Differences in the distribution of O-sulphate groups of cell-surface and secreted heparan sulphate produced by human neuroblastoma cells in culture

Confluent cultures of a human neuroblastoma cell line (CHP100) were incubated for 48 h with d-[1-³H]glucosamine and sodium [³⁵S]sulphate. Radioactive glycosaminoglycans were analysed in the growth medium, rapid trypsin digest of the cell monolayer and a 1% (w/v) Triton/0.5 M NaOH extract of the final cell pellet. Sulphated glycosaminoglycans co-chromatographed when eluted by NaCL gradient from DEAE-cellulose. The medium contained mainly chondroitin sulphates, whereas the cell surface was enriched in heparan sulphate. Heparan sulphate was isolated as chondroitinase ABC-resistant material and treated with nitrous acid. Analysis of the scission products on Bio-Gel P-10 yielded fragments varying in size from single disaccharides to glycans consisting of nine disaccharide units. Cell-surface and medium heparan sulphate had respectively 52% and 54% N-sulphated glucosamine residues distributed in similar patterns along the polymer chain. The N:O-sulphate ratio of neuroblastoma heparan sulphate was 1.1:1. Analysis by high-voltage electrophoresis of di- and tetrasaccharide products produced by nitrous acid treatment showed that the distribution of $\text{‘O’-}\text{sulphate}$ groups differed strikingly between heparan sulphates from the medium and cell-surface compartments. A di-O-sulphated tetrasaccharide was identified in both heparan sulphate species. The absence of detectable amounts of ³⁵[S]sulphate associated with fragments larger than tetrasaccharide supports the close topographical association of N-sulphate and O-sulphate groups.     

Domains of neuronal heparan sulphate proteoglycans involved in neurite growth on laminin

Summary A single neuronal cell assay of neurite growth was utilized to determine types and domains of neuronal proteoglycans involved in neurite growth on laminin. Perturbations of biosynthesis and processing, enzymatic digestion with specific lyases, and competition with glycosaminoglycan side chains produced complementary data consistent with a molecular model implicating glycosaminoglycan (GAG) residues of heparan sulphate proteoglycans (HSPGs) in neurite growth. The observations suggest that HSPGs promote neurite growth on laminin by bridging between binding domains for HSPGs on laminin and on the neuronal cell surface, and that the bridge is tethered at both ends by noncovalent interactions between the binding domains and GAG side chains. Sulphation of the GAGs of HSPGs appears to be critical to the tethering and/or neurite growth-promoting activity of neuronal HSPGs.     

High molecular weight proteoglycans biosynthesized in culture by pigeon aortas

The properties of aortic proteoglycans synthesized in vitro were examined to demonstrate synthesis of intact proteoglycans by aortic tissue in culture and to compare labeling and synthetic rates of two different populations of proteoglycan. Following 3, 6, or 9 h of incubation in medium containing [³⁵S]sodium sulfate and [³H]serine, the tissue was extracted with 4.0 M guanidine hydrochloride containing protease inhibitors. Extracts were chromatographed on Sepharose CL-4B and subjected to buoyant density centrifugation under dissociative conditions. Radioactive precursors were incorporated into two major populations of aortic proteoglycan, one of high molecular weight eluting near the void volume of Sepharose CL-4B (Protooglycan I) and one of lower molecular weight (Proteoglycan II) having a K_av of 0.40–0.44. The radioactively labeled proteoglycans were localized at densities 1.50–1.56 g/ml (Preparation 1) and 1.43–1.49 g/ml (Preparation 2) following CsCl buoyant density centrifugation. Both proteoglycan populations had increased incorporation of ³⁵S and ³H over time. At all times the lower molecular weight proteoglycan had a higher specific activity (dpm ³⁵S and ³H/μg hexuronic acid). At 3, 6, and 9 h, the specific activity of Proteoglycan II was 8.2-, 6.7- and 3.0-fold higher than Proteoglycan I using ³⁵S and 13.0-, 8.1- and 2.7-fold higher using ³H, suggesting different synthetic rates for the two proteoglycans. The results illustrate synthesis of intact proteoglycans during short-term artery culture. The proteoglycan types have size and buoyant density characteristics as described for artery, but based upon changes in specific activity ratios, the two proteoglycan populations differ in rates of synthesis.     

Mesenchymal stem cells,neural lineage potential,heparan sulfate proteoglycans and the matrix

Along with the tri-lineage of bone, cartilage and fat, human mesenchymal stem cells (hMSCs) retain neural lineage potential. Multiple factors have been described that influence lineage fate of hMSCs including the extracellular microenvironment or niche. The niche includes the extracellular matrix (ECM) providing structural composition, as well as other associated proteins and growth factors, which collectively influence hMSC stemness and lineage specification. As such, lineage specific differentiation of MSCs is mediated through interactions including cell–cell and cell–matrix, as well as through specific signalling pathways triggering downstream events. Proteoglycans (PGs) are ubiquitous within this microenvironment and can be localised to the cell surface or embedded within the ECM. In addition, the heparan sulfate (HS) and chondroitin sulfate (CS) families of PGs interact directly with a number of growth factors, signalling pathways and ECM components including FGFs, Wnts and fibronectin. With evidence supporting a role for HSPGs and CSPGs in the specification of hMSCs down the osteogenic, chondrogenic and adipogenic lineages, along with the localisation of PGs in development and regeneration, it is conceivable that these important proteins may also play a role in the differentiation of hMSCs toward the neuronal lineage. Here we summarise the current literature and highlight the potential for HSPG directed neural lineage fate specification in hMSCs, which may provide a new model for brain damage repair.     

人血清脂蛋白与糖胺聚糖及人主动脉蛋白聚糖的相互作用

本文报道了在[Ca~(2+)]=30mmol/L时,人血清或人血清脂蛋白与各种糖胺聚糖(GAG)及人主动脉两种蛋白聚糖(PG)的相互作用。GAG与血清的作用能力为6—硫酸软骨素(C6—S)>肝素(Hep)>4—硫酸软骨素(C4—S)>透明质酸(HA)>硫酸皮肤素(DS)。极低密度脂蛋白(VLDL)及低密度脂蛋白(LDL)可与肝素作用形成不溶性复合物,而高密度脂蛋白(HDL)则不能。人主动脉硫酸软骨素—PG(CS—PG)、硫酸皮肤素—硫酸软骨素—PG(DS—CS—PG)与血清形成不溶性复合物的曲线类型不同,后者的类型似有利于DS—CS—PG与血清脂蛋白结合从而使之在动脉壁沉积。     

Heterogeneity of heparan sulfate proteoglycans synthesized by PYS-2 cells

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

The critical interaction of the metallopeptidase PHEX with heparan sulfate proteoglycans

The PHEX gene (phosphate-regulating gene with homologies to endopeptidase on the X chromosome) identified as a mutated gene in patients with X-linked hypophosphatemia (XLH), encodes a protein (PHEX) that shows striking homologies to members of the M13 family of zinc metallopeptidases. In the present work the interaction of glycosaminoglycans with PHEX has been investigated by affinity chromatography, circular dichroism, protein intrinsic fluorescence analysis, hydrolysis of FRET substrates flow cytometry and confocal microscopy. PHEX was eluted from a heparin-Sepharose chromatography column at 0.8 M NaCl showing a strong interaction with heparin. Circular dichroism spectra and intrinsic fluorescence analysis showed that PHEX is protected by glycosaminoglycans against thermal denaturation. Heparin, heparan sulfate and chondroitin sulfate inhibited PHEX catalytic activity, however among them, heparin presented the highest inhibitory activity (K_i = 2.5 ± 0.2 nM). Flow cytometry analysis showed that PHEX conjugated to Alexa Fluor 488 binds to the cell surface of CHO-K1, but did not bind to glycosaminoglycans defective cells CHO-745. Endogenous PHEX was detected at the cell surface of CHO-K1 colocalized with heparan sulfate proteoglycans, but was not found at the cell surface of glycosaminoglycans defective cells CHO-745. In permeabilized cells, PHEX was detected in endoplasmic reticulum of both cells. In addition, we observed that PHEX colocalizes with heparan sulfate at the cell surface of osteoblasts. This is the first report that the metallopeptidase PHEX is a heparin binding protein and that the interaction with GAGs modulates its enzymatic activity, protein stability and cellular trafficking.     

The heparan sulfates of Swiss mouse 3T3 cells. The effect of transformation

Three major pools of heparan sulfate have been isolated from cultures of Swiss mouse 3T3 and SV40-transformed 3T3 cells: cell-surface, medium, and intracellular heparan sulfates. The cell-surface heparan sulfate is a high molecular weight proteogylcan which is partially degraded by pronase. Before pronase treatment, it has a peak molecular weight (as estimated by gel filtration) of appox. 7.2 · 10⁵ in contrast to only 2.4 · 10⁵ after pronase treatment. The medium heparan sulfate appears to be similar in structure to the cell-surface heparan sulfate, since they coelute on Bio-Gel A-15m and DEAE-cellulose, and are both proteoglycans. In contrast, the intracellular heparan sulfate has a low molecular weight (6.0 · 10³) and has little if any attached protein. Both the medium and intracellular heparan sulfate exhibit the transformation-associated change in structure reported earlier for cell-surface heparan sulfate (Underhill, C.B. and Keller, J.M. (1975) Biochem. Biophys. Res. Commun. 63, 448–454). This transformation-associated change, detected by DEAE-cellulose chromatography is not the result of changes in either molecular weight or protein core. Cellulose acetate electrophoresis of the cell-surface heparan sulfate at pH 1 suggests that the transformation-associated change in structure is due to a difference in sulfate content. Both types of heparan sulfate are produced in mixed cultures ot 3T3 and SV3T3 cells, indicating that neither serum factors in the culture medium nor secreted cell products are responsible for the transformation-associated change in heparan sulfate structure. The presented date are discussed with respect to the postulated role of heparan sulfate in cell social behavior.     

Differences in the distribution of O-sulphate groups of cell-surface and secreted heparan sulphate produced by human neuroblastoma cells in culture

Analogs of luteinizing hormone-releasing hormone (LHRH) having higher biological activity than LHRH itself are being mainly used to study the biological effects and the mechanism of action of LHRH. In the present study, conditions for the direct 3H-labelling at the histidine residue of analogs of LHRH were worked out, circumventing the synthesis of precursor peptides for labelling. [D-Phe6,desGly10]-LHRH ethylamide and [D-Ser(But)6,desGly10]-LHRH ethylamide were tritiated by tritium gas and a 10% Pd/Al2O3 catalyst to high specific radioactivities. The labelled peptides are sufficiently stable to be used in biochemical studies. The degradability of the analogs by homogenates of various tissues of rats was compared with that of the native LHRH. The analogs were shown to be distinctly degradable, but to a lower extent. The kidney homogenate degrades the analogs [D-Phe6,desGly10]- and [D-Ser(But)6,desGly10]-LHRH ethylamide with 35 and 50%, respectively, of the velocity observed with LHRH, whereas the degradation velocity of the analogs by a homogenate of the hypothalamus and pituitary is only 10% of that of LHRH. It is suggested that the lower degradability of the analogs at peripheral sites and target sites (pituitary, ovary) explains partly their higher biological activity.     

HSULF-1 inhibits ERK and AKT signaling and decreases cell viability in vitro in human lung epithelial cells

Background

Heparan sulfate proteoglycans (HSPGs) modulate the binding and activation of signaling pathways of specific growth factors, such as fibroblast growth factor-2 (FGF-2). Human endosulfatase 1 (HSULF-1) is an enzyme that selectively removes 6-O sulfate groups from HS side chains and alter their level and pattern of sulfation and thus biological activity. It is known that HSULF-1 is expressed at low levels in some cancer cell lines and its enhanced expression can inhibit cancer cell growth or induce apoptosis, but the mechanism(s) involved has not been identified.

Methods

HSULF-1 mRNA expression was assessed in five normal cells (primary human lung alveolar type 2 (hAT2) cells, adult lung fibroblasts (16Lu), fetal lung fibroblasts (HFL), human bronchial epithelial cells (HBE), and primary human lung fibroblasts (HLF)) and five lung cancer cell lines (A549, H292, H1975, H661, and H1703) using quantitative real time polymerase chain reaction (qRT-PCR). H292 and hAT2 cells over-expressing HSULF-1 were analyzed for cell viability, apoptosis, and ERK/Akt signaling, by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay, and Western Blot, respectively. Apoptosis pathway activation was confirmed by PCR array in hAT2, H292, and A549 cells.

Results

HSULF-1 was expressed at a significantly lower level in epithelial cancer cell lines compared to normal cells. Infection with recombinant adenovirus for HSULF-1 over-expression resulted in decreased cell viability in H292 cells, but not in normal hAT2 cells. HSULF-1 over-expression induced apoptosis in H292 cells, but not in hAT2 cells. In addition, apoptosis pathways were activated in both H292 and A549 cells, but not in hAT2 cells. HSULF-1 over-expression reduced ERK and Akt signaling activation in H292 cells, which further demonstrated its inhibitory effects on signaling related to proliferation.

Conclusions

These results indicate that HSULF-1 is expressed at lower levels in H292 lung cancer cells than in normal human alveolar cells and that its over-expression reduced cell viability in H292 cells by inducing apoptotic pathways, at least in part by inhibiting ERK/Akt signaling. We hypothesize that HSULF-1 plays important roles in cancer cells and functions to modify cell signaling, inhibit cancer proliferation, and promote cancer cell death.     

The kinetics of FGF-2 binding to heparan sulfate proteoglycans and MAP kinase signaling

Binding of growth factors to specific cell surface receptors is the first step in initiating cell signaling cascades that ultimately result in diverse activities such as proliferation, differentiation, and apoptosis. Dimerization and phosphorylation of tyrosine kinase transmembrane receptors is the typical paradigm for this activation but, for many growth factors, cell surface interactions are not limited to a single receptor type. In particular, heparin-binding growth factors, such as fibroblast growth factor-2 (FGF-2), bind to heparan sulfate proteoglycans (HSPG) on the cell surface and within the extracellular matrix (ECM), and these molecules have been viewed as accessory co-receptors serving to facilitate tyrosine kinase receptor binding. Recent studies, however, have indicated that HSPG can directly participate in signal transduction in response to FGF-2 binding. Thus, in the present study, we used mathematical modeling to examine whether the kinetics of formation of the various FGF-2 bound complexes on the cell surface correlate with the activation of the downstream mediators of FGF-2 response, Erk1/2. We find that FGF-2 binding to its receptor correlates well with Erk1/2 activation and that HSPG can modulate this response through its ability to stabilize these ligand receptor complexes. Moreover, we also observed that FGF-2 binding to HSPG correlates strongly with Erk1/2 activation under conditions where there is a loss of receptor activity, and we demonstrate that the relative amounts of signaling and non-signaling HSPG on the cell surface, as well as the presence of competing HSPG in the ECM, can impact the signal potential via this pathway. Thus, the selective regulation of specific HSPG might provide a mechanism for fine tuned modulation of heparin-binding growth factor signaling in cells where signal intensity and duration could direct cellular response toward growth, migration or differentiation.     

A spoonful of sugar makes the melanoma go: the role of heparan sulfate proteoglycans in melanoma metastasis

Heparan sulfate proteoglycans (HSPGs) have been shown to regulate signaling in many systems and are of increasing interest in cancer. While these are not the only sugars to drive melanoma metastasis, HSPGs play important roles in driving metastatic signaling cascades in melanoma. The ability of these proteins to modulate ligand-receptor interactions in melanoma has been quite understudied. Recent data from several groups indicate the importance of these ligands in modulating key signaling pathways including Wnt and fibroblast growth factor (FGF) signaling. In this review, we summarize the current knowledge regarding the structure and function of these proteoglycans and their role in melanoma. Understanding how HSPGs modulate signaling in melanoma could lead to new therapeutic approaches via the dampening or heightening of key signaling pathways.     

人主动脉壁硫酸乙酰肝素蛋白聚糖对培养的人主动脉平滑机细胞合成蛋白聚糖的影响

从人正常胸主动脉分离硫酸乙酰肝素蛋白聚糖（ＨＳＰＧ）,观察其对体外培养的人主动脉平滑肌细胞（ＨＡＳＭＣ）合成ＰＧ的影响。ＨＡＳＭＣ在不加（对照）或加ＨＳＰＧ（１９μｇ醛酸／ｍｌ）的￣（３５）Ｓ－硫酸钠培养液中培养,以标记ＰＧ。继之,培养液及细胞层的４ｍｏｌ／Ｌ盐酸胍提取液中的ＰＧｓ经离子交换及凝胶过滤柱层析分离,发现加ＨＳＰＧ后,培养液中的ＨＳＰＧ,硫酸软骨素ＰＧ（ＣＳＰＧ）及硫酸皮肤素－硫酸软骨素ＰＧ（ＤＳＣＳＰＧ）均明显增高,而细胞层中仅ＨＳＰＧ和ＣＳＰＧ增高,且加ＨＳＰＧ后细胞层的ＤＳＣＳＰＧ分子大小有所不同,进一步分析ＤＳＣＳＰＧ中ＤＳ及ＣＳ含量发现加ＨＳＰＧ组ＨＡＳＭＣ细胞层中的ＤＳ％含量略低于对照组。结果提示ＨＳＰＧ可刺激ＨＡＳＭＣ的ＰＧ合成,其可能与血管壁修复及动脉壁脂质沉积有关。     

Basement membrane and interstitial proteoglycans produced by MDCK cells correspond to those expressed in the kidney cortex

Multiple proteoglycans (PGs) are present in all basement membranes (BM) and may contribute to their structure and function, but their effects on cell behavior are not well understood. Their postulated functions include: a structural role in maintaining tissue histoarchitecture, or aid in selective filtration processes; sequestration of growth factors; and regulation of cellular differentiation. Furthermore, expression PGs has been found to vary in several disease states. In order to elucidate the role of PGs in the BM, a well-characterized model of polarized epithelium, Madin-Darby canine kidney (MDCK) cells has been utilized. Proteoglycans were prepared from conditioned medium by DEAE anion exchange chromatography. The eluted PGs were treated with heparitinase or chondroitinase ABC (cABC), separately or combined, followed by SDS-PAGE. Western blot analysis, using antibodies specific for various PG core proteins or CS stubs generated by cABC treatment, revealed that both basement membrane and interstitial PGs are secreted by MDCK cells. HSPGs expressed by MDCK cells are perlecan, agrin, and collagen XVIII. Various CSPG core proteins are made by MDCK cells and have been identified as biglycan, bamacan, and versican (PG-M). These PGs are also associated with mammalian kidney tubules in vivo.     

Heparan sulphate and the binding of lipoprotein lipase to porcine thoracic aorta endothelium

Purified bovine milk lipoprotein lipase was shown to bind to intact porcine aortic endothelium in a specific, saturable fashion. The binding was reversed by exogenous heparin. A single class of binding sites was involved and at saturation 1.24?10¹¹ molecules of lipoprotein lipase / cm² were bound. This represents 0.51?10⁶ enzyme molecules per endothelial cell at a density of 1.2?10³ molecules / μm². The enzyme binding was reduced by prior trypsinisation of the endothelial surface under conditions that removed cell surface glycosaminoglycan chains. The porcine endothelium was shown to have available at its surface 5.4?10¹¹ chains of heparan sulphate plus heparin-like glycosaminoglycans / cm². Such as excess suggests that lipoprotein lipase may interact with approximately one in four of the available heparan sulphate chains.     

The structural characterization of proteoglycans of culture aortic smooth muscle cells and arterial wall of the pig

Aortic proteoglycans, from the growth medium of cultured smooth muscle cells and from sequential associative and dissociative extracts of the tissue of origin, the pig aorta, were isolated and purified by precipitation with cetylpiridinium chloride. After isopycnic CsCl gradient centrifugation under associative conditions 94% of the cell-secreted proteoglycans were recuperated in the bottom one fifth (?_av = 1.62 g/ml) fraction. In contrast 80% of the tissue proteoglycans of both extracts, fractionated into two fractions: the bottom one fifth (?_av = 1.60 g/ml) fraction and three fifths (?_av = 1.42 g/ml) fraction. Fractionated tissue proteoglycans were composed predominantly of chondroitin sulfate-dermatan sulfate (83–90%) with lower proportions of heparan sulfate (5–11%) and hyaluronic acid (3–6%) whilst cell-secreted proteoglycans showed a similar glycosaminoglycan composition but with a higher proportion of hyaluronic acid (11–13%). Sepharose 2B and C1-2B chromatography of tissue proteoglycans of high buoyant density showed the presence of only subunit proteoglycans whilst those of intermediate density contained a complex species, partially dissociable in 4 M guanidinium chloride, along with K_av 0.50 subunit species. The latter was also observed for cell-secreted proteoglycans although obtained at high buoyant density. The cell-secreted subunit proteoglycans became separated into two distinct populations when chromatographed on Sepharose 4B and C1-4B, half of which eluted in the column V_o and the rest at a K_av of 0.34.. The majority of subunit macromolecules eluted at the V_o fractions of Sepharose 6B and C1-6B columns. Unlike the major species of cartilage proteoglycans, only approx. 20% of purified arterial proteoglycans formed complexes. This proportion could be increased by only 4–7% by interaction, of a mixture of subunit cell-secreted and tissue-extracted proteoglycans, with hyaluronic acid. These results suggest that proteoglycans secreted by cultured aortic smooth muscle cells and present in the aortic tissue possess certain similar physicochemical properties and are present in the form of complex and several subunit species.     

Bone marrow stromal proteoglycans regulate megakaryocytic differentiation of human progenitor cells

Adherence of hematopoietic progenitor cells (HPCs) to stroma is an important regulatory step in megakaryocytic differentiation. However, the mechanisms through which megakaryocytic progenitors are inhibited by stroma are poorly understood. We examined the role of sulfated glycoconjugates, such as proteoglycans (PGs), on human bone marrow stroma (hBMS). To this end, PG structure was altered by desulfation or enzymatic cleavage. PGs participated in adhesion of human HPC, as desulfation resulted in about 50% decline in adhesion to hBMS. Heparan sulfate proteoglycans (HSPGs) were found to be responsible by showing about 25% decline in adhesion after pre-incubation of HPC with heparin and about 15% decline in adhesion after enzymatic removal of HSPGs from hBMS. Furthermore, PGs were involved in binding cytokines. Both desulfation and enzymatic removal of stromal HSPGs increased release of megakaryocytopoiesis-inhibiting cytokines, that is, interleukin-8 (IL-8, 1.9-fold increase) and macrophage inflammatory protein-1alpha (MIP-1alpha, 1.4-fold increase). The megakaryocytic output of HPC grown in conditioned medium of desulfated stroma was decreased to 50% of the megakaryocytic output in CM of sulfated stroma. From these studies, it can be concluded that PGs in bone marrow, in particular HSPGs, are involved in binding HPC and megakaryocytopoiesis-inhibiting cytokines. Bone marrow stromal PGs thus reduce differentiation of HPC toward megakaryocytes.