Laurence Duchesne

Interactions of Multiple Heparin Binding Growth Factors with Neuropilin-1 and Potentiation of the Activity of Fibroblast Growth Factor-2

The hypothesis that neuropilin-1 (Npn-1) may interact with heparin-binding proteins other than vascular endothelial growth factor has been tested using an optical biosensor-based binding assay. The results show that fibroblast growth factor (FGF) 1, 2, 4, and 7, FGF receptor 1, hepatocyte growth factor/scatter factor (HGF/SF), FGF-binding protein, normal protease sensitive form of prion protein, antithrombin III, and Npn-1 itself are all able to interact with Npn-1 immobilized on the sensor surface. FGF-2, FGF-4, and HGF/SF are also shown to interact with Npn-1 in a solution assay. Moreover, these protein-protein interactions are dependent on the ionic strength of the medium and are inhibited by heparin, and the kinetics of binding of FGF-2, FGF-4 and HGF/SF to Npn-1 are characterized by fast association rate constants (270,000–1,600,000 m–1 s–1). These results suggest that Npn-1 possesses a “heparin” mimetic site that is able to interact at least in part through ionic bonding with the heparin binding site on many of the proteins studied. Npn-1 was also found to potentiate the growth stimulatory activity of FGF-2 on human umbilical vein endothelial cells, indicating that Npn-1 may not just bind but also regulate the activity of heparin-binding proteins.

N-Glycosylation of Fibroblast Growth Factor Receptor 1 Regulates Ligand and Heparan Sulfate Co-receptor Binding

The regulation of cell function by fibroblast growth factors (FGF) occurs through a dual receptor system consisting of a receptor-tyrosine kinase, FGFR and the glycosaminoglycan heparan sulfate (HS). Mutations of some potential N-glycosylation sites in human fgfr lead to phenotypes characteristic of receptor overactivation. To establish how N-glycosylation may affect FGFR function, soluble- and membrane-bound recombinant receptors corresponding to the extracellular ligand binding domain of FGFR1-IIIc were produced in Chinese Hamster Ovary cells. Both forms of FGFR1-IIIc were observed to be heavily N-glycosylated and migrated on SDS-PAGE as a series of multiple bands between 50 and 75 kDa, whereas the deglycosylated receptors migrated at 32 kDa, corresponding to the expected molecular weight of the polypeptides. Optical biosensor and quartz crystal microbalance-dissipation binding assays show that the removal of the N-glycans from FGFR1-IIIc caused an increase in the binding of the receptor to FGF-2 and to heparin-derived oligosaccharides, a proxy for cellular HS. This effect is mediated by N-glycosylation reducing the association rate constant of the receptor for FGF-2 and heparin oligosaccharides. N-Glycans were analyzed by mass spectrometry, which demonstrates a predominance of bi- and tri-antennary core-fucosylated complex type structures carrying one, two, and/or three sialic acids. Modeling of such glycan structures on the receptor protein suggests that at least some may be strategically positioned to interfere with interactions of the receptor with FGF ligand and/or the HS co-receptor. Thus, the N-glycans of the receptor represent an additional pathway for the regulation of the activity of FGFs.

Transport of Fibroblast Growth Factor 2 in the Pericellular Matrix Is Controlled by the Spatial Distribution of Its Binding Sites in Heparan Sulfate

A single-molecule imaging study reveals that heparan sulfate chains in the pericellular matrix present a structured network of binding sites that controls FGF2 transport.

Robust Ligand Shells for Biological Applications of Gold Nanoparticles

An important point regarding the development of stable biofunctional nanoparticles for biomedical applications is their potential for aspecific interactions with the molecules of the biological environment. Here we report a new self-assembled ligand monolayer system for gold nanoparticles called Mix-matrices, formed by a mixture of HS-PEG and alcohol peptides (peptidols) molecules. Stability of the Mix-capped nanoparticles prepared in various conditions was assessed using tests of increasing stringency. The results highlight the importance of identifying a concentration of ligands sufficiently high to obtain a compact matrix when preparing nanoparticles and that the stability of capped nanoparticles in biological environments cannot be predicted solely on their resistance to electrolyte-induced aggregation. The Mix-capped nanoparticles are resistant to aggregation induced by electrolytes and to aspecific interactions with proteins and ligand exchange. In addition, Mix-matrices allow the easy introduction of a single recognition function per nanoparticle, allowing the specific and stoichiometric labeling of proteins with gold nanoparticles. Therefore, the Mix-matrices provide a useful tool for the development of nanoparticle-based quantitative bioanalytical and imaging techniques, as well as for therapeutic purposes, such as the specific targeting of cancerous cells for photothermal destruction.

A generic approach to monofunctionalized protein-like gold nanoparticles based on immobilized metal ion affinity chromatography

Due to their chemical, optical and electronic properties, metal nanoparticles (NPs) are essential components of new biosensors and self-assembled nanodevices. The ability to vary and control the surface composition of NPs with molecular accuracy is crucial for many envisioned applications and has been a major focus of research. The average composition can be varied through synthesis or ligand exchange by using different capping-ligand mixtures. Pioneering work by Alivisatos’ group led to the separation of NPs with a defined number of DNA strands by gel electrophoresis. The drawback of gel electrophoresis is that it is not easily scalable and is limited to DNA. Monofunctionalization based on solid-phase coupling and solid-phase ligand-exchange reactions has recently been reported for small amphiphilic NPs. We have recently developed a route to protein-like metal NPs based on self-assembled monolayers of peptides. The protein-like behaviour of NPs enables a wide range of biochemistry techniques to be applied. Separation of proteins has been a major challenge in the past decades, and powerful tools have been created to achieve this task. In this communication, we report the power of immobilized metal ion affinity chromatography (IMAC), a method developed and optimized for the purification of recombinant proteins, to separate peptide-capped NPs with a given number of molecular labels. This simple, scalable and analytical strategy is applicable to the preparation of NPs bearing a predefined number of virtually any water-soluble chemical moieties. Peptide-capped NPs were prepared by mixing 6 nm gold NPs with peptide solutions. The excess peptide was removed by size-exclusion chromatography. The peptide solutions comprised different proportions of the peptide CALNN (“matrix” peptide) and of an extended peptide, namely CALNNGHHHHHHGKbiotinG (“functional” peptide). The extension of the functional peptide is composed of two independent entities: the His-tag (a sequence of 6 histidines), which is used for its ability to bind to immobilized chelated transition metal ions such as nickel, followed by a label (Figure 1). Because

Photothermal Absorption Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a popular technique, complementary to cell imaging for the investigation of dynamic processes in living cells. Based on fluorescence, this single molecule method suffers from artifacts originating from the poor fluorophore photophysics: photobleaching, blinking, and saturation. To circumvent these limitations we present here a new correlation method called photothermal absorption correlation spectroscopy (PhACS) which relies on the absorption properties of tiny nano-objects. PhACS is based on the photothermal heterodyne detection technique and measures akin FCS, the time correlation function of the detected signals. Application of this technique to the precise determination of the hydrodynamic sizes of different functionalized gold nanoparticles are presented, highlighting the potential of this method.

Supramolecular Domains in Mixed Peptide Self‐Assembled Monolayers on Gold Nanoparticles

Self‐organization in mixed self‐assembled monolayers of small molecules provides a route towards nanoparticles with complex molecular structures. Inspired by structural biology, a strategy based on chemical cross‐linking is introduced to probe proximity between functional peptides embedded in a mixed self‐assembled monolayer at the surface of a nanoparticle. The physical basis of the proximity measurement is a transition from intramolecular to intermolecular cross‐linking as the functional peptides get closer. Experimental investigations of a binary peptide self‐assembled monolayer show that this transition happens at an extremely low molar ratio of the functional versus matrix peptide. Molecular dynamics simulations of the peptide self‐assembled monolayer are used to calculate the volume explored by the reactive groups. Comparison of the experimental results with a probabilistic model demonstrates that the peptides are not randomly distributed at the surface of the nanoparticle, but rather self‐organize into supramolecular domains.

Protein-GAG interactions: new surface-based techniques, spectroscopies and nanotechnology probes

New approaches, rooted in the physical sciences, have been developed to gain a more fundamental understanding of protein-GAG (glycosaminoglycan) interactions. DPI (dual polarization interferometry) is an optical technique, which measures real-time changes in the mass of molecules bound at a surface and the geometry of the bound molecules. QCM-D (quartz crystal microbalance-dissipation), an acoustic technique, measures the mass and the viscoelastic properties of adsorbates. The FTIR (Fourier-transform IR) amide bands I, II and III, resulting from the peptide bond, provide insight into protein secondary structure. Synchrotron radiation CD goes to much shorter wavelengths than laboratory CD, allowing access to chromophores that provide insights into the conformation of the GAG chain and of beta-strand structures of proteins. To tackle the diversity of GAG structure, we are developing noble metal nanoparticle probes, which can be detected at the level of single particles and so enable single molecule biochemistry and analytical chemistry. These new approaches are enabling new insights into structure-function relationships in GAGs and together they will resolve many of the outstanding problems in this field.

The formation of ordered nanoclusters controls cadherin anchoring to actin and cell–cell contact fluidity

Visualization of single cadherins within cell membrane at nanometric resolution shows that E-cadherins arrange in ordered clusters and that these clusters control the anchoring of cadherin to actin and cell–cell contact fluidity.

The heparan sulfate co-receptor and the concentration of fibroblast growth factor-2 independently elicit different signalling patterns from the fibroblast growth factor receptor

BackgroundThe fibroblast growth factor receptor (FGFR) interprets concentration gradients of FGF ligands and structural changes in the heparan sulfate (HS) co-receptor to generate different cellular responses. However, whether the FGFR generates different signals is not known.ResultsWe have previously shown in rat mammary fibroblasts that in cells deficient in sulfation, and so in HS co-receptor, FGF-2 can only stimulate a transient phosphorylation of p42/44 MAPK and so cannot stimulate DNA synthesis. Here we demonstrate that this is because in the absence of HS, FGF-2 fails to stimulate the phosphorylation of the adaptor FGFR substrate 2 (FRS2). In cells possessing the HS co-receptor, FGF-2 elicits a bell-shaped dose response: optimal concentrations stimulate DNA synthesis, but supramaximal concentrations (≥ 100 ng/mL) have little effect. At optimal concentrations (300 pg/mL) FGF-2 stimulates a sustained dual phosphorylation of p42/44 MAPK and tyrosine phosphorylation of FRS2. In contrast, 100 ng/mL FGF-2 only stimulates a transient early peak of p42/44 MAPK phosphorylation and fails to stimulate appreciably the phosphorylation of FRS2 on tyrosine.ConclusionsThese results suggest that the nature of the FGFR signal produced is determined by a combination of the HS co-receptor and the concentration of FGF ligand. Both the phosphorylation of the adaptor FRS2, the kinetics (sustained or transient) of phosphorylation of p42/44(MAPK) are varied, and so differing cellular responses are produced.