title: Substrate specificity of the metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) assessed by mutagenesis and analysis of synthetic peptides: substrate residues distant from the scissile bond are critical for proteolysis.
abstract: Human pregnancy-associated plasma protein-A (PAPP-A) cleaves insulin-like growth factor (IGF) binding protein-4 (IGFBP-4), causing a dramatic reduction in its affinity for IGF-I and -II. Through this mechanism, PAPP-A is a regulator of IGF bioactivity in several systems, including the human ovary and the cardiovascular system. PAPP-A belongs to the metzincin superfamily of zinc metalloproteinases, and is the founding member of a fifth metzincin family, the pappalysins. Herein, we first determined that PAPP-A cleaves IGFBP-4 at a single site (Met-135/Lys-136), and we analysed the influence of ionic strength, pH and zinc ion concentration on the cleavage reaction. Secondly, we sought to delineate the role of substrate residues in PAPP-A-mediated cleavage by the construction and analysis of 30 IGFBP-4 mutants in which various residues were replaced by alanine, by the analysis of eight mutants of IGFBP-5 (found recently to be a second PAPP-A substrate), and by cleavage analysis of synthetic peptides derived from IGFBP-4. Our data reveal a complex mode of substrate recognition and/or binding, pointing at important roles for several basic residues located up to 16 residues N-terminal to the scissile bond. An unexpected parallel can be drawn with an intracellular enzyme, the mitochondrial processing peptidase, that may help us to understand properties of the pappalysins. Further, proteinase-resistant variants of IGFBP-4 and -5, presented here, will be useful tools for the study of proteolysis in cell-based systems, and our finding that a synthetic peptide can be cleaved by PAPP-A provides the basis for development of quantitative assays for the investigation of PAPP-A enzyme kinetics.
authors.affiliation: Department of Molecular and Structural Biology, Science Park, University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark.
authors.forename: Lisbeth S
authors.initials: LS
authors.lastname: Laursen
authors.forename: Michael T
authors.initials: MT
authors.lastname: Overgaard
authors.forename: Claus G
authors.initials: CG
authors.lastname: Nielsen
authors.forename: Henning B
authors.initials: HB
authors.lastname: Boldt
authors.forename: Kathrin H
authors.initials: KH
authors.lastname: Hopmann
authors.forename: Cheryl A
authors.initials: CA
authors.lastname: Conover
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: D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000378:metabolism; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000378:metabolism; D007477:Ions; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D011266:Pregnancy-Associated Plasma Protein-A / Q000737:chemistry* / Q000378:metabolism; D011506:Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014162:Transfection; D015032:Zinc / Q000378:metabolism
chemical_list: D018974:Insulin-Like Growth Factor Binding Protein 4; D018975:Insulin-Like Growth Factor Binding Protein 5; D007477:Ions; D010455:Peptides; D011506:Proteins; D011266:Pregnancy-Associated Plasma Protein-A; D015032:Zinc