Abstract
Most mammalian cells must adhere to the extracellular matrix (ECM) to maintain proper growth and development. Fibronectin is a predominant ECM protein that engages integrin cell receptors through its Arg-Gly-Asp (RGD) and Pro-His-Ser-Arg-Asn (PHSRN) peptide binding sites. To study the roles these motifs play in cell adhesion, proteins derived from the 9th (containing PHSRN) and 10th (containing RGD) type III fibronectin domains were engineered to be in frame with cutinase, a serine esterase that forms a site-specific, covalent adduct with phosphonate ligands. Self-assembled monolayers (SAMs) that present phosphonate ligands against an inert background of tri(ethylene glycol) groups were used as model substrates to immobilize the cutinase-fibronectin fusion proteins. Baby hamster kidney cells attached efficiently to all protein surfaces, but only spread efficiently on protein monolayers containing the RGD peptide. Cells on RGD-containing protein surfaces also displayed defined focal adhesions and organized cytoskeletal structures compared to cells on PHSRN-presenting surfaces. Cell attachment and spreading were shown to be unaffected by the presence of PHSRN when compared to RGD alone on SAMs presenting higher densities of protein, but PHSRN supported an increased efficiency in cell attachment when presented at low protein densities with RGD. Treatment of suspended cells with soluble RGD or PHSRN peptides revealed that both peptides were able to inhibit the attachment of FN10 surfaces. These results support a model wherein PHSRN and RGD bind competitively to integrins rather than a two-point synergistic interaction and the presence of PHSRN serves to increase the density of ligand on the substrate and therefore enhance the sticking probability of cells during attachment.
Original language | English (US) |
---|---|
Pages (from-to) | 13942-13951 |
Number of pages | 10 |
Journal | Langmuir |
Volume | 25 |
Issue number | 24 |
DOIs | |
State | Published - Dec 15 2009 |
Funding
ASJC Scopus subject areas
- Condensed Matter Physics
- Spectroscopy
- General Materials Science
- Surfaces and Interfaces
- Electrochemistry