Self-assembling multimeric integrin alpha5beta1 ligands for cell attachment and spreading.
Substrates utilising clustered arginine-glycine-aspartic acid (RGD) ligand displays support greater cell adhesion over random displays. However, cell adhesion to integrin alpha5beta1 requires the synergy site on the 9th type III fibronectin domain (FIII) in addition to RGD on the 10th FIII domain. Here, we have designed and expressed soluble protein chimeras consisting of an N-terminal 9th-10th FIII domain pair, IgG-derived hinge and leucine zipper-derived helix; the latter mutated to yield di-, tri- and tetrameric coiled coils and thus self-assembling, multimeric integrin alpha5beta1 ligands. A unique C-terminal cysteine was appended to the helix to facilitate 'anchoring' of the chimeras with a defined orientation on a surface. Size-exclusion chromatography and circular dichroism demonstrated that the chimeras self-assembled as multimers in solution with defined secondary structures predicted from theoretical calculations. Biotinylation via a thioether bond was used to selectively bind the chimeras to streptavidin-coated surfaces, each of which was then shown to bind integrin alpha5beta1 by surface plasmon resonance. Spreading of fibroblasts to surfaces derivatised with the chimeras was found to proceed in the order: tetramer > trimer > dimer > monomer. Thus, we describe novel polyvalent integrin alpha5beta1 ligands for facile derivatisation of substrates to improve cell adhesion in vitro.
Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow, UK.
M
M
Kreiner
Z
Z
Li
J
J
Beattie
S M
SM
Kelly
H J
HJ
Mardon
C F
CF
van der Walle
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002448:Cell Adhesion / Q000502:physiology; D002460:Cell Line; D048430:Cell Shape / Q000502:physiology; D019281:Dimerization; D005353:Fibronectins / Q000737:chemistry* / Q000378:metabolism; D039081:Integrin alpha5beta1 / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism
D005353:Fibronectins; D039081:Integrin alpha5beta1; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; C501364:fibronectin type III like peptide, human; C047981:arginyl-glycyl-aspartic acid