Rabia Sadir

Heparan Sulfate/Heparin Oligosaccharides Protect Stromal Cell-derived Factor-1 (SDF-1)/CXCL12 against Proteolysis Induced by CD26/Dipeptidyl Peptidase IV

Stromal cell-derived factor-1 (SDF-1) is a CXC chemokine that is constitutively expressed in most tissues and displayed on the cell surface in association with heparan sulfate (HS). Its numerous biological effects are mediated by a specific G protein-coupled receptor, CXCR4. A number of cells inactivate SDF-1 by specific processing of the N-terminal domain of the chemokine. In particular, CD26/dipeptidyl peptidase IV (DPP IV), a serine protease that co-distributes with CXCR4 at the cell surface, mediates the selective removal of the N-terminal dipeptide of SDF-1. We report here that heparin and HS specifically prevent the processing of SDF-1 by DPP IV expressed by Caco-2 cells. The level of processing increases with the level of differentiation of these cells, which correlates with an increase of DPP IV activity. A mutant SDF-1 that does not interact with HS is readily cleaved by DPP IV, a process that is not inhibited by HS, demonstrating that a productive interaction between HS and SDF-1 is required for the protection to take place. Moreover, we found that protection depends on the degree of polymerization of the HS sulfated S-domains. Finally a structural model of SDF-1, in complex with HS oligosaccharides of defined length, rationalizes the experimental data. The mechanisms by which HS regulates SDF-1 may thus include, in addition to its ability to locally concentrate the chemokine at the cell surface, a control of selective protease cleavage events that directly affect the chemokine activity.

The Novel CXCL12γ Isoform Encodes an Unstructured Cationic Domain Which Regulates Bioactivity and Interaction with Both Glycosaminoglycans and CXCR4

Background CXCL12α, a chemokine that importantly promotes the oriented cell migration and tissue homing of many cell types, regulates key homeostatic functions and pathological processes through interactions with its cognate receptor (CXCR4) and heparan sulfate (HS). The alternative splicing of the cxcl12 gene generates a recently identified isoform, CXCL12γ, which structure/function relationships remain unexplored. The high occurrence of basic residues that characterize this isoform suggests however that it could feature specific regulation by HS. Methodology/Principal Findings Using surface plasmon resonance and NMR spectroscopy, as well as chemically and recombinantly produced chemokines, we show here that CXCL12γ first 68 amino acids adopt a structure closely related to the well described α isoform, followed by an unfolded C-terminal extension of 30 amino acids. Remarkably, 60 % of these residues are either lysine or arginine, and most of them are clustered in typical HS binding sites. This provides the chemokine with the highest affinity for HP ever observed (Kd = 0.9 nM), and ensures a strong retention of the chemokine at the cell surface. This was due to the unique combination of two cooperative binding sites, one strictly required, found in the structured domain of the protein, the other one being the C-terminus which essentially functions by enhancing the half life of the complexes. Importantly, this peculiar C-terminus also regulates the balance between HS and CXCR4 binding, and consequently the biological activity of the chemokine. Conclusions/Significance Together these data describe an unusual binding process that gives rise to an unprecedented high affinity between a chemokine and HS. This shows that the γ isoform of CXCL12, which features unique structural and functional properties, is optimized to ensure its strong retention at the cell surface. Thus, depending on the chemokine isoform to which it binds, HS could differentially orchestrate the CXCL12 mediated directional cell kinesis.

Polypyrrole oligosaccharide array and surface plasmon resonance imaging for the measurement of glycosaminoglycan binding interactions.

In order to construct tools able to screen oligosaccharide-protein interactions, we have developed a polypyrrole-based oligosaccharide chip constructed via a copolymerization process of pyrrole and pyrrole-modified oligosaccharide. For our study, GAG (glycosaminoglycans) or GAG fragments, which are involved in many fundamental biological processes, were modified by the pyrrole moiety on their reducing end and then immobilized on the chip. The parallel binding events on the upperside of the surface can be simultaneously monitored and quantified in real time and without labeling by surface plasmon resonance imaging (SPRi). We show that electrocopolymerization of the oligosaccharide-pyrrole above a gold surface enables the covalent immobilization of multiple probes and the subsequent monitoring of their binding capacities using surface plasmon resonance imaging. Moreover, a biological application was made involving different GAG fragments and different proteins, including stromal cell-derived factor-1alpha (SDF-1alpha), interferon-gamma (IFN-gamma), and monoclonal antibody showing different affinity pattern.

The CXCL12γ Chemokine Displays Unprecedented Structural and Functional Properties that Make It a Paradigm of Chemoattractant Proteins

The CXCL12γ chemokine arises by alternative splicing from Cxcl12, an essential gene during development. This protein binds CXCR4 and displays an exceptional degree of conservation (99%) in mammals. CXCL12γ is formed by a protein core shared by all CXCL12 isoforms, extended by a highly cationic carboxy-terminal (C-ter) domain that encompass four overlapped BBXB heparan sulfate (HS)-binding motifs. We hypothesize that this unusual domain could critically determine the biological properties of CXCL12γ through its interaction to, and regulation by extracellular glycosaminoglycans (GAG) and HS in particular. By both RT-PCR and immunohistochemistry, we mapped the localization of CXCL12γ both in mouse and human tissues, where it showed discrete differential expression. As an unprecedented feature among chemokines, the secreted CXCL12γ strongly interacted with cell membrane GAG, thus remaining mostly adsorbed on the plasmatic membrane upon secretion. Affinity chromatography and surface plasmon resonance allowed us to determine for CXCL12γ one of the higher affinity for HS (Kd = 0.9 nM) ever reported for a protein. This property relies in the presence of four canonical HS-binding sites located at the C-ter domain but requires the collaboration of a HS-binding site located in the core of the protein. Interestingly, and despite reduced agonist potency on CXCR4, the sustained binding of CXCL12γ to HS enabled it to promote in vivo intraperitoneal leukocyte accumulation and angiogenesis in matrigel plugs with much higher efficiency than CXCL12α. In good agreement, mutant CXCL12γ chemokines selectively devoid of HS-binding capacity failed to promote in vivo significant cell recruitment. We conclude that CXCL12γ features unique structural and functional properties among chemokines which rely on the presence of a distinctive C-ter domain. The unsurpassed capacity to bind to HS on the extracellular matrix would make CXCL12γ the paradigm of haptotactic proteins, which regulate essential homeostatic functions by promoting directional migration and selective tissue homing of cells.

Cytokines and growth factors cross-link heparan sulfate

The glycosaminoglycan heparan sulfate (HS), present at the surface of most cells and ubiquitous in extracellular matrix, binds many soluble extracellular signalling molecules such as chemokines and growth factors, and regulates their transport and effector functions. It is, however, unknown whether upon binding HS these proteins can affect the long-range structure of HS. To test this idea, we interrogated a supramolecular model system, in which HS chains grafted to streptavidin-functionalized oligoethylene glycol monolayers or supported lipid bilayers mimic the HS-rich pericellular or extracellular matrix, with the biophysical techniques quartz crystal microbalance (QCM-D) and fluorescence recovery after photobleaching (FRAP). We were able to control and characterize the supramolecular presentation of HS chains—their local density, orientation, conformation and lateral mobility—and their interaction with proteins. The chemokine CXCL12α (or SDF-1α) rigidified the HS film, and this effect was due to protein-mediated cross-linking of HS chains. Complementary measurements with CXCL12α mutants and the CXCL12γ isoform provided insight into the molecular mechanism underlying cross-linking. Fibroblast growth factor 2 (FGF-2), which has three HS binding sites, was also found to cross-link HS, but FGF-9, which has just one binding site, did not. Based on these data, we propose that the ability to cross-link HS is a generic feature of many cytokines and growth factors, which depends on the architecture of their HS binding sites. The ability to change matrix organization and physico-chemical properties (e.g. permeability and rigidification) implies that the functions of cytokines and growth factors may not simply be confined to the activation of cognate cellular receptors.

Insights into the Mechanism by Which Interferon-γ Basic Amino Acid Clusters Mediate Protein Binding to Heparan Sulfate

The extensive functional repertoire of heparin and heparan sulfate, which relies on their ability to interact with a large number of proteins, has recently emerged. To understand the forces that drive such interactions the binding of heparin to interferon-γ (IFNγ), used as a model system, was investigated. NMR-based titration experiments demonstrated the involvement of two adjacent cationic domains (D1: KTGKRKR and D2: RGRR), both of which are present within the carboxy-terminal sequence of the cytokine. Kinetic analysis showed that these two domains contribute differently to the interaction: D1 is required to form a complex and constitutes the actual binding site, whereas D2, although unable to associate with heparin by itself, increased the association rate of the binding. These data are consistent with the view that D2, through nonspecific electrostatic forces, places the two molecules in favorable orientations for productive binding within the encounter complex. This mechanism was supported by electrostatic potential analysis and thermodynamic investigations. They showed that D1 association to heparin is driven by both favorable enthalpic and entropic contributions, as expected for a binding sequence, but that D2 gives rise to entropic penalty, which opposes binding in a thermodynamic sense. The binding mechanism described herein, by which the D2 domain kinetically drives the interaction, has important functional consequences and gives a structural framework to better understand how specific are the interactions between proteins and heparin.

Functional Characterization of the Recombinant Human C1 Inhibitor Serpin Domain: Insights into Heparin Binding

Variants of the human C1 inhibitor serpin domain containing three N-linked carbohydrates at positions 216, 231, and 330 (C1inhΔ97), a single carbohydrate at position 330 (C1inhΔ97DM), or no carbohydrate were produced in a baculovirus/insect cells system. An N-terminally His-tagged C1inhΔ97 variant was also produced. Removal of the oligosaccharide at position 330 dramatically decreased expression, precluding further analysis. All other variants were characterized chemically and shown to inhibit C1s activity and C1 activation in the same way as native C1 inhibitor. Likewise, they formed covalent complexes with C1s as shown by SDS-PAGE analysis. C1 inhibitor and its variants inhibited the ability of C1r-like protease to activate C1s, but did not form covalent complexes with this protease. The interaction of C1 inhibitor and its variants with heparin was investigated by surface plasmon resonance, yielding KD values of 16.7 × 10−8 M (C1 inhibitor), 2.3 × 10−8 M (C1inhΔ97), and 3.6 × 10−8 M (C1inhΔ97DM). C1s also bound to heparin, with lower affinity (KD = 108 × 10−8 M). Using the same technique, 50% inhibition of the binding of C1 inhibitor and C1s to heparin was achieved using heparin oligomers containing eight and six saccharide units, respectively. These values roughly correlate with the size of 10 saccharide units yielding half-maximal potentiation of the inhibition of C1s activity by C1 inhibitor, consistent with a “sandwich” mechanism. Using a thermal shift assay, heparin was shown to interact with the C1s serine protease domain and the C1 inhibitor serpin domain, increasing and decreasing their thermal stability, respectively.

HSulf sulfatases catalyze processive and oriented 6-O-desulfation of heparan sulfate that differentially regulates fibroblast growth factor activity

Sulfs are extracellular sulfatases that have emerged recently as critical regulators of heparan sulfate (HS) activities through their ability to catalyze specific 6‐O‐desulfation of the polysaccharide. Consequently, Sulfs have been involved in many physiological and pathological processes, and notably for Sulf‐2, in the development of cancers with poor prognosis. Despite growing interest, little is known about the structure and activity of these enzymes and the way they induce dynamic remodeling of HS 6‐O‐sulfation status. Here, we have combined an array of analytical approaches, including mass spectrometry, NMR, HS oligosaccharide sequencing, and FACS, to dissect HSulf‐2 sulfatase activity, either on a purified octasaccharide used as a mimic of HS functional domains, or on intact cell‐surface HS chains. In parallel, we have studied the functional consequences of HSulf‐2 activity on fibroblast growth factor (FGF)‐induced mitogenesis and found that the enzyme could differentially regulate FGF1 and FGF2 activities. Notably, these data supported the existence of precise 6‐O‐sulfation patterns for FGF activation and provided new insights into the saccharide structures involved. Altogether, our data bring to light an original processive enzymatic mechanism, by which HSulfs catalyze oriented alteration of HS 6‐O‐desulfation patterns and direct fine and differential regulation of HS functions.—Seffouh, A., Milz, F., Przybylski, C., Laguri, C., Oosterhof, A., Bourcier, S., Sadir, R., Dutkowski, E., Daniel, R., van Kuppevelt, T. H., Dierks, T., Lortat‐Jacob, H., Vivès, R. R. HSulf sulfatases catalyze processive and oriented 6‐O‐desulfation of heparan sulfate that differentially regulates fibroblast growth factor activity. FASEB J. 27, 2431–2439 (2013). www.fasebj.org

NMR characterization of the interaction between the C-terminal domain of interferon-γ and heparin-derived oligosaccharides

Interferons are cytokines that play a complex role in the resistance of mammalian hosts to pathogens. IFNgamma (interferon-gamma) is secreted by activated T-cells and natural killer cells. IFNgamma is involved in a wide range of physiological processes, including antiviral activity, immune response, cell proliferation and apoptosis, as well as the stimulation and repression of a variety of genes. IFNgamma activity is modulated by the binding of its C-terminal domain to HS (heparan sulphate), a glycosaminoglycan found in the extracellular matrix and at the cell surface. In the present study, we analysed the interaction of isolated heparin-derived oligosaccharides with the C-terminal peptide of IFNgamma by NMR, in aqueous solution. We observed marked changes in the chemical shifts of both peptide and oligosaccharide compared with the free state. Our results provide evidence of a binding through electrostatic interactions between the charged side chains of the protein and the sulphate groups of heparin that does not induce specific conformation of the C-terminal part of IFNgamma. Our data also indicate that an oligosaccharide size of at least eight residues displays the most efficient binding.

The effect of delivering the chemokine SDF-1α in a matrix-bound manner on myogenesis

Several chemokines are important in muscle myogenesis and in the recruitment of muscle precursors during muscle regeneration. Among these, the SDF-1α chemokine (CXCL12) is a potent chemoattractant known to be involved in muscle repair. SDF-1α was loaded in polyelectrolyte multilayer films made of poly(L-lysine) and hyaluronan to be delivered locally to myoblast cells in a matrix-bound manner. The adsorbed amounts of SDF-1α were tuned over a large range from 100 ng/cm(2) to 5 μg/cm(2), depending on the initial concentration of SDF-1α in solution, its pH, and on the film crosslinking extent. Matrix-bound SDF-1α induced a striking increase in myoblast spreading, which was revealed when it was delivered from weakly crosslinked films. It also significantly enhanced cell migration in a dose-dependent manner, which again depended on its presentation by the biopolymeric film. The low-crosslinked film was the most efficient in boosting cell migration. Furthermore, matrix-bound SDF-1α also increased the expression of myogenic markers but the fusion index decreased in a dose-dependent manner with the adsorbed amount of SDF-1α. At high adsorbed amounts of SDF-1α, a large number of Troponin T-positive cells had only one nucleus. Overall, this work reveals the importance of the presentation mode of SDF-1α to emphasize its effect on myogenic processes. These films may be further used to provide insight into the role of SDF-1α presented by a biomaterial in physiological or pathological processes.