title: Identification of a negatively charged peptide motif within the catalytic domain of progelatinases that mediates binding to leukocyte beta 2 integrins.
abstract: 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.
authors.affiliation: Department of Biosciences, Division of Biochemistry, Viikinkaari 5, University of Helsinki, FIN-00014 Helsinki, Finland.
authors.forename: Michael
authors.initials: M
authors.lastname: Stefanidakis
authors.forename: Mikael
authors.initials: M
authors.lastname: Bjorklund
authors.forename: Eveliina
authors.initials: E
authors.lastname: Ihanus
authors.forename: Carl G
authors.initials: CG
authors.lastname: Gahmberg
authors.forename: Erkki
authors.initials: E
authors.lastname: Koivunen
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D018821:CD18 Antigens / Q000378:metabolism*; D020134:Catalytic Domain; D002448:Cell Adhesion; D002460:Cell Line; D002465:Cell Movement; D004305:Dose-Response Relationship, Drug; D004792:Enzyme Precursors / Q000737:chemistry*; D005340:Fibrinogen / Q000378:metabolism; D018093:Gelatinases / Q000737:chemistry*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D016023:Integrins / Q000737:chemistry / Q000378:metabolism; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D019169:Jurkat Cells; D007962:Leukocytes / Q000378:metabolism*; D008024:Ligands; D020778:Matrix Metalloproteinase 2 / Q000737:chemistry; D020780:Matrix Metalloproteinase 9 / Q000737:chemistry / Q000378:metabolism; D008666:Metalloendopeptidases / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry*; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014407:Tumor Cells, Cultured
chemical_list: D018821:CD18 Antigens; D004792:Enzyme Precursors; D016023:Integrins; D008024:Ligands; D019151:Peptide Library; D010455:Peptides; D018799:Intercellular Adhesion Molecule-1; D005340:Fibrinogen; D005982:Glutathione Transferase; D018093:Gelatinases; D008666:Metalloendopeptidases; C069860:progelatinase; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9