Identification of a Negatively Charged Peptide Motif within the Catalytic Domain of Progelatinases That Mediates Binding to Leukocyte β2 Integrins

The αMβ2 integrin of leukocytes can bind a variety of ligands. We screened phage display libraries to isolate peptides that bind to the αM 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 β2 integrin ligands and also in the catalytic domain of matrix metalloproteinases (MMPs). We show that purified β2 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 β2 integrin-binding site. Furthermore, a synthetic DDGW-containing phage display peptide inhibited the ability of β2 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 αMβ2 and αLβ2 integrins in leukocytes, and pro-MMP-9 colocalized with αMβ2 in cell surface protrusions. The DDGW peptide and the gelatinase-specific inhibitor peptide CTTHWGFTLC blocked β2 integrin-dependent leukocyte migration in a transwell assay. These results suggest that leukocytes may move in a progelatinase-β2 integrin complex-dependent manner.