Identification of a negatively charged peptide motif within the catalytic domain of progelatinases that mediates binding to leukocyte beta 2 integrins

• Published in: The Journal of biological chemistry 2003-09, Vol.278 (36), p.34674
• Main Authors: Stefanidakis, Michael, Bjorklund, Mikael, Ihanus, Eveliina, Gahmberg, Carl G, Koivunen, Erkki
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Binding Sites; Catalytic Domain; CD18 Antigens - metabolism; Cell Adhesion; Cell Line; Cell Movement; Dose-Response Relationship, Drug; Enzyme Precursors - chemistry; Fibrinogen - metabolism; Gelatinases - chemistry; Glutathione Transferase - metabolism; Humans; Immunoblotting; Integrins - chemistry; Integrins - metabolism; Intercellular Adhesion Molecule-1 - metabolism; Jurkat Cells; Leukocytes - metabolism; Ligands; Matrix Metalloproteinase 2 - chemistry; Matrix Metalloproteinase 9 - chemistry; Matrix Metalloproteinase 9 - metabolism; Metalloendopeptidases - chemistry; Microscopy, Fluorescence; Molecular Sequence Data; Peptide Library; Peptides - chemistry; Precipitin Tests; Protein Binding; Protein Structure, Tertiary; Time Factors; Tumor Cells, Cultured
• ISSN: 0021-9258

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 and 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.