title: The synthesis and coupling of photoreactive collagen-based peptides to restore integrin reactivity to an inert substrate, chemically-crosslinked collagen.
abstract: Collagen is frequently advocated as a scaffold for use in regenerative medicine. Increasing the mechanical stability of a collagen scaffold is widely achieved by cross-linking using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). However, this treatment consumes the carboxylate-containing amino acid sidechains that are crucial for recognition by the cell-surface integrins, abolishing cell adhesion. Here, we restore cell reactivity to a cross-linked type I collagen film by covalently linking synthetic triple-helical peptides (THPs), mimicking the structure of collagen. These THPs are ligands containing an active cell-recognition motif, GFOGER, a high-affinity binding site for the collagen-binding integrins. We end-stapled peptide strands containing GFOGER by coupling a short diglutamate-containing peptide to their N-terminus, improving the thermal stability of the resulting THP. A photoreactive Diazirine group was grafted onto the end-stapled THP to allow covalent linkage to the collagen film upon UV activation. Such GFOGER-derivatized collagen films showed restored affinity for the ligand-binding I domain of integrin α2β1, and increased integrin-dependent cell attachment and spreading of HT1080 and Rugli cell lines, expressing integrins α2β1 and α1β1, respectively. The method we describe has wide application, beyond collagen films or scaffolds, since the photoreactive diazirine will react with many organic carbon skeletons.
authors: Department of Biochemistry, University of Cambridge, Downing Site, Cambridge, CB2 1QW, UK.
authors: Jean-Daniel
authors: JD
authors: Malcor
authors: Department of Materials Science and Metallurgy, University of Cambridge, 27 Charles Babbage Road, Cambridge, CB3 0FS, UK.
authors: Daniel
authors: D
authors: Bax
authors: Department of Biochemistry, University of Cambridge, Downing Site, Cambridge, CB2 1QW, UK.
authors: Samir W
authors: SW
authors: Hamaia
authors: Department of Materials Science and Metallurgy, University of Cambridge, 27 Charles Babbage Road, Cambridge, CB3 0FS, UK.
authors: Natalia
authors: N
authors: Davidenko
authors: Department of Materials Science and Metallurgy, University of Cambridge, 27 Charles Babbage Road, Cambridge, CB3 0FS, UK.
authors: Serena M
authors: SM
authors: Best
authors: Department of Materials Science and Metallurgy, University of Cambridge, 27 Charles Babbage Road, Cambridge, CB3 0FS, UK.
authors: Ruth E
authors: RE
authors: Cameron
authors: Department of Biochemistry, University of Cambridge, Downing Site, Cambridge, CB2 1QW, UK. Electronic address: rwf10@cam.ac.uk.
authors: Richard W
authors: RW
authors: Farndale
authors: Department of Biochemistry, University of Cambridge, Downing Site, Cambridge, CB2 1QW, UK.
authors: Dominique
authors: D
authors: Bihan
mesh_terms: D001665:Binding Sites; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D024042:Collagen Type I / Q000737:chemistry*; D003978:Diazomethane / Q000494:pharmacology; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000737:chemistry; D006801:Humans; D039222:Integrin alpha1beta1 / Q000737:chemistry*; D038982:Integrin alpha2beta1 / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D013388:Succinimides / Q000737:chemistry; D054457:Tissue Scaffolds / Q000737:chemistry
chemical_list: D024042:Collagen Type I; D039222:Integrin alpha1beta1; D038982:Integrin alpha2beta1; D010455:Peptides; D013388:Succinimides; D003978:Diazomethane; C001426:N-hydroxysuccinimide; D005022:Ethyldimethylaminopropyl Carbodiimide
keywords: Biomimetic material; Cell adhesion; Cell spreading; HT1080; Photoreactive triple-helical peptide; Rugli