title: Proteinase inhibition by proform of eosinophil major basic protein (pro-MBP) is a multistep process of intra- and intermolecular disulfide rearrangements.
abstract: The metzincin metalloproteinase pregnancy-associated plasma protein A (PAPP-A, pappalysin-1) promotes cell growth by the cleavage of insulin-like growth factor-binding proteins-4 and -5, causing the release of bound insulin-like growth factors. The proteolytic activity of PAPP-A is inhibited by the proform of eosinophil major basic protein (pro-MBP), which forms a covalent 2:2 proteinase-inhibitor complex based on disulfide bonds. To understand the process of complex formation, we determined the status of cysteine residues in both of the uncomplexed molecules. A comparison of the disulfide structure of the reactants with the known disulfide structure of the PAPP-A.pro-MBP complex reveals that six cysteine residues of the pro-MBP subunit (Cys-51, Cys-89, Cys-104, Cys-107, Cys-128, and Cys-169) and two cysteine residues of the PAPP-A subunit (Cys-381 and Cys-652) change their status from the uncomplexed to the complexed states. Upon complex formation, three disulfide bonds of pro-MBP, which connect the acidic propiece with the basic, mature portion, are disrupted. In the PAPP-A.pro-MBP complex, two of these form the basis of both two interchain disulfide bonds between the PAPP-A and the pro-MBP subunits and two disulfide bonds responsible for pro-MBP dimerization, respectively. Based on the status of the reactants, we investigated the role of individual cysteine residues upon complex formation by mutagenesis of specific cysteine residues of both subunits. Our findings allow us to depict a hypothetical model of how the PAPPA.pro-MBP complex is formed. In addition, we have demonstrated that complex formation is greatly enhanced by the addition of micromolar concentrations of reductants. It is therefore possible that the activity in vivo of PAPP-A is controlled by the redox potential, and it is further tempting to speculate that such mechanism operates under pathological conditions of altered redox potential.
authors.affiliation: Department of Molecular Biology, Science Park, University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark.
authors.forename: Simon
authors.initials: S
authors.lastname: Glerup
authors.forename: Henning B
authors.initials: HB
authors.lastname: Boldt
authors.forename: Michael T
authors.initials: MT
authors.lastname: Overgaard
authors.forename: Lars
authors.initials: L
authors.lastname: Sottrup-Jensen
authors.forename: Linda C
authors.initials: LC
authors.lastname: Giudice
authors.forename: Claus
authors.initials: C
authors.lastname: Oxvig
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D049109:Cell Proliferation; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D016207:Cytokines / Q000737:chemistry; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D047093:Eosinophil Major Basic Protein / Q000502:physiology*; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D008956:Models, Chemical; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010447:Peptide Hydrolases / Q000494:pharmacology*; D010455:Peptides / Q000737:chemistry; D011266:Pregnancy-Associated Plasma Protein-A / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011498:Protein Precursors / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014162:Transfection
chemical_list: D016207:Cytokines; D004220:Disulfides; D004791:Enzyme Inhibitors; D010455:Peptides; D011498:Protein Precursors; C119358:prepro-major basic protein homolog; D047093:Eosinophil Major Basic Protein; D010447:Peptide Hydrolases; D011266:Pregnancy-Associated Plasma Protein-A; D005978:Glutathione; D003545:Cysteine; D010100:Oxygen