Structure of a heparin-linked biologically active dimer of fibroblast growth factor

The fibroblast growth factors (FGFs) form a large family of structurally related, multifunctional proteins that regulate various biological responses. They mediate cellular functions by binding to transmembrane FGF receptors, which are protein tyrosine kinases. FGF receptors are activated by oligomerization, and both this activation and FGF-stimulated biological responses require heparin-like molecules as well as FGF. Heparins are linear anionic polysaccharide chains; they are typically heterogeneously sulphated on alternating L-iduronic and D-glucosamino sugars, and are nearly ubiquitous in animal tissues as heparan sulphate proteoglycans on cell surfaces and in the extracellular matrix. Although several crystal structures have been described for FGF molecules in complexes with heparin-like sugars, the nature of a biologically active complex has been unknown until now. Here we describe the X-ray crystal structure, at 2.9 A resolution, of a biologically active dimer of human acidic FGF in a complex with a fully sulphated, homogeneous heparin decassacharide. The dimerization of heparin-linked acidic FGF observed here is an elegant mechanism for the modulation of signalling through combinatorial homodimerization and heterodimerization of the 12 known members of the FGF family.     

High-performance liquid chromatographic separation and electrochemical detection of cephalosporins

It was found that both normal human myometrium and uterine leiomyoma contain several glycosaminoglycans. In contrast to many normal and tumour tissues the amount of hyaluronic acid is very low and the proportional amount of sulphated glycosaminoglycans is distinctly higher. It is of interest that heparan sulphate is the major glycosaminoglycan component both in normal myometrium, and in leiomyoma. The amount of hyaluronic acid in myometrium and in the leiomyoma is very low. No significant change in hyaluronate content was observed during the tumour growth. In contrast to that the amount of some sulphated glycosaminoglycans (heparan sulphate, keratan sulphate, chondroitin sulphates and heparin) distinctly increased. It is suggested that some of the GAGs participate in the creation of a storage depot for biologically active molecules (growth factors, enzymes) which are thereby stabilized and protected. Hydrolytic degradation of some GAGs may result in the release of some cytokines which may promote the tumour growth and stimulate collagen biosynthesis by tumour cells.     

Age-dependent modulation of heparan sulfate structure and function

Heparan sulfate interacts with growth factors, matrix components, effectors and modulators of enzymatic catalysis as well as with microbial proteins via sulfated oligosaccharide domains. Although a number of such domains have been characterized, little is known about the regulation of their formation in vivo. Here we show that the structure of human aorta heparan sulfate is gradually modulated during aging in a manner that gives rise to markedly enhanced binding to isoforms of platelet-derived growth factor A and B chains containing polybasic cell retention sequences. By contrast, the binding to fibroblast growth factor 2 is affected to a much lesser extent. The enhanced binding of aorta heparan sulfate to platelet-derived growth factor is suggested to be due to an age-dependent increase of GlcN 6-O-sulfation, resulting in increased abundance of the trisulfated L-iduronic acid (2-OSO3)-GlcNSO3(6-OSO3) disaccharide unit. Such units have been shown to hallmark the platelet-derived growth factor A chain-binding site in heparan sulfate.     

Empirically supported treatments for children with phobic and anxiety disorders: current status

Although interactions of proteins with glycosaminoglycans (GAGs), such as heparin and heparan sulphate, are of great biological importance, structural requirements for protein-GAG binding have not been well-characterised. Ionic interactions are important in promoting protein-GAG binding. Polyelectrolyte theory suggests that much of the free energy of binding comes from entropically favourable release of cations from GAG chains. Despite their identical charges, arginine residues bind more tightly to GAGs than lysine residues. The spacing of these residues may determine protein-GAG affinity and specificity. Consensus sequences such as XBBBXXBX, XBBXBX and a critical 20 A spacing of basic residues are found in some protein sites that bind GAG. A new consensus sequence TXXBXXTBXXXTBB is described, where turns bring basic interacting amino acid residues into proximity. Clearly, protein-GAG interactions play a prominent role in cell-cell interaction and cell growth. Pathogens including virus particles might target GAG-binding sites in envelope proteins leading to infection.     

The role of stromal cell heparan sulphate in regulating haemopoiesis

Intimate contact between haemopoietic progenitor cells and elements of the bone marrow stroma is required for progenitor cell proliferation and differentiation. It is believed that the stroma provides particular niches for the development of haemopoietic cells of different lineages. Cytokines, stromal cell surface molecules and molecules of the stromal extracellular matrix all contribute to defining these microenvironmental niches. Data obtained using an in vitro model of haemopoiesis support the view that progenitor cell adhesion to stroma is mediated by multiple receptor-ligand interactions. The possibility of a tethering step, mediated by the engagement of stromal cell heparan sulphate with its ligands on the progenitor cells, preceding stable cell adhesion is discussed. The role of stromal cell heparan sulphate is likely to include cytokine presentation to progenitors as well as the tethering of progenitors to stroma. It is proposed that intracellular signals induced by progenitor cell adhesion to stroma act in association with cytokine induced signals to regulate progenitor cell proliferation and differentiation.     

Heparan sulfate upregulates platelet-derived growth factor receptors on human lung fibroblasts

Heparan sulfate is a molecule that possesses a large structural variability and which has been shown to inhibit the proliferation of fibroblasts in vitro. The aim of this study was to determine whether the anti-proliferative effects of heparan sulfate were exerted by regulation of the activity of the platelet-derived growth factor and/or of the platelet-derived growth factor receptors. Both l-iduronate-rich, anti-proliferative and the l-iduronate-poor, non-anti-proliferative heparan sulfate species, were incubated with confluent human embryonic lung fibroblasts for 24 h. The mRNA levels for PDGF-AA, PDGF-BB, and their receptors were measured. Binding studies were performed with [125I]-PDGF-BB and [125I]-EGF for 2 h at 4 degreesC in cultures preincubated with both types of heparan sulfate for 24 h. In separate experiments, cultures were incubated together with heparan sulfate and [125I]-PDGF-BB for 2 h at 4 degreesC. Increases of two- to threefold in the mRNA levels for both the alpha- and the beta-receptors of PDGF was obtained after treatment with both types of heparan sulfate, whereas the mRNA levels of both the PDGF-AA and the PDGF-BB were essentially unaffected. A sixfold increase in binding was only noted for [125I]-PDGF-BB in cultures pre-treated with the anti-proliferative heparan sulfate for 24 h, whereas no effect was noted with use of the non-anti-proliferative heparan sulfate. Incubating the [125I]-PDGF-BB and the anti-proliferative heparan sulfate together for 2 h resulted in a smaller, threefold increase in binding. This indicates that the anti-proliferative heparan sulfate both stabilizes and increases expression of the PDGF receptors. To investigate whether the increased number of PDGF receptors could affect cell activity, cells were preincubated with anti-proliferative heparan sulfate and then treated with PDGF-BB. This resulted in an increase in mitogenicity compared to cells treated only with PDGF-BB. Neither an increase in binding for [125I-EGF] nor an increase in the mitogenic response of EGF could be observed in cultures pre-treated with the anti-proliferative heparan sulfate. The results indicate that the extracellular matrix itself may regulate important biological phenomena such as cell proliferation and matrix production through affecting the expression of receptors of PDGF, which initiate both stimulatory and inhibitory signals.     

Formation of promutagenic methylation damage in tissue-DNA of mice treated with antischistosomal agents

BACKGROUND: Cultured bovine corneal endothelial cells (CEC) synthesize heparan sulfate and dermatan sulfate containing proteoglycans and distribute them between different compartments. METHODS AND RESULTS: [35S]sulfate labelled proteoglycans are found associated with the cell layer, secreted into the culture medium and deposited into the underlaying extracellular matrix. In the presence of basic fibroblast growth factor (bFGF)-a strong mitogen for CEC-subconfluent cells incorporate [35S]sulfate into the sulfated proteoglycans at a rate three times higher as compared with the proteoglycans of CEC in the absence of bFGF. The enhanced proteoglycan synthesis is accompanied with a shift in the proteoglycan distribution pattern. While in control cells the cell-associated heparan sulfate accounts for about 30% of the total glycosaminoglycans under the influence of bFGF the HS percentage increases to approximately 60%. CONCLUSIONS: CEC synthesize and deposit endogenous bFGF into the extracellular matrix. Heparitinase treatment of the extracellular matrix releases bFGF activity which is able to stimulate the 35S incorporation into proteoglycans in a comparable manner as exogenous bFGF but does not influence the proteoglycan distribution pattern. Pretreatment of the matrix-bound bFGF activity with polyclonal antibodies against bFGF abolishes its stimulating activity.     

Quantitative assay system for specific enzyme activity using antibody: the case of protease, subtilisin BPN''

Syndecan-1 is a cell surface heparan sulphate proteoglycan, which binds to the extracellular matrix (ECM), growth factors and antithrombin III. The early expression of syndecan-1 during mouse embryonic development suggests a potential role in the communication between the embryo and the ECM of decidua. Using immunohistochemical methods, the present study showed that the expression of syndecan-1 in the trophoblast cells changes along trophoblast differentiation. The syncytiotrophoblasts in the chorionic villi exhibited an apical expression of syndecan-1. This suggests that the expression is restricted to non-migrating, non-proliferating trophoblasts. The mode of syndecan-1 expression by human placental trophoblasts is independent of gestational age. The expression is not changed in miscarriages. In pre-eclampsia, the staining for syndecan-1 on the villous syncytiotrophoblast is weaker compared to normal pregnancy, but in placental bed the expression is similar. The unique apical localization of syndecan-1 in chorionic villi, not detected in any other tissues, suggests a potential role in fetomaternal communication probably via growth factor binding and in anticoagulation of intervillous circulation.     

Multiple heparan sulfate chains are required for optimal syndecan-1 function

Syndecans have three highly conserved sites available for heparan sulfate attachment. To determine if all three sites are required for normal function, a series of mutated syndecans having two, one, or no heparan sulfate chains were expressed in ARH-77 cells. Previously, we demonstrated that expression of wild-type syndecan-1 on these myeloma cells mediates cell-matrix and cell-cell adhesion and inhibits cell invasion into collagen gels. Here we show that to optimally mediate each of these activities, all three sites of heparan sulfate attachment are required. Generally, an increasing loss of syndecan-1 function occurs as the number of heparan sulfate attachment sites decreases. This loss of function is not the result of a decrease in either the total amount of cell surface heparan sulfate or syndecan-1 core protein. In regard to cell invasion, cells expressing syndecan-1 bearing a single heparan sulfate attachment site exhibit a hierarchy of function based upon the position of the site within the core protein; the presence of an available attachment site at serine 47 confers the greatest level of activity, while serine 37 contributes little to syndecan-1 function. However, when all three heparan sulfate chains are present, significantly greater biological activity is observed than is predicted by the sum of the activities occurring when the chains act individually. This synergy provides a functional basis for the evolutionary conservation of the three heparan sulfate attachment sites on syndecans and supports the idea that molecular heterogeneity, which is characteristic of proteoglycans, contributes to their functional diversity.     

UV-B radiometry and dosimetry for solar measurements

Glycosaminoglycan administration has favourable effects on morphological and functional renal abnormalities in different models. The possibility that exogenous glycosaminoglycans modulate glomerular matrix synthesis was explored in both primary and SV40-MES13 murine mesangial cell cultures. On both cell types, both low-molecular-weight heparin and different glycosaminoglycans showed dose-dependent inhibition of proliferation and increase of 35SO4(2)-uptake. After 36 h the cell compartment contained a spectrum of 35S-molecules of less than 200 kDa; under heparin treatment, the two main 35SO4(2)-components (high and medium MW) increased by 16 and 37% respectively. Susceptibility to glycosidases revealed that heparin promotes the expression of heparan sulphate and increases that of chondroitin sulphate. Moreover, heparin modifies the expression of decorin and biglycan, involved in adhesion and fibrillogenesis, while not affecting perlecan. The extracellular matrix modulation in renal cells, for which the sulphation type and ratio of heparin are crucial, may thus explain the beneficial renal effects of heparin.     

Glycosaminoglycans can modulate extracellular localization of the wingless protein and promote signal transduction

Wingless, the Drosophila homologue of the proto-oncogene Wnt-1, encodes a secreted glycoprotein that regulates differentiation and proliferation of nearby cells. Here we report on the biochemical mechanism(s) by which the wingless signal is transmitted from cell to cell. When expressed in S2 cells, the majority (approximately 83%) of secreted wingless protein (WG) is bound to the cell surface and extracellular matrix through specific, noncovalent interactions. The tethered WG can be released by addition of exogenous heparan sulfate and chondroitin sulfate glycosaminoglycans. WG also binds directly to heparin agarose beads with high affinity. These data suggest that WG can bind to the cell surface via naturally occurring sulfated proteoglycans. Two lines of evidence indicate that extracellular glycosaminoglycans on the receiving cells also play a functional role in WG signaling. First, treatment of WG-responsive cells with glycosaminoglycan lyases reduced WG activity by 50%. Second, when WG-responsive cells were preincubated with 1 mM chlorate, which blocks sulfation, WG activity was inhibited to near-basal levels. Addition of exogenous heparin to the chlorate-treated cells was able to restore WG activity. Based on these results, we propose that WG belongs to the group of growth factor ligands whose actions are mediated by extracellular proteoglycan molecules.     

Growth factors and biological supports for endothelial cell lining: in vitro study

Endothelial cell covering over the vascular prosthesis luminal surface is a process that may require the presence of growth factors (GFs) and extracellular matrix supports. Endothelialization could be improved by combining both GFs and an extracellular matrix analog. In the present study, different biological substrates made of type I or IV collagens, gelatin, fibronectin, fibrin, laminin, chondroitin sulphate, heparan sulphate, heparin or hyaluronic acid were used to support endothelial cell culture. An endothelial cell growth supplement (ECGS) was incorporated in (group 1) or overlaid on (group 2) the substrates; or present in medium (group 3); or absent (group 4). GF binding assay using 125I bFGF showed that more GF remained combined to the substrates in group 2 than those in group 1. Growth and morphology of human umbilical vein endothelial cells were sequentially analyzed in vitro for 8 days using DNA (nuclei counts) and F-actin labelings. Growth was relatively stable for the first 48 hours, later in groups 1, 2 and 4, cell death was observed on all the substrates except for fibronectin. Growth failure could be related to the degradation or inefficient release of ECGS. In group 3, growth increased and confluency was reached within 5-8 days on all the substrates except for gelatin and type I collagen. Confluent cells containing actin filaments were organized on glycoproteins and disorganized on glycosaminoglycans and fibrin. Despite that glycoproteins can enhance cell adhesion and lining pattern, GFs continually delivered in a fresh soluble form seem to be the appropriate condition to obtain an endothelial cell lining.     

Crystal structure of the angiogenesis inhibitor endostatin at 1.5 A resolution

A number of extracellular proteins contain cryptic inhibitors of angiogenesis. Endostatin is a 20 kDa C-terminal proteolytic fragment of collagen XVIII that potently inhibits endothelial cell proliferation and angiogenesis. Therapy of experimental cancer with endostatin leads to tumour dormancy and does not induce resistance. We have expressed recombinant mouse endostatin and determined its crystal structure at 1.5 A resolution. The structure reveals a compact fold distantly related to the C-type lectin carbohydrate recognition domain and the hyaluronan-binding Link module. The high affinity of endostatin for heparin is explained by the presence of an extensive basic patch formed by 11 arginine residues. Endostatin may inhibit angiogenesis by binding to the heparan sulphate proteoglycans involved in growth factor signalling.     

Pseudobacteriuria associated with a urine flow rate transducer

Normal wound healing in skin embraces several reparative processes, many of which directly involve components of the extracellular matrix and the cutaneous basement membrane zone. Proteoglycans are a group of extracellular matrix macromolecules that have both structural and regulatory properties. In wound healing, certain proteoglycans fulfill a mechanical function of absorbing water and preventing tissue compression. However, proteoglycans may also have other roles in wound healing including a direct influence on inflammation, cell attachment and migration, and growth factor binding. Furthermore, proteoglycans may help to determine other aspects of the long-term quality of wound healing in skin through regulation of basement membrane permeability, epidermal hyperproliferation, and dermal fibrosis.     

Case 1--1993. Emergency use of cardiopulmonary bypass for resuscitation from CPR-resistant cardiac arrest

Basement membrane-associated heparan sulphate proteoglycans have been demonstrated immunohistochemically in organs from patients afflicted with various types of amyloidosis. In a recent report, we were able to isolate and partly characterize a basement membrane-associated heparin sulphate proteoglycan from human hepatic amyloid. In the present study proteoglycans were extracted with guanidine from human amyloid-laden kidney, spleen and lymph nodes. All tissues extracted with guanidine contained both heparan sulphate proteoglycan (HSPG) and galactosaminoglycan (CS/DS) free chains. Tissue staining using a monoclonal antibody against basement membrane HSPG revealed the presence of HSPG in amyloid deposits in kidney and spleen. Furthermore, following SDS-PAGE of HSPG from kidney after deaminative cleavage of the HS chains, a 15-kDa and 80-kDa protein appeared, probably representing the core protein(s). In lymph node HSPG, three core proteins of 65, 30 and 25 kDa could be demonstrated on SDS-PAGE, the first reacting with the anti-basement membrane HSPG antibody when subjected to Western blotting subsequent to SDS-PAGE. By immunohistochemistry, we failed to demonstrate any staining of the renal and splenic tissue sections employing an antibody against the decorin core protein.     

Physical activity before and after exercise in women with chronic fatigue syndrome

Placenta growth factor (PlGF) is a homodimeric glycoprotein, 46-50 kDa in size, belonging to the vascular endothelial growth factor (VEGF) sub-family. It exists as two isoforms, PlGF-1 and -2, the latter having a heparin-binding domain. Like VEGF, it is a potent angiogenic factor; however, PlGF homodimers interact with the VEGF receptor Flt-1 (fms-like tyrosine kinase), but not with the kinase domain-containing region (KDR). Since PlGF is made by the human placenta and extravillous trophoblast (EV-T) cells of the human placenta express Flt-1 in situ, these cells may be responsive to PlGF. Therefore, this study examined whether first trimester EVT cells propagated in vitro expressed the mRNA or the protein of Flt-1 and PlGF, and whether exogenous PlGF-1 had any effect on EVT cell proliferation, migration or invasiveness. Immunocytochemical and RT-PCR analyses revealed that both normal and SV40 Tag-immortalized EVT cells expressed the protein and mRNA for Flt-1, but not for PlGF-1 or -2. Exogenous PlGF-1 stimulated proliferation (measured by 3H-thymidine uptake) of normal EVT cells in a concentration-dependent manner, but only in the presence of excess heparan sulphate proteoglycans (HSPGs). These results raise two possibilities: that exogenous PlGF-1 (in spite of having a low affinity for heparin) was sequestered away from its receptor because of binding to heparan sulphate proteoglycans on the EVT cell surface or the ECM, or that HSPGs could modify the interaction between Flt-1 and PlGF. PlGF-1, in the presence or absence of HSPGs, however, had no effect on EVT migration or invasiveness, when measured with a transwell invasion (in the presence of Matrigel) or migration (in the absence of Matrigel) assay. These findings place PlGF amongst a large group of growth factors that promote EVT cell proliferation without influencing their migratory or invasive behaviours, and suggest that PlGF-Flt-1 interactions may be regulated by HSPGs in situ.     

Molecular cloning of a lobster Gbeta subunit and Gbeta expression in olfactory receptor neuron dendrites and brain neuropil

BACKGROUND: Basic fibroblast growth factor (bFGF) promotes angiogenesis and healing of gastric ulcers in rats, and bFGF expression is up regulated in such ulcers. However, little is known about expression of bFGF in human gastric mucosa. AIMS: To investigate bFGF expression in intact human gastric mucosa and gastric ulcers and to determine whether low bFGF content or altered binding by mucosa is associated with ulceration. SUBJECTS: Endoscopy outpatients, gastrectomy patients, and organ donors. METHODS: bFGF was isolated by heparin affinity chromatography and characterised by western blotting and endothelial cell bioassay. bFGF was measured by immunoassay and its distribution defined by immunohistochemistry and in situ hybridisation. Binding of bFGF by heparan sulphate proteoglycans was investigated by sodium chloride and heparin extraction. RESULTS: Bioactive bFGF (19 kDa) was detected in normal mucosa but bFGF mRNA was not found. bFGF expression was up regulated in granulation tissue endothelial cells, mononuclear cells, and epithelial cells at the ulcer rim. Gastric ulcer patients had constitutively low bFGF concentrations in intact antral mucosa which were not explained by changes in binding to heparan sulphate proteoglycans. CONCLUSIONS: bFGF expression is up regulated in human gastric ulcers. Low intact mucosal bFGF content is associated with gastric ulceration.