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In an effort to rationalize and optimize an antiapoptotic coating combining chondroitin sulfate (CS) and epidermal growth factor (EGF) for vascular applications, the authors here report the comparison of two grafting strategies aiming to display EGF in an oriented fashion on CS. For that purpose, the authors produced, purified, and characterized a chimeric protein corresponding to EGF that was N-terminally fused to a cysteine and a coil peptide. The chimera was covalently immobilized via its free thiol group or captured via coiled-coil interactions at the surface of a biosensor or on a chondroitin sulfate coating in multiwell plates, mimicking the coating that was previously developed by them for stent-graft surfaces. The interactions of grafted EGF with the soluble domain of its receptor or the impact of grafted EGF upon vascular smooth muscle survival in proapoptotic conditions indicated that the coiled-coil based tethering was the best approach to display EGF. These results, combined to direct enzyme-linked immunosorbent assay measurements, indicated that the coiled-coil tethering approach allowed increasing the amount of bioavailable EGF when compared to covalent coupling, rather than the total amount of grafted EGF, while using much lower concentrations of tagged EGF during incubation.

In an effort to evaluate the impact of various epidermal growth factor (EGF) grafting strategies upon cell surface receptor activation and cell adhesion, we generated low-fouling surfaces by homogeneously grafting carboxymethylated dextran (CMD) on amino-coated glass substrate. By preventing nonspecific cell adhesion while providing reactive groups facilitating subsequent protein grafting, CMD allowed achieving specific cell/tethered EGF interactions and therefore deriving unambiguous conclusions about various EGF grafting strategies. We demonstrate here that A-431 cell response to immobilized EGF is highly dependent on the bioactivity of the tagged protein being tethered, its proper orientation, and its surface density. Among all the approaches we tested, the oriented tethering of fully bioactive EGF via a de novo-designed coiled-coil capture system was shown to be the most efficient. That is, it led to the most intense and sustained phosphorylation of EGF receptors as well as to strong A-431 cell adhesion, the latter being comparable to that observed with amino-coated surfaces in the absence of CMD.

The de novo designed heterodimeric E/K coiled-coil system has been previously demonstrated to be an excellent capture/dimerization system applicable to various needs in both biotechnology and pharmaceutical fields. Those include controlled protein dimerization, capture, purification and Western-blot detection. We here report the development of a new generation of ELISA test based on coiled-coil interactions for the direct quantitation of coil-tagged epidermal growth factor (EGF). The new approach was evaluated for its specificity, plate storability and reusability as well as for convenience when compared to commercially available systems. Our results show a similar affinity/sensitivity to standard capturing antibody-based ELISA systems and an improved affinity/sensitivity when compared to the commercially available Ni-NTA capture system. The E/K coiled-coil ELISA system was validated with respect to recovery, intra- and inter-assay variations. The practical working range was estimated to be between 5.2 and 34,000 pM. Furthermore, the storability and reusability of the plates was greater than the two aforementioned systems, suggesting that the E/K coiled-coil system is a good alternative to traditional tags such as poly-histidine for the development of ELISA tests aiming at quantitating coil-tagged proteins., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The development of new strategies for protein immobilization to control cell adhesion, growth and differentiation is of prime interest in the field of tissue engineering. Here we propose a versatile approach based on the interaction between two de novo designed peptides, Ecoil and Kcoil, for oriented immobilization of epidermal growth factor (EGF) on polyethylene terephthalate (PET) films. After amination of PET surfaces by ammonia plasma treatment, Kcoil peptides were covalently grafted in an oriented fashion using succinimidyl 6-[30-(2-pyridyldithio)-propionamido] hexanoate (LC-SPDP) linker, and the Kcoil-functionalized films were characterized by X-ray photoelectron spectroscopy (XPS). Bioactivity of Ecoil-EGF captured on Kcoil-functionalized PET via coiled-coil interactions was confirmed by EGF receptor phosphorylation analysis following A-431 cell attachment. We also demonstrated cell biological effects where tethered EGF enhanced adhesion, spreading and proliferation of human corneal epithelial cells compared to EGF that was either physically adsorbed or present in solution. Tethered EGF effects were most likely linked to the prolonged activation of both mitogen-activated protein kinase and phosphoinositidine 3-kinase pathways. Taken together, our results indicate that coiled-coil-based oriented immobilization is a powerful method to specifically tailor biomaterial surfaces for tissue engineering applications., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

We propose an approach for the detection of proteins by Western blot that takes advantage of the high-affinity interaction occurring between two de novo designed peptides, the E and K coils. As a model system, K coil-tagged epidermal growth factor (EGF) was revealed with secreted alkaline phosphatase (SeAP) tagged with E coil (SeAP-Ecoil) as well as with biotinylated E coil. In that respect, we first produced purified SeAP-Ecoil and verified its ability to interact with K coil peptides by surface plasmon resonance biosensing. We demonstrated that protein detection with Ecoil-biotin was more specific than with SeAP-Ecoil. We then showed that our approach is as sensitive as conventional detection strategies relying on nickel-nitrilotriacetic acid-horseradish peroxidase (Ni-NTA-HRP), anti-His-HRP, or anti-EGF. Altogether, our results indicate that the E/K coiled-coil system is a good alternative for protein detection by Western blot., (Crown Copyright 2009. Published by Elsevier Inc. All rights reserved.)

Mounting evidence indicates that mesenchymal stem cells (MSC) are pivotal to vascular repair and neointima formation in various forms of vascular disease. Yet, the mechanisms that allow MSC to resist apoptosis at sites where other cell types, such as endothelial cells (EC), are dying are not well defined. In the present work, we demonstrate that apoptotic EC actively release paracrine mediators which, in turn, inhibit apoptosis of MSC. Serum-free medium conditioned by apoptotic EC increases extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation and inhibits apoptosis (evaluated by Bcl-xL protein levels and poly (ADP-ribose) polymerase cleavage) of human MSC. A C-terminal fragment of perlecan (LG3) released by apoptotic EC is one of the mediators activating this antiapoptotic response in MSC. LG3 interacts with beta1-integrins, which triggers downstream ERK1/2 activation in MSC, albeit to a lesser degree than medium conditioned by apoptotic EC. Hence, other mediators released by apoptotic EC are probably required for induction of the full antiapoptotic phenotype in MSC. Adopting a comparative proteomic strategy, we identified epidermal growth factor (EGF) as a novel mediator of the paracrine component of the endothelial apoptotic program. LG3 and EGF cooperate in triggering beta1-integrin and EGF receptor-dependent antiapoptotic signals in MSC centering on ERK1/2 activation. The present work, providing novel insights into the mechanisms facilitating the survival of MSC in a hostile environment, identifies EGF and LG3 released by apoptotic EC as central antiapoptotic mediators involved in this paracrine response.

We have elaborated and validated a novel approach for the oriented tethering of proteins such as the epidermal growth factor (EGF) on aminated surfaces. The grafting reactions were optimized to generate a dense and homogeneous EGF layer. Impact of EGF orientation on A-431 cellular response was investigated. Our results demonstrate that, in sharp contrast to responses obtained with soluble EGF supply or with randomly grafted EGF, oriented immobilization of EGF via a de novo designed coiled-coil capture system leads to a sustained phosphorylation of A-431 cell surface EGF receptors. Our results thus indicate that oriented protein immobilization via coiled-coil interactions is an efficient and versatile method to control tethering of bioactive molecules for future applications in the field of regenerative medicine and tissue engineering.

For tissue engineering applications, growth factor immobilization on cell culture scaffolds bears the potential to stimulate cell proliferation while minimizing costs associated to soluble growth factor supply. In order to evaluate the potential of a de novo-designed heterodimerization peptide pair, namely the E and K coils, for epidermal growth factor (EGF) grafting on various scaffolds, production of coil-tagged EGF chimeras using a mammalian cell expression system as well as their purification have been performed. The influence of the type of coil (E or K) upon EGF bioactivity, assessed in an in vitro cell assay, was compared to that of the fragment crystallizable (Fc) domain of immunoglobulin G by monitoring phosphorylation of EGF receptor (EGFR) upon chimeric EGF exposure. Our results demonstrate that the type and the location of the tag have a strong impact on growth factor bioactivity (EC50 ranging from 5.5 to 63 nM). Additional surface plasmon resonance-based biosensor experiments were conducted to test the ability of captured chimeric EGF to bind to their receptor ectodomain in vitro. These experiments indicated that the oriented coiled-coil-mediated immobilization of EGF was significantly more efficient than a random approach as coil-tagged EGF displayed enhanced affinities for artificially dimerized EGFR ectodomain when compared to Fc-tagged EGF (apparent KD of 5 pM vs. 16 nM). Altogether, our results highly suggest that coil-tagged chimeras represent an attractive avenue for the oriented immobilization of growth factors for tissue engineering applications and that HEK293 cells offer a robust platform for their expression in a bioactive form.