Impact of Epidermal Growth Factor Tethering Strategy on Cellular Response Benoı ˆt Liberelle, † Cyril Boucher, †,‡ Jingkui Chen, † Mario Jolicoeur, † Yves Durocher, ‡ and Gregory De Crescenzo* ,† Department of Chemical Engineering, Groupe de Recherche en Sciences et Technologies Biome ´dicales, Bio-P 2 Research Unit, E ´ cole Polytechnique de Montre ´al, P.O. Box 6079, succ. Centre-Ville, Montre ´al (Qc), Canada H3C 3A7, and Animal Cell Technology Group, Bioprocess Sector, Biotechnology Research Institute, National Research Council Canada, Montre ´al (Qc), Canada H4P 2R2. Received June 8, 2010; Revised Manuscript Received October 15, 2010 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 carboxy- methylated 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. INTRODUCTION Bioengineered implants are becoming extremely promising with respect to the development of biocompatible surfaces aiming at promoting and sustaining tissue regeneration. Within the last three decades, in an effort to mimic nature’s mechanisms that are involved in tissue development and healing processes, the immobilization of growth factors on various substrates has been extensively studied by several groups (1–3). Such an interest from the scientific community may be ascribed to the direct implication of growth factors in almost all fundamental cellular processes, including cell survival, proliferation, migra- tion, differentiation, and apoptosis. Furthermore, growth factor immobilization is believed to limit their clearance through endocytosis/degradation pathways and their diffusive mobility, thus restraining delivery to the local implant region (4). In that context, the epidermal growth factor (EGF), a soluble 6 kDa polypeptide that mediates its biological function by promoting the oligomerization (5, 6) and the phosphorylation (7–9) of cell surface EGF receptors (EGFRs), has attracted a lot of attention and has already shown great promises for the generation of smart corneal implants promoting re-epithelialization (10, 11) as well as for ex ViVo stem cell expansion (12–15). Nonspecific adsorption of EGF on substrate has been demonstrated to drastically diminish EGF bioactivity, most likely due to the occurrence of adsorption-induced conformational changes or inappropriate orientation of EGF (4). In contrast, covalent immobilization strategies via photoimmobilization or wet chemistry route resulted in unambiguous effects upon cell adhesion, phenotypic changes, and DNA synthesis (4, 16). However, for human EGF (hEGF), amine chemistry has been acknowledged to be suboptimal since the lysine side chains of hEGF extensively contribute to receptor binding; their involve- ment in the generation of an amide bound during EGF immobilization has been shown to greatly decrease its bioactivity (4, 17, 18). To counteract this caveat, oriented and stable tethering of hEGF has been successfully achieved through the use of recombinant EGF chimeras. That is, EGF has been fused to various tags or domains including the Fc region of immunoglobulin G (19), poly histidine (12), or coil peptide tags (18), in order to subsequently tether these chimeras on surfaces on which Protein A/G, nitrilotriacetate (NTA), or complementary coil peptide had been immobilized in a covalent fashion, respectively. Although the noncovalent but stable capture of EGF via tags hold great promise, this strategy should be examined with caution. We indeed recently demonstrated that the nature and the position of the tags both influence EGF bioactivity (20). More specifically, an Fc tag was shown to have a deleterious effect on EGF bioactivity when fused at its N- or C-terminus, as deduced from the ability of these constructs to induce A-431 cell receptor phosphorylation in Vitro. In this assay, EGFR phosphorylation was not detected when the cells were treated with soluble Fc-EGF, while EC 50 values of 17 nM versus 4.3 nM for EGF-Fc and EGF, respectively, were determined when these ligands were provided to the cells in their soluble forms. In stark contrast, EGF being N-terminally tagged with a short (i.e., 35 amino-acid-long) peptide (the Ecoil) was found to be as active as untagged EGF (EC 50 of 5.5 nM). The effect of these various tags upon EGF signaling, when captured in an oriented fashion, has never been rigorously compared, although several independent studies have demon- strated that the EGF moiety of these chimeric proteins was able to promote a cellular response. Another point to be considered with great care in growth factor immobilization resides in the choice of the spacer arm ensuring proper display and attachment of the growth factor of interest. So far, homo- (21) and heterobifunctional (22) poly- ethylene glycol (PEG) chains have been extensively used. However, as outlined by Kuhl et al. (4) and Klenkler et al. (21), * To whom correspondence should be addressed. E-mail: gregory. decrescenzo@polymtl.ca. Tel: (514) 340-4711 (#7428). Fax: (514) 340- 2990. † E ´ cole Polytechnique de Montre ´al. ‡ National Research Council Canada. Bioconjugate Chem. 2010, 21, 2257–2266 2257 10.1021/bc1002604  2010 American Chemical Society Published on Web 11/08/2010