Chemistry and biology at the interface: Tailored materials for biotechnology

This thesis describes studies that use models of the extracellular matrix to understand the receptor-mediated adhesion of mammalian cells. This work developed self-assembled monolayers of alkanethiolates on gold as a flexible model surface to present ligand for cell adhesion. This methodology was applied to studies of cell adhesion to fibronectin and angiopoietin.; This work investigated the role for the synergy peptide PHSRN in mediating the adhesion of cells. The attachment of baby hamster kidney cells and 3T3 Swiss fibroblasts to model substrates presenting either the peptide GRGDS or PHSRN was evaluated. Both cell types attached efficiently to monolayers presenting either the RGD or the PHSRN peptide. Cell attachment was comparable on substrates presenting either peptide ligand, but less efficient than on substrates presenting the protein fibronectin. The degree of cell spreading, however, was substantially higher on substrates presenting RGD relative to PHSRN. Staining of 3T3 fibroblasts revealed clear cytoskeletal filaments and focal adhesions for cells attached by way of either RGD or PHSRN. Inhibition experiments showed that the attachment of 3T3 fibroblasts to monolayers presenting RGD could be inhibited completely by a soluble RGD peptide and partially by a soluble PHSRN peptide. IMR 90 fibroblast attachment to monolayers presenting PHSRN can be inhibited with either anti-integrin α₅ or anti-integrin β ₁ antibody. No cells attached to monolayers presenting scrambled peptide GRDGS or HRPSN. This work demonstrated unambiguously that PHSRN alone can support the attachment of cells and that the RGD and PHSRN bind competitively to the integrin receptors.; In a second project, this methodology was used to identify a cell-binding sequence in Ang-1, EMAQIQQ. A peptide array presenting twelve overlapping peptides spanning the Ang-1 cell-binding domain was prepared and used to show that both HUVECs and 3T3 Swiss fibroblasts bind only to one of these peptides, KSEMAQIQQNAVQNH. Cell adhesion was evaluated for several related peptides—obtained by truncating the original sequence and introducing point mutations—to show that EMAQIQQ is the cell-binding site in Ang-1. Further studies with scrambled and mutant peptides confirmed EMAQIQQ as the minimum cell-binding sequence in Ang-1. Three cell lines, HUVECs, 3T3 Swiss fibroblasts and CHO B2 cells, were shown to bind to monolayers presenting this short 7-residue sequence. This work demonstrated that the model substrate approach could be a robust tool in mechanistic cell biology studies.