Mutational analysis verifies that "kneed" sequence of fibronectin participates in cell-substrate interactions

Interactions between cell-surface integrins and extracellular matrix (ECM) proteins underlie a versatile recognition system providing cells with anchorage and traction for migration or matrix remodeling. Short peptide sequences of fibronectin (FN), most notably RGD found on a loop in the tenth type III domain, are effective in promoting cell adhesion when immobilized to a biomaterial scaffold. Using bioinformatics, we identified a candidate cell-binding peptide sequence, KNEED, located on the loop region of the 8^th domain of fibronectin that from in vitro studies appears to participate in cell attachment and spreading. To demonstrate its importance, the KNEED sequence of Fibronectin type III domains 7-10 (FN7-10) was mutated to AAAAA or SNSSS (FN7-10 AAAAA and FN7-10 SNSSS respectively). Wildtype and mutant proteins were expressed in E. coli, purified by conventional chromatography and tested in cell adhesion assays. Results indicate that the extent of cell spreading on surfaces coated with either FN7-10 AAAAA or FN7-10 SNSSS was measurably less than on surfaces coated with wildtype FN7-10. Mutational analysis may aid in the characterization of new targets for applications where biorecognition plays a key role.