Chondroitin sulfates and their binding molecules in the central nervous system.

Chondroitin sulfate (CS) is the most abundant glycosaminoglycan (GAG) in the central nervous system (CNS) matrix. Its sulfation and epimerization patterns give rise to different forms of CS, which enables it to interact specifically and with a significant affinity with various signalling molecules in the matrix including growth factors, receptors and guidance molecules. These interactions control numerous biological and pathological processes, during development and in adulthood. In this review, we describe the specific interactions of different families of proteins involved in various physiological and cognitive mechanisms with CSs in CNS matrix. A better understanding of these interactions could promote a development of inhibitors to treat neurodegenerative diseases.

Desorption electrospray ionization mass spectrometry of glycosaminoglycans and their protein noncovalent complex.

Glycosaminoglycans heparin and heparan sulfate are biologically active polysulfated carbohydrates that are among the most challenging biopolymers with regards to their structural analysis and functional assessment. Fragmentation of oligosaccharides and sulfate loss are important hindrance to their analysis by mass spectrometry (MS), requiring thus soft ionization methods. The recently introduced soft ionization method desorption electrospray ionization (DESI) has been applied here to heparin and heparan sulfate oligosaccharides, showing that DESI-MS is well suited for the detection of such fragile biomolecules in their intact form. Characterization of complicated oligosaccharides such as synthetic heparin octadecasulfated dodecasaccharide was successfully achieved. The use of water for a spray solvent instead of denaturing organic solvents allowed the first DESI-MS detection of noncovalent biomolecular complexes between heparin oligosaccharides and the chemokine Stromal Cell-derived Factor-1. The hyphenation of the DESI ion source with the high-resolution LTQ-Orbitrap MS analyzer led to high accuracy of mass measurement and enabled unambiguous determination of the protein-bound sulfated oligosaccharide.

The adenovirus type 3 dodecahedron's RGD loop comprises an HSPG binding site that influences integrin binding.

Human type 3 adenovirus dodecahedron (a virus like particle made of twelve penton bases) features the ability to enter cells through Heparan Sulphate Proteoglycans (HSPGs) and integrins interaction and is used as a versatile vector to deliver DNA or proteins. Cryo-EM reconstruction of the pseudoviral particle with Heparan Sulphate (HS) oligosaccharide shows an extradensity on the RGD loop. A set of mutants was designed to study the respective roles of the RGD sequence (RGE mutant) and of a basic sequence located just downstream. Results showed that the RGE mutant binding to the HS deficient CHO-2241 cells was abolished and unexpectedly, mutation of the basic sequence (KQKR to AQAS) dramatically decreased integrin recognition by the viral pseudoparticle. This basic sequence is thus involved in integrin docking, showing a close interplay between HSPGs and integrin receptors.

Endocan or endothelial cell specific molecule-1 (ESM-1): a potential novel endothelial cell marker and a new target for cancer therapy.

Endocan, previously called endothelial cell specific molecule-1, is a soluble proteoglycan of 50 kDa, constituted of a mature polypeptide of 165 amino acids and a single dermatan sulphate chain covalently linked to the serine residue at position 137. This dermatan sulphate proteoglycan, which is expressed by the vascular endothelium, has been found freely circulating in the bloodstream of healthy subjects. Experimental evidence is accumulating that implicates endocan as a key player in the regulation of major processes such as cell adhesion, in inflammatory disorders and tumor progression. Inflammatory cytokines such as TNF-alpha, and pro-angiogenic growth factors such as VEGF, FGF-2 and HGF/SF, strongly increased the expression, synthesis or the secretion of endocan by human endothelial cells. Endocan is clearly overexpressed in human tumors, with elevated serum levels being observed in late-stage lung cancer patients, as measured by enzyme-linked immunoassay, and with its overexpression in experimental tumors being evident by immunohistochemistry. Recently, the mRNA levels of endocan have also been recognized as being one of the most significant molecular signatures of a bad prognosis in several types of cancer including lung cancer. Overexpression of this dermatan sulphate proteoglycan has also been shown to be directly involved in tumor progression as observed in mouse models of human tumor xenografts. Collectively, these results suggest that endocan could be a biomarker for both inflammatory disorders and tumor progression as well as a validated therapeutic target in cancer. On the basis of the recent successes of immunotherapeutic approaches in cancer, the preclinical data on endocan suggests that an antibody raised against the protein core of endocan could be a promising cancer therapy.

High-affinity binding of interferon-gamma to a basement membrane complex (matrigel).

Recently it was demonstrated that growth factors are bound to the extracellular matrix, and can regulate cell behavior. Using three different types of binding assays, we have examined the interaction of interferon-gamma with a basement membrane produced by the Engelbreth-Holm-Swarm tumor. Basement membrane was found to bind interferon-gamma in both a time- and concentration-dependent manner. Equilibrium binding analysis revealed a high-affinity site with a dissociation constant of 1.5 10(-9) M and a maximum binding capacity of 1.6 10(9) sites/mm2 of basement membrane. Competition studies show that the binding is inhibited by heparan sulfate, suggesting that basement membrane-heparan sulfate proteoglycan could be the binding site. This interaction was clearly confirmed by native polyacrylamide gel electrophoresis and dot-blot analysis with purified basement membrane molecules. Furthermore, the carboxy-terminal part of the interferon-gamma molecule contains an amino acid cluster, very closely related to a consensus sequence, present in more than 20 proteins known to bind sulfated glycosaminoglycans such as heparin. These data demonstrate a possible role of extracellular matrix components in storing cytokines and in modulating the cellular response to such factors.

Binding of interferon-gamma to heparan sulfate is restricted to the heparin-like domains and involves carboxylic--but not N-sulfated--groups.

Interferon-gamma binds to the glycosaminoglycan part of basement membrane proteoglycan. To obtain a greater insight into this interaction, different glycosaminoglycans and their subfractions were used in various binding assays. High affinity binding occurs with heparin and heparan sulfate only, the latter being the predominant basement membrane glycosaminoglycan. Furthermore, using heparan sulfate and heparin treated with heparinases I and III, we have shown that the interferon-gamma binding sites are localized on the N-sulfated glucosamine rich domains of the molecule. Interestingly, interferon-gamma and fibroblast growth factor compete for the same binding domain on heparan sulfate, although they are unrelated proteins. This last point is discussed in the light of the conformational flexibility of the glycosaminoglycan molecules.

[Virus and heparan sulphate: from adsorption mechanism to cellular entry]. / Virus et héparane sulfate : des mécanismes d'adsorption cellulaire à l'entrée virale.

Heparan sulphates are complex polysaccharides that belong to a class of molecules called glycosaminoglycans. Linked to different core proteins, they are ubiquitously expressed at most cells' surface. These molecules interact with a huge number of distinct proteins and regulate their biological activities. In particular different viruses make use of heparan sulphate interactive properties to dock themselves at the surface of their cellular targets. This interaction enables the viruses to concentrate at the close proximity of others molecules that act as co-receptors, and as such increases viral entry. Recent progresses in the structural characterisation of glycosaminoglycans have helped to understand the relationship between the structure of these molecules and their ability to recognise viral capside or envelope glycoproteins. These works also showed the direct role of these molecules in viral tropism and mechanism of entry, and suggest medical applications as biotechnological strategies.

Interferon-gamma binds to heparan sulfate by a cluster of amino acids located in the C-terminal part of the molecule.

Using three different approaches (domain mapping with monoclonal antibodies, limited enzymatic digestion and competition with synthetic peptides), we demonstrated that a cluster of basic amino acids on interferon-gamma is involved in its binding to heparan sulfate. This cluster (Lys125-Arg131) is localized in the C-terminal part of IFN-gamma. Once bound to heparin sulfate, IFN-gamma is protected against protease attack.

Interferon-gamma inhibits 35S incorporation in heparan sulfate synthesized by human skin fibroblasts.

Glycosaminoglycans synthesized by human skin fibroblasts were simultaneously radiolabelled with D-[1-(3H)]glucosamine and Na2(35)SO4. Considering 3H incorporation, we found that IFNgamma increased the production of glycosaminoglycan synthesis, including hyaluronic acid, heparan and chondroitin/dermatan sulfate. In contrast, the production of heparan and chondroitin/dermatan sulfate was slightly decreased on the basis of the 35S signal. Furthermore, when heparan sulfate was treated with nitrous acid, the release of free 35S was greater in control than in treated cells, although the 3H patterns of depolymerization with this agent were similar. These data demonstrate that IFNgamma inhibits the incorporation of sulfate from extracellular medium into heparan sulfate.

Matrix receptors to cytokines: from concept to control of tissue fibrosis dynamics.

Fibrous tissue formation is one of the main elements of tissue repair and maintains the organ functional integrity after any aggression. When this connective tissue excessively accumulates, leading to tissue fibrosis, it induces serious impairment in the considered organ. The extracellular matrix--whose structure displays a great polymorphism--binds various active molecules (such as FGF, TGF beta, IFN gamma ...) and therefore locally regulates cellular activities. The latest studies both on fibrosis and on matrix receptors to cytokines have led to the idea of a matricial specificity to the cell's close surrounding.

Interferon-gamma, an anti-fibrogenic cytokine which binds to heparan sulfate.

Interferon-gamma is a T cell secreted cytokine. It has a great number of biological activities, among which is the down regulation of collagen molecules. IFN-gamma acts through cell surface receptors, but also binds to heparan sulfate proteoglycans. In vivo, it is postulated that the heparan sulfate/IFN-gamma interaction can modulate the activity and availability of the cytokine.

Interferon and heparan sulphate.

In 1954, substances that protected cells from viral infection were discovered and named IFN (interferon). This family of cytokines, which were the first to be used in clinical therapy, is classified into type I and II IFNs. Type I mainly consists of IFNalpha and IFNbeta subtypes, which are structurally related and bind to a common receptor. IFNgamma, the sole type II IFN, is structurally unrelated, binds to a different receptor and, as a dimer, strongly interacts with HS (heparan sulphate). In addition to its antiviral activity, it modulates nearly all phases of immune and inflammatory responses. IFNgamma binding to HS controls the blood clearance, the subsequent tissue targeting and the local accumulation of the cytokine. It also regulates IFNgamma activity by a unique mechanism involving a controlled processing of the C-terminal peptide. The binding site encompasses an N-acetylated glucosamine-rich domain separating two highly sulphated sequences that each binds to one IFNgamma monomer. Based on this template, a set of glycoconjugate mimetics that would mimic the IFNgamma binding site has been synthesized. One of these molecules displays high affinity for the cytokine and inhibits binding to both HS and IFNgammaR (IFNgamma receptor), the cell-surface receptor. These results validate the HS structural determinants for IFNgamma recognition, and provide a new strategy to inhibit IFNgamma in a number of diseases in which the cytokine has been identified as a target.

[The extracellular matrix: from supporting tissue to regulation of cytokines]. / La matrice extracellulaire: du tissu de soutien à la régulation des cytokines.

Extracellular matrix is a polymorphic structure composed of at least thirty molecules (collagens, glycoproteins, elastin, and proteoglycans) associated in a complex network. This insoluble structured framework ensures tissue cohesiveness, and allows cells to adhere, migrate and interact. Together with cytokines extracellular matrix is also involved in the control of various aspects of cell activities. Cytokines mediate cell to cell communication. Their broad effect result from their pleiotropic and overlapping activities. Once secreted, these molecules diffuse between cells--i.e. across extracellular matrix--to reach their target cells. An increasing number of cytokines are now known to bind to the extracellular matrix. These interactions change the availability of cytokines on effector cells, activate some growth factors, localize and/or increase the duration of the expected effect. Matrix-cytokines interactions are a new field of investigation which has changed our view on extracellular matrix. First considered as a supporting tissue, extracellular matrix appears now to be critically involved in the regulation of cytokine activities.

Interferon-gamma C-terminal function: new working hypothesis. Heparan sulfate and heparin, new targets for IFN-gamma, protect, relax the cytokine and regulate its activity.

Structure and function of the interferon-gamma C-terminal extremity has been widely studied. A basic amino acid cluster located in this domain is involved in the tridimentional structure of the protein and is essential for the biological activity. This specific group of amino acid is also involved in the binding of interferon-gamma to basement membrane or cell surface heparan sulfate. Once bound to heparan sulfate, interferon-gamma is protected from proteolytic cleavage and it is suggested that the protein folds in a new relaxed conformation, with increased stability.

Internalization and nuclear translocation of IFN-gamma and IFN-gammaR: an ultrastructural approach.

IFN-gamma signalling involves the Jak-STAT pathway. However, several hypothesis have been proposed where the receptor and its ligand itself took an active role within the cell. Using a quantitative immunogold approach, we found that both IFN-gamma and its receptor are rapidly internalized and translocated in the nucleus. We found that cell surface heparan sulfate, which binds IFN-gamma, delayed the nuclear accumulation of IFN-gamma suggesting that these molecules serve as storage depot around the cell for local delivery of the cytokine.

Molecular organization of the interferon gamma-binding domain in heparan sulphate.

Interferon (IFN)-gamma, in common with a number of cytokines or growth factors, strongly interacts with heparan sulphate (HS). It has been shown previously that one of the C-terminal basic clusters of amino acids (a regulatory element of IFN-gamma activity) is involved in this interaction. The structural organization of the HS domain that binds to human IFN-gamma has been investigated here. IFN-gamma-affinity chromatography of HS oligosaccharides released by either enzymic or chemical cleavage showed that the binding site is not found in a domain that is resistant to either heparinase or heparitinase or exclusively N-sulphated or N-acetylated. This led us to take a 'footprinting' approach in which HS was depolymerized in the presence of IFN-gamma and the cytokine-protected sequences were separated from the digested fragments. Using this strategy we consistently isolated an IFN-gamma-protected domain (IPD; approx. 10 kDa) which displayed the same affinity as full-length HS for the cytokine. Treatment of IPD with either heparinase or heparitinase strongly reduced its affinity, confirming that the high-affinity binding site encompassed a mixture of HS structural domains. Patterns of depolymerization with either enzymic or chemical agents were consistent with IPD being composed of an extended internal domain (approx. 7 kDa) which is predominantly N-acetylated and GlcA-rich, flanked by small N-sulphated oligosaccharides (mainly hexa- to octasaccharides). This is the first description of an HS protein-binding sequence with this type of molecular organization. Furthermore, using a cross-linking strategy, we demonstrated that one HS molecule bound to an IFN-gamma dimer. Together these results lead us to propose a novel model for the interaction of HS with a protein, in which two sulphated terminal sequences of the binding domain interact directly with the two IFN-gamma C-termini and bridge the two cytokine monomers through an internal N-acetyl-rich sequence.

Interferon gamma differentially affects the synthesis of chondroitin/dermatan sulphate and heparan sulphate by human skin fibroblasts.

Interferon gamma (IFN gamma) is often considered to be an antifibrotic cytokine because it inhibits collagen synthesis in fibroblasts. Here we report the effects of recombinant human IFN gamma on sulphated glycosaminoglycan chains produced by normal skin fibroblasts from adult donors. IFN gamma (250 i.u./ml) induced an increase in incorporation of D-[1-3H]glucosamine into glycosaminoglycans, either secreted into the culture medium or associated with the cell layer. The structures of these molecules were analysed by using various cleavage agents (heparinases I and II, heparitinase/chondroitinases ABC and AC/periodate oxidation) followed by size-exclusion and anion-exchange HPLC. No modification was detected in the structure of the heparan sulphate chains. In contrast, the cytokine induced changes in the microcomposition of chondroitin/dermatan sulphate chains. More precisely, we found a decrease in the iduronic acid content, associated with down-regulation of the 4-O-sulphation on the GalNAc residues. In contrast, the 6-O-sulphation on these GalNAc residues was potentiated by the cytokine. These results indicate that IFN gamma is able to modulate not only collagen but also the structure of galactosaminoglycans synthesized by human skin fibroblasts.

Caveolae and clathrin-coated vesicles: two possible internalization pathways for IFN-gamma and IFN-gamma receptor.

Interferon-gamma (IFN-gamma) elicits a variety of activities following binding to its cell-surface-specific receptor (IFN-gammaR). This complex formation leads to the activation of the Jak-STAT pathway. Several hypotheses have been proposed to explain the role and location of the receptor and its ligand in the signalling pathway. In vivo as well as in vitro, the present study shows that IFN-gamma and its receptor were internalized in different cellular compartments including cytoplasmic matrix, mitochondria and nucleus. In order to analyse the internalization pathway of IFN-gamma and its receptor, we have study in vivo and in vitro their colocalization with clathrin and caveolin by using double immunogold-labelling experiments using electron microscopy. We demonstrate that IFN-gamma and IFN-gammaR were colocalized in the caveolin-containing structures and the clathrin-coated pits suggesting that both internalization pathways may be used. This indicates that IFN-gamma and IFN-gammaR were internalized by these two different pathways, suggesting two different intracellular routes probably for different target cell-compartments.

The heparan sulfate binding sequence of interferon-gamma increased the on rate of the interferon-gamma-interferon-gamma receptor complex formation.

Interferon-gamma (IFNgamma), in common with a number of growth factors, binds both to heparan sulfate or heparin-related molecules and to a specific high affinity receptor (IFNgammaR). Using surface plasmon resonance technology, kinetic analysis of the IFNgamma. IFNgammaR complex formation was performed with the extracellular part of IFNgammaR immobilized on a sensor chip. At the sensor chip surface, IFNgamma was bound by two IFNgammaR molecules with an affinity in the nanomolar range (0.68 nM). This binding was characterized by an important on rate, kon = 7.3 x 10(6) M-1.s-1, and an off rate, koff = 5 x 10(-3).s-1. This binding assay was used to investigate a possible role of heparin in the IFNgamma.IFNgammaR complex formation. In contrast to growth factors for which binding to heparin is usually required for high affinity receptor interaction, we found in this study that IFNgamma bound to heparin displayed a strongly reduced affinity for its receptor. This is consistent with the fact that a cluster of basic amino acids (KTGKRKR, called the C1 domain) in the carboxyl-terminal sequence of the cytokine was involved both in heparin and receptor recognition. To understand how a single domain of IFNgamma could be implicated in two discrete functions (i.e. binding to heparin and to IFNgammaR), we also analyzed in a detailed manner the role of the IFNgamma carboxyl-terminal sequence in receptor binding. Using forms of IFNgamma, with carboxyl terminus truncations of defined regions of the heparin binding sequence, we found that the C1 domain functioned by increasing the on rate of the IFNgamma.IFNgammaR binding reaction but was not otherwise required for the stability of the complex. Interactions between the IFNgamma carboxyl-terminal domain and IFNgammaR could increased the association rate of the reaction either by increasing the number of encounters between the two molecules or by favoring productive collisions. The mechanisms by which heparan sulfate regulates IFNgamma activity may thus include both control of selective protease cleavage events, which directly affect the cytokine activity, and also an ability to modulate the interaction of IFNgamma with the IFNgammaR via competitive binding to the C1 domain.

Non-receptor-mediated tissue localization of human interferon-gamma: role of heparan sulfate/heparin-like molecules.

In addition to its cellular receptor, interferon-gamma (IFN-gamma) displays a high affinity for heparan sulfate. This glycosaminoglycan found in the extracellular matrix and at the surface of some cells was studied here as a possible in vivo binding site for IFN-gamma. For this purpose, rats were injected with [125I]-labelled human IFN-gamma, which does not bind to murine IFN-gamma receptors, but binds to murine heparan sulfate. It was found, first, that [125I]-IFN-gamma does not have equal access to all tissues, accumulating mainly in the spleen, liver and kidney, but not in muscles. Furthermore, [125I]-IFN-gamma was detected by autoradiographic analysis only in restricted areas within tissues, which correlates with the known locations of heparan sulfate. Such local concentrations were detected in the liver sinusoids and in the kidney glomerulus, for example. Heparin bound to [125I]-IFN-gamma was also used to block the heparan sulfate binding site of the cytokine. In this case, blood clearance and tissue accumulation in the liver and spleen were strongly inhibited, while in the kidney the distribution, but not the accumulation, of [125I]-IFN-gamma was affected by the presence of heparin. Kinetic analysis of the binding showed that [125I]-IFN-gamma accumulated in tissues between 5 and 20 min after injection, and was then quickly cleared. Taken together, these data demonstrate that heparan sulfate molecules are involved in blood clearance and in the subsequent tissue targeting, accumulation, and localization of [125I]-IFN-gamma.