title: Mutational analysis of the proteolytic domain of pregnancy-associated plasma protein-A (PAPP-A): classification as a metzincin.
abstract: The bioavailability of insulin-like growth factor (IGF)-I and -II is controlled by six IGF-binding proteins (IGFBPs 1-6). Bound IGF is not active, but proteolytic cleavage of the binding protein causes release of IGF. Pregnancy-associated plasma protein-A (PAPP-A) has recently been found to cleave IGFBP-4 in an IGF-dependent manner. To experimentally support the hypothesis that PAPP-A belongs to the metzincin superfamily of metalloproteinases, all containing the elongated zinc-binding motif HEXXHXXGXXH (His-482-His-492 in PAPP-A), we expressed mutants of PAPP-A in mammalian cells. Substitution of Glu-483 with Ala causes a complete loss of activity, defining this motif as part of the active site of PAPP-A. Interestingly, a mutant with Glu-483 replaced by Gln shows residual activity. Known metzincin structures contain a so-called Met-turn, whose strictly conserved Met residue is thought to interact directly with residues of the active site. By further mutagenesis we provide experimental evidence that Met-556 of PAPP-A, 63 residues from the zinc-binding motif, is located in a Met-turn of PAPP-A. Our hypothesis is also supported by secondary-structure prediction, and the ability of a 55-residue deletion mutant (d[S498-Y552]) to express and retain antigenecity. However, because PAPP-A differs in the features defining the individual established metzincin families, we suggest that PAPP-A belongs to a separate family. We also found that PAPP-A can undergo autocleavage, and that autocleaved PAPP-A is inactive. A lack of unifying elements in the sequences around the found cleavage sites of PAPP-A and a variant suggests steric regulation of substrate specificity.
authors.affiliation: Department of Molecular and Structural Biology, University of Aarhus, Science Park, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark.
authors.forename: H B
authors.initials: HB
authors.lastname: Boldt
authors.forename: M T
authors.initials: MT
authors.lastname: Overgaard
authors.forename: L S
authors.initials: LS
authors.lastname: Laursen
authors.forename: K
authors.initials: K
authors.lastname: Weyer
authors.forename: L
authors.initials: L
authors.lastname: Sottrup-Jensen
authors.forename: C
authors.initials: C
authors.lastname: Oxvig
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D004252:DNA Mutational Analysis; D006801:Humans; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000378:metabolism; D008666:Metalloendopeptidases / Q000737:chemistry; D008969:Molecular Sequence Data; D011266:Pregnancy-Associated Plasma Protein-A / Q000737:chemistry* / Q000145:classification* / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D015032:Zinc / Q000737:chemistry
chemical_list: D018974:Insulin-Like Growth Factor Binding Protein 4; D008666:Metalloendopeptidases; D011266:Pregnancy-Associated Plasma Protein-A; D015032:Zinc