title: A monoclonal antibody inhibits gelatinase B/MMP-9 by selective binding to part of the catalytic domain and not to the fibronectin or zinc binding domains.
abstract: Gelatinase B/matrix metalloproteinase-9 (MMP-9) is a multidomain enzyme functioning in acute and chronic inflammatory and neoplastic diseases. It belongs to a family of more than 20 related zinc proteinases. Therefore, the discovery and the definition of the action mechanism of selective MMP inhibitors form the basis for future therapeutics. The monoclonal antibody REGA-3G12 is a most selective inhibitor of human gelatinase B. REGA-3G12 was found to recognize the aminoterminal part and not the carboxyterminal O-glycosylated and hemopexin protein domains. A variant of gelatinase B, lacking the two carboxyterminal domains, was expressed in insect cells and fragmented with purified proteinases. The fragments were probed by one- and two-dimensional Western blot and immunoprecipitation experiments with REGA-3G12 to map the interactions between the antibody and the enzyme. The interaction unit was identified by Edman degradation analysis as the glycosylated segment from Trp(116) to Lys(214) of gelatinase B. The sequence of this segment was analysed by hydrophobicity/hydrophilicity, accessibility and flexibility profiling. Four hydrophilic peptides were chemically synthesized and used in binding and competition assays. The peptide Gly(171)-Leu(187) in molar excess inhibited partially the binding of MMP-9 to REGA-3G12 and thus refines the structure of the conformational binding site. These results define part of the catalytic domain of gelatinase B/MMP-9, and not the zinc-binding or fibronectin domains, as target for the development of selective inhibitors.
authors.affiliation: Rega Institute for Medical Research, Laboratory of Immunobiology, University of Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
authors.forename: Erik
authors.initials: E
authors.lastname: Martens
authors.forename: An
authors.initials: A
authors.lastname: Leyssen
authors.forename: Ilse
authors.initials: I
authors.lastname: Van Aelst
authors.forename: Pierre
authors.initials: P
authors.lastname: Fiten
authors.forename: Helene
authors.initials: H
authors.lastname: Piccard
authors.forename: Jialiang
authors.initials: J
authors.lastname: Hu
authors.forename: Francis J
authors.initials: FJ
authors.lastname: Descamps
authors.forename: Philippe E
authors.initials: PE
authors.lastname: Van den Steen
authors.forename: Paul
authors.initials: P
authors.lastname: Proost
authors.forename: Jo
authors.initials: J
authors.lastname: Van Damme
authors.forename: Grazia Maria
authors.initials: GM
authors.lastname: Liuzzi
authors.forename: Paolo
authors.initials: P
authors.lastname: Riccio
authors.forename: Eugenia
authors.initials: E
authors.lastname: Polverini
authors.forename: Ghislain
authors.initials: G
authors.lastname: Opdenakker
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D001665:Binding Sites; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005353:Fibronectins / Q000737:chemistry / Q000378:metabolism; D014644:Genetic Variation; D006801:Humans; D007313:Insecta; D020780:Matrix Metalloproteinase 9 / Q000737:chemistry / Q000235:genetics; D061965:Matrix Metalloproteinase Inhibitors; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D015032:Zinc / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D005353:Fibronectins; D061965:Matrix Metalloproteinase Inhibitors; D010446:Peptide Fragments; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D020780:Matrix Metalloproteinase 9; D015032:Zinc