Chimeric fibronectin matrix mimetic as a functional growth- and migration-promoting adhesive substrate

Abstract

Therapeutic protein engineering combines genetic, biochemical, and functional information to improve existing proteins or invent new protein technologies. Using these principles, we developed an approach to deliver extracellular matrix (ECM) fibronectin-specific signals to cells. Fibronectin matrix assembly is a cell-dependent process that converts the inactive, soluble form of fibronectin into biologically-active ECM fibrils. ECM fibronectin stimulates cell functions required for normal tissue regeneration, including cell growth, spreading, migration, and collagen reorganization. We have developed recombinant fibronectin fragments that mimic the effects of ECM fibronectin on cell function by coupling the cryptic heparin-binding fragment of fibronectin’s first type III repeat (FNIII1H) to the integrin-binding domain (FNIII8-10). GST/III1H,8-10 supports cell adhesion and spreading and stimulates cell proliferation to a greater extent than plasma fibronectin. Deletion and site-specific mutant constructs were generated to identify the active regions in GST/III1H,8-10 and reduce construct size. A chimeric construct in which the integrin-binding, RGDS loop was inserted into the analogous site in FNIII8 (GST/III1H,8RGD), supported cell adhesion and migration, and enhanced cell proliferation and collagen gel contraction. GST/III1H,8RGD was expressed in bacteria and purified from soluble lysate fractions by affinity chromatography. Fibronectin matrix assembly is normally up-regulated in response to tissue injury. Decreased levels of ECM fibronectin are associated with non-healing wounds. Engineering fibronectin matrix mimetics that bypass the need for cell-dependent fibronectin matrix assembly in chronic wounds is a novel approach to stimulating cellular activities critical for tissue repair.

Recombinant Fibronectin Matrix Mimetics Specify Integrin Adhesion and Extracellular Matrix Assembly

“…17,35 Tissue culture plates (48-well) were precoated with recombinant fibronectin proteins or collagen I at the indicated concentrations. FN-null MEFs were seeded at 2.5 · 10 3 cells/cm 2 and incubated for 4 h at 37°C to allow for adhesion and spreading.…”

“…GST/III1H,8-10, GST/III1H,8,10, GST/ III1H,8 RGD , GST/III1H, and FNIII11C were produced in Escherichia coli and purified as described. 32,35,37 The R1R2 peptide was produced by synthesis of an artificial gene (Integrated DNA Technologies) encoding amino acids Gly 195 -Thr 253 (bases 694-870) from the bacterial adhesin protein, SFS. 38 NcoI and PstI restriction sites were engineered on the 5¢ and 3¢ ends of the SFS sequence by addition of a 5¢ Met and 3¢ Ala-Gly, respectively.…”

“…10,31 We engineered fibronectin matrix mimetics that directly couple the open heparin-binding FNIII1 fragment (FNIII1H) to the integrin-binding domain (FNIII8-10) to provide cells with regulatory signals similar to those from ECM fibronectin. 32,35 We have shown that as adhesive substrates, the fibronectin matrix mimetics GST/III1H,8-10, GST/III1H,8,10, and GST/III1H,8…”

“…35 Smart biomaterials are currently being engineered in an attempt to generate tissues and organs in vitro. Examples include the perfusion-decellularized whole organs to generate intact ECM scaffolds that anatomically mimic native tissue.…”

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

“…In addition to these adhesive cues 9 , soluble factors [10][11][12] such as chemokines and growth factors can induce directed cell migration through chemoattractant receptors and their downstream motogenic signalling. Currently, it is not known whether engagement and signalling through adhesion receptors (i.e.…”