| Inhibition of leukocyte infiltration to the site of injury is an important therapeutic target against inflammatory diseases. Our laboratory has successfully used synthetic peptides derived from larger extracellular matrix proteins as anti-inflammatory agents. One such peptide derived from fibronectin, FN-C/H V-Y (sequence WQPPRARIY), has substantial anti-inflammatory activity in vivo as measured, in part, by inhibition of leukocyte infiltration to the site of injury. Previous studies in vitro demonstrated that FN- C/H V-Y inhibited leukocyte cell adhesion, however the specific cell adhesion molecule(s) recognized by FN-C/H V-Y were not identified. My thesis project has focused on identifying the molecular target for FN-C/H V-Y in an effort to understand the molecular mechanism of action of this peptide. In chapter 2, it is established that FN-C/H V-Y specifically inhibits {dollar}beta{dollar}1 integrins, which are important cell adhesion molecules. Furthermore, the peptide is determined to directly bind {dollar}alphabeta{dollar}1 integrin heterodimers thereby inhibiting integrin/ligand binding. Structure/activity relationship studies of the primary amino acids within FN-C/H V-Y are presented in chapter 3. Here, the C-terminal isoleucine-tyrosine residues of FN-C/H V-Y are demonstrated to be necessary and sufficient for inhibition of {dollar}alpha4beta{dollar}1 and {dollar}alpha5beta{dollar}1 integrin-mediated ligand binding and cell adhesion. Furthermore, conservative amino acid substitutions were used to demonstrate that C-terminal isoleucine-tyrosine represents a novel di-amino acid motif consisting of a penultimate branched, hydrophobic amino followed by an aromatic ring-containing amino acid. Peptides containing this C-terminal motif are active at inhibiting {dollar}beta{dollar}1 integrin-mediated cell adhesion and ligand binding. Finally, in chapter 4, the in vivo neuroprotective activity of C-terminal isoleucine-tyrosine is confirmed using a rat model of cerebral ischemia/reperfusion injury. Collectively, these studies define a novel anti-cell adhesion motif that may serve as a lead compound for the development of anti-adhesive peptidomimetics. |
