nuclei. Hence the degradation products were generated by nitric oxide-dependent cleavage. Accordingly, nitric oxide depletion reduced nuclear localization of the degradation products and counteracted the growth-inhibitory effect of the xyloside. We propose that 2-(6-hydroxynaphthyl)-beta-D-xylopyranoside entered cells and primed synthesis of heparan sulfate chains that were subsequently degraded by nitric oxide into products that accumulated in the nucleus. In vivo experiments demonstrated that the xyloside administered subcutaneously, perorally, or intraperitoneally was adsorbed and made available to tumor cells located subcutaneously. Treatment with the xyloside reduced the average tumor load by 70-97% in SCID mice. The present xyloside may serve as a lead compound for the development of novel antitumor strategies.