Identification of a negatively charged peptide motif within the catalytic domain of progelatinases that mediates binding to leukocyte β2 integrins

The alpha(M)beta(2) integrin of leukocytes can bind a variety of ligands. We screened phage display libraries to isolate peptides that bind to the alpha(M) I domain, the principal ligand binding site of the integrin. Only one peptide motif, (D/E)(D/E)(G/L)W, was obtained with this approach despite the known ligand binding promiscuity of the I domain. Interestingly, such negatively charged sequences are present in many known beta(2) integrin ligands also in the catalytic domain of matrix metalloproteinases (MMPs). We show that purified beta(2) integrins bind to pro-MMP-2 and pro-MMP-9 gelatinases and that that the negatively charged sequence of the MMP catalytic domain is an active beta(2) integrin-binding site. Furthermore, a synthetic DDGW-containing phage display peptide inhibited the ability of beta(2) integrin to bind progelatinases but did not inhibit the binding of cell adhesion-mediating substrates such as intercellular adhesion molecule-1, fibrinogen, or an LLG-containing peptide. Immunoprecipitation and cell surface labeling demonstrated complexes of pro-MMP-9 with both the alpha(M)beta(2) and alpha(L)beta(2) integrins in leukocytes, and pro-MMP-9 colocalized with alpha(M)beta(2) in cell surface protrusions. The DDGW peptide and the gelatinase-specific inhibitor peptide CTTHWGFTLC blocked beta(2) integrin-dependent leukocyte migration in a transwell assay. These results suggest that leukocytes may move in a progelatinase-beta(2) integrin complex-dependent manner.