Impact of epidermal growth factor tethering strategy on cellular response.
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.
Department of Chemical Engineering, Groupe de Recherche en Sciences et Technologies Biomédicales, Bio-P2 Research Unit, École Polytechnique de Montréal, P.O. Box 6079, succ. Centre-Ville, Montréal (Qc), Canada.
Benoît
B
Liberelle
Cyril
C
Boucher
Jingkui
J
Chen
Mario
M
Jolicoeur
Yves
Y
Durocher
Gregory
G
De Crescenzo
D000588:Amines / Q000737:chemistry; D002448:Cell Adhesion / Q000187:drug effects; D045744:Cell Line, Tumor; D003911:Dextrans / Q000737:chemistry; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism*; D015870:Gene Expression; D005898:Glass / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D013499:Surface Properties / Q000187:drug effects; D023822:Tissue Engineering / Q000379:methods
D000588:Amines; D003911:Dextrans; D055767:Immobilized Proteins; D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor; C014392:carboxymethyl dextran; D066246:ErbB Receptors