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PEDS Advance Access originally published online on May 30, 2008
Protein Engineering Design and Selection 2008 21(9):553-560; doi:10.1093/protein/gzn032
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Self-assembling multimeric integrin {alpha}5β1 ligands for cell attachment and spreading

M. Kreiner1, Z. Li2, J. Beattie1, S.M. Kelly3, H.J. Mardon2 and C.F. van der Walle1,4

1Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow 2Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital, Headington, Oxford 3Faculty of Biomedical and Life Sciences, Joseph Black Building, University of Glasgow, Glasgow, UK

4 To whom correspondence should be addressed. E-mail: chris.walle{at}strath.ac.uk

Substrates utilising clustered arginine–glycine–aspartic acid (RGD) ligand displays support greater cell adhesion over random displays. However, cell adhesion to integrin {alpha}5β1 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 {alpha}5β1 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 {alpha}5β1 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 {alpha}5β1 ligands for facile derivatisation of substrates to improve cell adhesion in vitro.

Keywords: cell adhesion/coiled coil/fibronectin/integrin/polyvalency

Received January 28, 2008; revised April 3, 2008; accepted May 7, 2008.


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