Biomedical subjects

H Lortat-Jacob

Publications and source records attributed to H Lortat-Jacob.

28 records · Page 2Linked to original sources

PubMed1995

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.

Animals↗

PubMed1994

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.

Animals↗

PubMed1994

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.

Animals↗

PubMed1994

[The extracellular matrix: from supporting tissue to regulation of 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.

Cell Communication↗

PubMed1992

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.

Binding Sites↗

PubMed1992

Interleukin-6 does not regulate interstitial collagenase, stromelysin and tissue inhibitor of metalloproteinases synthesis by cultured human fibroblasts.

We have examined the effect of interleukin-6 (IL-6) on the synthesis of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMP) by human skin fibroblasts, both at protein and messenger RNA levels. IL-6 did not modulate the production of MMPs and TIMP. Furthermore IL-6 did not modify the stimulatory effect exerted by interleukin-1 (IL-1) on the expression of MMPs and TIMP. These results strongly suggest that IL-6 is not involved in the extracellular matrix breakdown, either directly by acting on cell production of MMPs or TIMP, or indirectly by modulating the effect of IL-1 on the synthesis of MMPs and TIMP.

Cells, Cultured↗

PubMed1991

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.

Amino Acid Sequence↗

PubMed1991

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.

Antibodies, Monoclonal↗

PubMed1991

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.

Amino Acid Sequence↗

PubMed1990

[Connective matrix and localization of a biological signal: demonstration of a matrix receptor for interferon-gamma in basement membranes].

Extracellular matrix has a variety of biological effects on cells, including increased cell adhesion, migration, division and differentiation. Cells are also regulated by soluble factors such as cytokines. We have investigated the binding of interferon-gamma to basement membrane. Interferon-gamma binds basement membrane with a high affinity (kD = 1.5 x 10(-9) M). The binding site is located on the glycosaminoglycan part of the heparan sulfate proteoglycan. This result suggests that extracellular matrix interacts with interferon-gamma and could modulate the cellular response to such factors.

Basement Membrane↗