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

• Published in: Biochimica et biophysica acta Vol. 1770; no. 2; pp. 178 - 186
• Main Authors: Martens, Erik, Leyssen, An, Van Aelst, Ilse, Fiten, Pierre, Piccard, Helene, Hu, Jialiang, Descamps, Francis J., Van den Steen, Philippe E., Proost, Paul, Van Damme, Jo, Liuzzi, Grazia Maria, Riccio, Paolo, Polverini, Eugenia, Opdenakker, Ghislain
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.02.2007
• Subjects: Amino Acid Sequence; Animals; Antibodies, Monoclonal - pharmacology; Binding Sites; Catalytic Domain; Cloning, Molecular; DNA, Complementary - genetics; Domain mapping; Enzyme Inhibitors - pharmacology; Fibronectins - chemistry; Fibronectins - metabolism; Gelatinase B; Genetic Variation; Humans; Hydrophilicity; Insecta; Matrix Metalloproteinase 9 - chemistry; Matrix Metalloproteinase 9 - genetics; Matrix Metalloproteinase Inhibitors; Matrix metalloproteinase-9; Molecular Sequence Data; Monoclonal antibody; Peptide Fragments - chemistry; Protease Inhibitors - pharmacology; Recombinant Proteins - antagonists & inhibitors; Recombinant Proteins - chemistry; Zinc - metabolism; Matrix metalloproteinase-9; MMP; ΔOG; SDS-PAGE; Hydrophilicity; ΔOGHem; Monoclonal antibody; EDTA; ΔHem; PVDF; Gelatinase B; Domain mapping
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2006.10.012

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.