Hybrid Proteoglycan Research Articles

Novel Biomarkers of Health and Degeneration in Human Intervertebral Discs: In-depth Proteomic Analysis of Collagen Framework of Fetal, Healthy, Scoliotic, Degenerate, and Herniated Discs.

Profiling proteins expressed in the nucleus pulposus (NP) of intervertebral discs (IVDs) in five different biological states. To evaluate the molecular complexity of the collagen (COL) framework and its role in the health and disease of human IVDs. Changes in COL composition have been linked to degenerative disk disease (DDD). Despite the fact that humans have 28 different types of COLs, most of the literature focuses solely on COL-1 and COL-2. This study used high-end proteomic technology to examine the entire COL composition of the human IVD across fetal (developmental-FD), normal (healthy-ND), scoliotic (early degeneration-SD), herniated (degenerate-DH), and degenerated (DD) disk phenotypes. Forty NP tissues were snap-frozen in liquid nitrogen (-196°C) immediately before being subjected to proteomic and bioinformatic analyses from five different disk phenotypes (eight each). Tandem mass spectrometric analysis revealed a total of 1,050 proteins in FDs, 1,809 in ND, 1,487 in SD, 1,859 in DH, and 1,538 in the DD group. Of 28 major collagens reported in the human body, this study identified 24 different collagens with 34 subtypes in NP. Fibril-forming collagens (COL-1, 2, and 11A1) and fibril-associated collagens with interrupted triple helices (COL-9A1, 12A1, and 14A1) were abundantly expressed in FDs, representing their role in the development of NP. Multiplexin (COL-15), a hybrid proteoglycan-collagen molecule, was discovered only in FDs. Degeneration was associated with COL2A1 downregulation and COL-10A1 upregulation. COL10 was discovered to be a new biomarker for disk degeneration. Besides COL-1 and 2, other important COLs (6, 9, 11, 12, 14, 15) with anabolic potential and abundant expression in the fetal phenotype could be investigated for tissue engineering and novel DDD therapy.

Pentosan Polysulfate, a Semisynthetic Heparinoid Disease-Modifying Osteoarthritic Drug with Roles in Intervertebral Disc Repair Biology Emulating the Stem Cell Instructive and Tissue Reparative Properties of Heparan Sulfate.

This review highlights the attributes of pentosan polysulfate (PPS) in the promotion of intervertebral disc (IVD) repair processes. PPS has been classified as a disease-modifying osteoarthritic drug (DMOAD) and many studies have demonstrated its positive attributes in the countering of degenerative changes occurring in cartilaginous tissues during the development of osteoarthritis (OA). Degenerative changes in the IVD also involve inflammatory cytokines, degradative proteases, and cell signaling pathways similar to those operative in the development of OA in articular cartilage. PPS acts as a heparan sulfate (HS) mimetic to effect its beneficial effects in cartilage. The IVD contains small cell membrane HS proteoglycans (HSPGs) such as syndecan, and glypican and a large multifunctional HS/chondroitin sulfate (CS) hybrid proteoglycan (HSPG2/perlecan), that have important matrix-stabilizing properties and sequester, control, and present growth factors from the FGF, VEGF, PDGF, and BMP families to cellular receptors to promote cell proliferation, differentiation, and matrix synthesis. HSPG2 also has chondrogenic properties and stimulates the synthesis of extracellular matrix (ECM) components and expansion of cartilaginous rudiments, and has roles in matrix stabilization and repair. Perlecan is a perinuclear and nuclear proteoglycan (PG) in IVD cells with roles in chromatin organization and control of transcription factor activity, immunolocalizes to stem cell niches in cartilage, promotes escape of stem cells from quiescent recycling, differentiation and attainment of pluripotency and migratory properties. These participate in tissue development and morphogenesis, ECM remodeling and repair. PPS also localizes in the nucleus of stromal stem cells, promotes development of chondroprogenitor cell lineages, ECM synthesis and repair and discal repair by resident disc cells. The availability of recombinant perlecan and PPS offers new opportunities in repair biology. These multifunctional agents offer welcome new developments in repair strategies for the IVD.

Easy HPLC-based separation and quantitation of chondroitin sulphate and hyaluronan disaccharides after chondroitinase ABC treatment

The sulphation patterns of glycosaminoglycan (GAG) chains are decisive for the biological activity of their proteoglycan (PG) templates for sugar chain polymerization and sulphation. The amounts and positions of sulphate groups are often determined by HPLC analysis of disaccharides resulting from enzymatic degradation of the GAG chains. While heparan sulphate (HS) and heparin are specifically degraded by heparitinases, chondroitinases not only degrade chondroitin sulphate (CS) and dermatan sulphate (DS), but also the protein-free and unsulphated GAG hyaluronan (HA). Thus, disaccharide preparations derived by chondroitinase degradation may be contaminated by HA disaccharides. The latter will often comigrate in HPLC chromatograms with unsulphated disaccharides derived from CS. We have investigated how variation of pH, amount of enzyme, and incubation time affects disaccharide formation from CS and HA GAG chains. This allowed us to establish conditions where chondroitinase degrades CS completely for quantification of all the resulting disaccharides, with negligible degradation of HA, allowing subsequent HA analysis. In addition, we present simple methodology for disaccharide analysis of small amounts of CS attached to a hybrid PG carrying mostly HS after immune isolation. Both methods are applicable to small amounts of GAGs synthesized by polarized epithelial cells cultured on permeable supports.

Glycosaminoglycan chains from α5β1integrin are involved in fibronectin-dependent cell migrationDedicated to the memory of Professor Carl P. Dietrich.

Alpha5beta1 integrin from both wild-type CHO cells (CHO-K1) and deficient in proteoglycan biosynthesis (CHO-745) is post-translationally modified by glycosaminoglycan chains. We demonstrated this using [35S]sulfate metabolic labeling of the cells, enzymatic degradation, immunoprecipitation reaction with monoclonal antibody, fluorescence microscopy, and flow cytometry. The alpha5beta1 integrin heterodimer is a hybrid proteoglycan containing both chondroitin and heparan sulfate chains. Xyloside inhibition of sulfate incorporation into alpha5beta1 integrin also supports that integrin is a proteoglycan. Also, cells grown with xyloside adhered on fibronectin with no alteration in alpha5beta1 integrin expression. However, haptotactic motility on fibronectin declined in cells grown with xyloside or chlorate as compared with controls. Thus, alpha5beta1 integrin is a proteoglycan and the glycosaminoglycan chains of the integrin influence cell motility on fibronectin. Similar glycosylation of alpha5beta1 integrin was observed in other normal and malignant cells, suggesting that this modification is conserved and important in the function of this integrin. Therefore, these glycosaminoglycan chains of alpha5beta1 integrin are involved in cellular migration on fibronectin.

High glucose-induced alterations in subendothelial matrix perlecan leads to increased monocyte binding.

Hyperglycemia is an independent risk factor for cardiovascular disease in diabetic patients, although the link between the two is unknown. These studies were designed to model effects of high glucose on an early event in atherogenesis: the binding of monocytes to subendothelial matrix (SEM). SEM was prepared from human aortic endothelial cells (HAECs) and bovine aortic endothelial cells (BAECs) cultured in the presence of low (5 mmol/L) or high (30 mmol/L) glucose for 3 to 5 days. Monocyte binding was significantly higher (P<0.05) to the SEM of both HAEC and BAEC exposed to high glucose. This increase was a result of changes in SEM heparan sulfate proteoglycans (HSPGs). Metabolic radiolabeling of BAEC demonstrated a 24% decrease in [35S]sulfate incorporation into SEM HSPG produced by cells incubated in 30 mmol/L versus 5 mmol/L glucose, whereas no glucose-associated differences were measured in [35S]methionine incorporation into proteoglycans (PGs) or non-PG proteins. Autoradiography revealed 2 high-molecular weight SEM HSPGs. One was a hybrid PG that contained both heparan sulfate and chondroitin sulfate/dermatan sulfate chains. Both PGs were identified by Western blotting as perlecan. These results illustrate that hyperglycemia-induced structural changes in perlecan may result in a SEM that is more favorable to retention of monocytes.

Structural Characterization of Heparan Sulfate and Chondroitin Sulfate of Syndecan-1 Purified from Normal Murine Mammary Gland Epithelial Cells: COMMON PHOSPHORYLATION OF XYLOSE AND DIFFERENTIAL SULFATION OF GALACTOSE IN THE PROTEIN LINKAGE REGION TETRASACCHARIDE SEQUENCE

Syndecan-1, present on the surfaces of normal murine mammary gland epithelial cells, is a transmembrane hybrid proteoglycan, which bears glycosaminoglycan (GAG) side chains of heparan sulfate (HS) and chondroitin sulfate (CS). Purified syndecan-1 ectodomains were analyzed for disaccharide composition and the GAG-protein linkage region after digestion with bacterial lyases. The HS chains contained predominantly a nonsulfated unit with smaller proportions of two monosulfated, two disulfated, and a trisulfated unit, whereas CS chains were demonstrated for the first time to bear GlcUA-GalNAc(4-O-sulfate) as a major component as well as GlcUA-GalNAc, GlcUA-GalNAc(6-O-sulfate), and an E disaccharide unit GlcUA-GalNAc(4,6-O-disulfate) as minor yet appreciable components. Two kinds of linkage region tetrasaccharides, GlcUA-Gal-Gal-Xyl and GlcUA-Gal-Gal-Xyl(2-O-phosphate), were found for the HS chains in a molar ratio of 55:45. In marked contrast, an additional sulfated tetrasaccharide, GlcUA-Gal(4-O-sulfate)-Gal-Xyl, was demonstrated only for the CS chains, and the unmodified phosphorylated and sulfated components were present at a molar ratio of 55:26:19. The present study thus provided conclusive evidence for the hypothesis that 4-O-sulfation of Gal is peculiar to CS chains in contrast to the phosphorylation of Xyl, which is common to both HS and CS chains. These modifications may be required for biosynthetic maturation of the linkage region tetrasaccharide sequence, which is a prerequisite for creating the repeating disaccharide region of GAG chains and/or biosynthetic selective chain assembly of CS and HS chains.

Mouse Mastocytoma Cells Synthesize Undersulfated Heparin and Chondroitin Sulfate in the Presence of Brefeldin A

In order to study the subcellular localization and organization of the enzymes involved in the glycosylation of the hybrid proteoglycan serglycin, mouse mastocytoma cells were metabolically labeled with [35S]sulfate or [3H]glucosamine in the absence or presence of brefeldin A. This drug is known to induce a disassembly of the proximal part of the Golgi complex, resulting in a redistribution of cis-, medial-, and trans-Golgi resident enzymes back to the endoplasmic reticulum, and to block the anterograde transport of proteins to the trans-Golgi network. Although the total incorporation of [3H]glucosamine into glycosaminoglycan chains was reduced to about 25% in brefeldin A-treated cells compared to control cells, both control cells and cells treated with brefeldin A synthesized heparin as well as chondroitin sulfate chains. Therefore, enzymes involved in the biosynthesis of both types of glycosaminoglycan chains seem to be present proximal to the trans-Golgi network in these cells. Chondroitin sulfate and heparin synthesized in cells exposed to brefeldin A were undersulfated, as demonstrated by ion-exchange chromatography, compositional analyses of disaccharides, as well as by a lower [35S]sulfate/[3H]glucosamine ratio compared to controls. In heparin biosynthesis, both N- and O-sulfation reactions were impaired, with a larger relative decrease in 2-O-sulfation than in 6-O-sulfation. Despite undersulfation, the heparin chains synthesized in the presence of brefeldin A were larger (30 kDa) than the heparin synthesized by control cells (20 kDa). The reduced [3H]glucosamine incorporation in brefeldin A-treated cells was partly due to decreased number of glycosaminoglycan chains synthesized, but also to the biosynthesis of chondroitin sulfate chains of smaller molecular size (8 versus 15 kDa in control cells). Brefeldin A had no effect on the glycosaminoglycan synthesis when used in a cell-free, microsomal fraction, indicating that brefeldin A does not interfere directly with the enzymes involved in the biosynthesis of glycosaminoglycans.

Synthesis of sulphated proteoglycans by primary cultures of rat hepatocytes--modulation by matrix substratum.

Primary cultures of rat hepatocytes maintained on different matrix proteins such as collagen (Co IV) fibronectin (Fn), Laminin (Ln) or different tissue biomatrices were metabolically labelled with 35[S]-SO4 and the synthesis of sulphated proteoglycans was studied. The incorporation of the label into total glycosaminoglycan (GAG) was significantly higher in cells maintained on Co IV compared to those maintained on Fn or Ln. Similarly the incorporation of label was maximum in those cells maintained on the aortic biomatrix compared to liver or mammary gland biomatrix. About 80-95% of the GAG synthesised and secreted by cells maintained on individual matrix proteins and liver biomatrix was heparan sulphate (HS). But in the case of cells maintained on collagen IV aortic or mammary biomatrix in addition to HS, significant amount of chondroitin sulphate (CS) was also found. Nearly 50% of the total 35[S]-GAG was associated with the cell layer after 24 h in culture in the case of cells maintained on individual matrix protein while those maintained on tissue biomatrix, retained about 70% of the 35[S]-labelled proteoglycans (PG) with the cell layer. Analysis of the cell surface 35[S]-labelled proteoglycans isolated from cells maintained on different biomatrix showed that it is a hybrid proteoglycan consisting of CS and HS. While the PG isolated from cells maintained on liver biomatrix consists of HS and CS in the ratio of 3:2 that from cells maintained on aorta or mammary gland matrix was about 2:3 indicating an alteration in the nature of the cell surface PGs produced by cells maintained on different tissue biomatrix. These results indicate that depending on the nature of the matrix substratum with which the cells are in contact, the nature and quantity of sulphated proteoglycans produced by hepatocytes vary.

Heparin proteoglycans synthesized by mouse mastocytoma contain chondroitin sulphate

Proteoglycans (PGs), biosynthetically labelled with [35S]sulphate, were isolated from mouse mastocytoma tissue. Chromatography on antithrombin (AT)-Sepharose resulted in the separation of the 35S-labelled PGs into three fractions: PGs with no affinity for the gel (NA-PGs), PGs with low affinity (LA-PGs), and PGs with high affinity (HA-PGs) for antithrombin. Whereas NA-PGs contained almost exclusively chondroitin sulphate (CS), the AT-binding PGs contained 80-85% heparin and 15-20% CS. [35S]CS-containing macromolecules obtained from the HA-PG fraction after removal of the heparin polysaccharide chains were rechromatographed on AT-Sepharose. A majority of these 35S-labelled macromolecules no longer showed affinity for AT. These experiments indicate that the [35S]CS recovered in the AT-binding PGs is present in hybrid PGs. Polysaccharide chain-length determination demonstrated that the heparin chains were somewhat larger (M(r) approximately 30,000) than the CS chains in the NA-PGs (M(r) approximately 25,000). CS chains in the hybrid PGs were slightly smaller (M(r) approximately 20,000). Characterization of the sulphated CS disaccharides from NA- and HA-PGs showed that they contained similar amounts (20%) of disulphated disaccharides of [GlcA-GalNAc(4,6-di-OSO3)] type. The monosulphated CS-disaccharides were O-sulphated at C-4 of the galactosamine units. Analysis by gel chromatography of the [35S]CS components isolated from HA-PGs after heparinase treatment showed that a major portion of these contained one CS chain only. Calculations of the number of CS and heparin chains in AT-binding PGs, based on polysaccharide composition and polysaccharide chain length, indicate that all heparin-containing PGs are hybrids.

Modulated glycosylation of proteoglycans during differentiation of human B lymphocytes

Proteoglycans are mediators of cellular adhesion and regulate growth factor activities. Proteoglycans of B lymphocytes undergo structural changes during B cell ontogeny which may correspond to the specific requirements of the respective microenvironment of the maturing cell. We analyzed three human B cell lines representing pre-B cells (Nalm-6), activated B cells (Jok-1) and plasma cells (U266) for their cellular proteoglycans. Gel filtration of the 35S-labeled macromolecules of the three cell lines revealed an increase in size in the order Nalm-6 < Jok-1 < U266. In Jok-1 and U266 cells the major pool of proteoglycans consisted of proteochondroitin sulfates of 50 to 90 kDa. These proteoglycans carried a protein core of approx. 30 kDa to which 1 to 3 glycosaminoglycan chains in the range of 28 to 32 kDa were attached. In Nalm-6 cells only free chondroitin sulfate chains of 23 kDa, but no intact proteoglycans, were detected. Chondroitin sulfate chains were predominantly composed of chondroitin-4-sulfate, those of Nalm-6 and U266 cells additionally contained 10–20% of unsulfated disaccharides. In U266 cells 30% of glycosaminoglycans consisted of heparan sulfate either bound to pure proteoheparan sulfate or to chondroitin sulfate/heparan sulfate hybrid-proteoglycans. Earlier, syndecan-1 was described as a hybrid proteoglycan containing heparan sulfate/chondroitin sulfate chains which is transcribed by murine B cells at early and late maturation stages. In order to see whether syndecan is transcribed by the human B cell lines used here, we measured expression of syndecan mRNA by the reverse transcriptase polymerase chain reaction. Similar to murine lymphocytes, syndecan-specific mRNA was detected in Nalm-6 and U266 cells, equivalent to early and late B cells, but not in lymphoblastoid Jok-1 cells. However, Nalm-6 cells do not produce proteoheparan sulfate. In these cells, syndecan synthesis may be blocked at the translational level. Also, the proteoglycans of U266 are different from syndecan-1 in their composition of glycosaminoglycans and in size of protein cores. Together, these results indicate that the major pool of proteoglycans produced by human B cells consists of proteochondroitin sulfate and additionally in later stages of a smaller proportion of proteoheparan sulfate which is not identical to syndecan-1. During distinct phases of B cell differentiation, modulations in the glycosaminoglycan moiety concerning size and sulfation of glycosaminoglycan chains were also found.

Membrane associated proteoglycans in rat testicular peritubular cells.

Confluent testicular peritubular cells derived from immature rats were used to study membrane associated proteoglycans (PG). Peripheral material (heparin releasable), membrane and intracellular material (Triton X-100 releasable) were collected, purified by anion exchange chromatography then characterized by gel filtration and by hydrophobic interaction chromatography, followed by enzymatic digestion and chemical treatment. The peripheral material was constituted of two populations of PG (Kav = 0 and 0.10 on Superose 6 column), each containing both heparan sulfate proteoglycans (HSPG) and chondroitin proteoglycans (CSPG) and perhaps a hybrid PG (HSCSPG). These PG being not retained on an octyl Sepharose column, they were devoided of hydrophobic properties. The integral membrane proteoglycans isolated on the basis of their hydrophobic properties represented 20% of the Triton X-100 releasable material, and were exclusively constituted of proteoheparan sulfate. There were no relationships between this membrane HSPG and the peripheral HSPG as evidenced by pulse chase experiments. The mode of intercalation of the hydrophobic HSPG in the cell membrane was studied. The majority of these macromolecules (80%) were sensitive to trypsin and only a minor proportion (20%) were sensitive to phosphatidylinositol specific phospholipase C. Thus, about 80% of the hydrophobic HSPG were intercalated in the cell membrane by a hydrophobic segment of the core protein whereas about 20% were associated with the cell membrane via a phosphatidylinositol residue covalently bound to the core protein of the PG.

Syndecan-1 expressed in Schwann cells causes morphological transformation and cytoskeletal reorganization and associates with actin during cell spreading

To investigate the biological functions of transmembrane proteoglycans we have produced clonal cell lines of rat Schwann cells that express the hybrid proteoglycan syndecan-1. This was done by transfection of newborn rat Schwann cells with a plasmid vector bearing the rat syndecan-1 cDNA sequence under transcriptional control of the constitutively active cytomegalovirus promoter, and a neomycin resistance gene. Stably expressing cells were selected by growth in G418. Expression of syndecan-1 was verified by Northern and immunoblot analysis and immunoprecipitation of 35SO4-labeled proteoglycans. The syndecan-1 expressing cells exhibited significantly enhanced spreading on several different substrata, including fibronectin and laminin, and an altered morphology. The enhanced spreading appeared to result from the presence of syndecan-1, based on the observation that anti-syndecan-1 antibodies inhibited the enhanced substratum spreading. There was also a reorganization of cytoskeletal structures and formation of focal adhesions, visualized by anti-vinculin staining, which were absent from control Schwann cells. There was no apparent stable association of cell surface syndecan-1 with focal contact sites, as determined by dual staining with anti-syndecan-1 and anti-vinculin antibodies. Colocalization of patches of cell surface syndecan-1 with actin was observed, but only during cell spreading. These findings provide evidence for a role of transmembrane proteoglycans in cellular morphogenesis, and suggest that transient association of syndecans with microfilaments may be an important aspect of their biological function.

Membrane-intercalated proteoglycan of a stroma-inducing clone from Lewis lung carcinoma binds to fibronectin via its heparan sulfate chains.

A Lewis lung carcinoma-derived low metastatic clone, P29, with a capacity to induce a fibrotic stromal response of host tissue, exhibits tumorigenesis depending on an interstitial matrix formed by the induced stromal cells. Using this clone, in the present study we isolated and characterized a membrane-intercalated proteoglycan that mediates interaction between the tumor cells and interstitial matrix. The tumor cells were cultured in the presence of [3H]glucosamine and [35S]sulfate or [35S]methionine, and hydrophobic proteoglycans were isolated by chromatography on DEAE-Sephacel and then Octyl-Sepharose CL-4B. Proteoglycans with high affinity to the octylresidue were obtained from the cell layer but not to any significant extent from the medium. By CsCl density gradient centrifugation, they were separated into bottom, middle, and top subfractions, which were shown to consist of homogeneous species with estimated M(r) values of 270,000 (named CPGIIIB), 200,000 (CPGIIIM), and 195,000 (CPGIIIT), respectively, by gel filtration on Sepharose CL-4B. These proteoglycans were intercalated into phosphatidylcholine liposomes, suggesting that they are all membrane-intercalated proteoglycans. Analyses of their glycosaminoglycans with chondroitinase ABC and heparitinase I plus II demonstrated that they all contain heparan sulfate as a major glycosaminoglycan (58-85%) and chondroitin 4-sulfate as a minor one (15-42%). Of these three proteoglycans, only CPGIIIB proteoglycan bound specifically to fibronectin-Sepharose 4B under physiological conditions. Molecular analyses of this proteoglycan by Sepharose CL-4B or SDS-PAGE before and after treatments with glycosaminoglycan degradation enzymes or trifluoromethanesulfonic acid demonstrated that CPGIIIB proteoglycan is a hybrid proteoglycan having heparan sulfate and chondroitin 4-sulfate chains on the same core protein with an M(r) of 40,000. Affinity chromatographies of the CPGIIIB proteoglycan on fibronectin-Sepharose 4B after treatments with these enzymes demonstrated that it bound to fibronectin via its heparan sulfate chains. On the basis of the above results, we propose that the CPGIIIB proteoglycan mediates the interaction between the tumor cells and interstitial matrix.

Testican, a multidomain testicular proteoglycan resembling modulators of cell social behaviour.

The molecular characterization of a human testicular proteoglycan, the progenitor of a seminal plasma glycosaminoglycan-bearing peptide, was achieved by cDNA cloning. Its protein core encompasses several domains encountered in various proteins associated with adhesion, migration and cell proliferation. An osteonectin-like domain, a Kazal-like sequence and a 46-amino-acid motif around a Cys-Trp-Cys-Val peptide encountered in cell-surface antigens, cell-adhesion molecules and growth-factor-binding proteins are distributed within the testican protein core. Testican is the progenitor of the unique heparan/chondroitin-sulfate-bearing peptide present in human seminal plasma, a feature which might confer additional potentialities to this hybrid proteoglycan.

Regulated expression of syndecan in vascular smooth muscle cells and cloning of rat syndecan core protein cDNA.

cDNA encoding the core protein of rat syndecan was cloned from a neonatal rat aortic cDNA library by polymerase chain reaction amplification. Expression of syndecan mRNA in rat aortic vascular smooth muscle (VSM) cells was demonstrated by reverse transcriptase-linked polymerase chain reaction amplification of syndecan sequences using total RNA from rat aortic VSM cells as templates. Polyclonal antibodies against rat syndecan core protein were produced by immunizing rabbits with a recombinant fusion protein containing a fragment of the extracellular domain. The anti-syndecan antibodies immunoprecipitated a large 35SO4-labeled molecule synthesized by cultured rat aortic VSM cells. The immunoprecipitated molecule was identified as a hybrid proteoglycan, based on results of alkaline, nitrous acid, and chondroitinase ABC digestions. On immunoblots the antibodies recognized a proteoglycan of greater than 200 kDa, with a core protein size after deglycosylation of approximately 50 kDa. The anti-syndecan antibodies stained cultured rat aortic VSM cells as well as tissue sections of neonatal and adult rat aortas in the medial, smooth muscle layer. On Northern blots of RNA isolated from cultured VSM cells, a syndecan cDNA probe hybridized to a major RNA species of 2.6 kilobases. Quantitative Northern blot analysis of total RNA isolated from VSM cells harvested at different cell densities revealed a decrease in syndecan mRNA levels with increased cell density. These results demonstrate the regulated synthesis of syndecan by rat VSM cells.

Establishment of a Cell Line from the EHS Tumor: Biosynthesis of Basement Membrane Constituents and Characterization of a Hybrid Proteoglycan Containing Heparan and Chondroitin Sulfate Chains

We have established a continuous cell line from the Engelbreth-Holm-Swarm (EHS) tumor, a transplantable murine neoplasm that has been extensively utilized to investigate basement membrane constituents. The EHS-derived cells, designated BAM cells, have been subcultured for over 40 passages and have maintained phenotypic and biological properties of the parent EHS tumor cells. BAM cells have retained an epithelioid morphology and the ability to induce EHS-like tumors in mice. Biochemical and immunochemical studies demonstrated that BAM cells synthesize laminin A and B chains, collagen type IV, entactin and the basement membrane specific heparan sulfate proteoglycan. Interestingly, the proteoglycan synthesized by BAM cells was a hybrid molecule containing 2–3 heparan sulfate chains of 25–35 kDa and 1 chondroitin sulfate chain of ≈ 17 kDa attached to a 400-kDa protein core. This cell line will be useful to investigations concerning biosynthesis of basement membrane constituents and will be a valuable source of extracellular matrix for testing cellular properties such as attachment, locomotion and differentiation.

The phosphorylated and/or sulfated structure of the carbohydrate – protein‐linkage region isolated from chondroitin sulfate in the hybrid proteoglycans of Engelbreth‐Holm‐Swarm mouse tumor

The structure of the linkage region of chondroitin sulfate chains attached to the hybrid proteoglycans of the Engelbreth-Holm-Swarm mouse tumor was investigated. The peptidoglycan fraction which contains oversulfated chondroitin sulfate rich in the GlcA beta 1-3GalNAc-4,6-diO-sulfate unit and undersulfated heparan sulfate rich in GlcA beta 1-4GlcNAc and GlcA beta 1-4GlcN-2N-sulfate units was isolated after exhaustive protease digestion of the acetone powder of the tumor tissue, (GlcA, glucuronic acid; GalNAc, 2-deoxy-2-N-acetylamino-D-galactose). Glycosaminoglycans were released by beta-elimination using NaB3H4 and digested with chondroitinase ABC. The linkage region fraction was separated from heparan sulfate by gel filtration and fractionated by HPLC on an amine-bound silica column. Six radiolabeled compounds (L1-L6) were obtained and structurally analyzed by cochromatography with authentic hexasaccharide alditols recently isolated by us from the linkage region, and by digestion using chondroitinase ACII, alkaline phosphatase and beta-galactosidase in conjugation with HPLC. These compounds shared the conventional hexasaccharide backbone structure: delta GlcA beta 1-3GalNAc beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl-ol, (delta GlcA, delta 4.5-GlcA or D-gluco-4-enepyranosyluronic acid). L1 was not sulfated or phosphorylated. L2 and L4 were monosulfated at C-6 and C-4 of the GalNAc residue, respectively. Upon alkaline phosphatase digestion, L3, L5 and L6 were converted to L1, L2 and L4, respectively. Analysis of the periodate oxidation products indicated that the phosphate group in L3, L5 and L6 is located at C-2 of Xyl-ol. These results suggest that Xyl-2-O-phosphate is associated with both 4-O-sulfated and 6-O-sulfated GalNAc units and does not directly determine the sulfation pattern of chondroitin sulfate.

Syndecan--a cell surface proteoglycan that selectively binds extracellular effector molecules.

Syndecan is a cell surface proteoglycan (Saunders et al., 1989), which was originally isolated from mouse mammary epithelial (NMuMG) cells as a hybrid proteoglycan containing both heparan sulfate and chondroitin sulfate glycosaminoglycan (GAG) chains (Rapraeger et al., 1985). The ratio and total amount of GAG bound to syndecan is known to vary in different tissues (Sanderson & Bernfield, 1988; Salmivirta et al., 1991) and cells (Rapraeger 1989). Syndecan binds several extracellular matrix molecules such as fibrillar collagens of interstitial matrix (Koda et al., 1985), fibronectin (Saunders & Bernfield, 1988) and thrombospondin (Sun et al., 1989) but shows no binding to some other heparin binding matrix molecules, like vitronectin and laminin (Koda et al., 1985; Elenius et al., 1990). Syndecan also binds growth factors, like bFGF (Kiefer et al., 1990), and could therefore play a major role in the regulation of cell adhesion, growth and differentiation (for a review see Jalkanen et al., 1991).

Differential expression of cell surface heparan sulfate proteoglycans in human mammary epithelial cells and lung fibroblasts.

Treating the liposome-intercalatable heparan sulfate proteoglycans from human lung fibroblasts and mammary epithelial cells with heparitinase and chondroitinase ABC revealed different core protein patterns in the two cell types. Lung fibroblasts expressed heparan sulfate proteoglycans with core proteins of approximately 35, 48/90 (fibroglycan), 64 (glypican), and 125 kDa and traces of a hybrid proteoglycan which carried both heparan sulfate and chondroitin sulfate chains. The mammary epithelial cells, in contrast, expressed large amounts of a hybrid proteoglycan and heparan sulfate proteoglycans with core proteins of approximately 35 and 64 kDa, but the fibroglycan and 125-kDa cores were not detectable in these cells. Phosphatidylinositol-specific phospholipase C and monoclonal antibody (mAb) S1 identified the 64-kDa core proteins as glypican, whereas mAb 2E9, which also reacted with proteoglycan from mouse mammary epithelial cells, tentatively identified the hybrid proteoglycans as syndecan. The expression of syndecan in lung fibroblasts was confirmed by amplifying syndecan cDNA sequences from fibroblastic mRNA extracts and demonstrating the cross-reactivity of the encoded recombinant core protein with mAb 2E9. Northern blots failed to detect a message for fibroglycan in the mammary epithelial cells and in several other epithelial cell lines tested, while confirming the expression of both glypican and syndecan in these cells. Confluent fibroblasts expressed higher levels of syndecan mRNA than exponentially growing fibroblasts, but these levels remained lower than observed in epithelial cells. These data formally identify one of the cell surface proteoglycans of human lung fibroblasts as syndecan and indicate that the expression of the cell surface proteoglycans varies in different cell types and under different culture conditions.

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- 35SO 4 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- 35SO 4 proteoglycan eluted with the column void volume and at a K av of 0.26 from Sepharose CL-4B columns. The most abundant medium heparan- 35SO 4 proteoglycan was a high buoyant density proteoglycan similar in hydrodynamic size (Sepharose CL-6B K av 0.23) to those previously described in glomerular basement membranes and isolated glomeruli. Heparan- 35SO 4 chains from both proteoglycans were 36 kDa. A smaller proportion of Sepharose CL-6B excluded dermatan- 35SO 4 proteoglycan was also synthesized by these cells. The predominant protein cores of both medium heparan- 35SO 4 proteoglycans were approximately 230 and 180 kDa. A hybrid chondroitin/dermatan-heparan- 35SO 4 proteoglycan with an 80-kDa protein core copurified with the smaller medium heparan- 35SO 4 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 K av 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.