title: Complex of pregnancy-associated plasma protein-A and the proform of eosinophil major basic protein. Disulfide structure and carbohydrate attachment.
abstract: Pregnancy-associated plasma protein-A (PAPP-A) is a metzincin superfamily metalloproteinase responsible for cleavage of insulin-like growth factor-binding protein-4, thus causing release of bound insulin-like growth factor. PAPP-A is secreted as a dimer of 400 kDa but circulates in pregnancy as a disulfide-bound 500-kDa 2:2 complex with the proform of eosinophil major basic protein (pro-MBP), recently shown to function as a proteinase inhibitor of PAPP-A. Except for PAPP-A2, PAPP-A does not share global similarity with other proteins. Three lin-notch (LNR or LIN-12) modules and five complement control protein modules (also known as SCR modules) have been identified in PAPP-A by sequence similarity with other proteins, but no data are available that allow unambiguous prediction of disulfide bonds of these modules. To establish the connectivities of cysteine residues of the PAPP-A.pro-MBP complex, biochemical analyses of peptides derived from purified protein were performed. The PAPP-A subunit contains a total of 82 cysteine residues, of which 81 have been accounted for. The pro-MBP subunit contains 12 cysteine residues, of which 10 have been accounted for. Within the 2:2 complex, PAPP-A is dimerized by a single disulfide bond; pro-MBP is dimerized by two disulfides, and each PAPP-A subunit is connected to a pro-MBP subunit by two disulfide bonds. All other disulfides are intrachain bridges. We also show that of 13 potential sites for N-linked carbohydrate substitution of the PAPP-A subunit, 11 are occupied. The large number of disulfide bonds of the PAPP-A.pro-MBP complex imposes many restraints on polypeptide folding, and knowledge of the disulfide pattern of PAPP-A will facilitate structural studies based on recombinant expression of individual, putative PAPP-A domains. Furthermore, it will allow rational experimental design of functional studies aimed at understanding the formation of the PAPP-A.pro-MBP complex, as well as the inhibitory mechanism of pro-MBP.
authors.affiliation: Department of Molecular Biology, Science Park, University of Aarhus, Gustav Wieds Vej 10C, Denmark.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Overgaard
authors.forename: Esben S
authors.initials: ES
authors.lastname: Sorensen
authors.forename: Damian
authors.initials: D
authors.lastname: Stachowiak
authors.forename: Henning B
authors.initials: HB
authors.lastname: Boldt
authors.forename: Lene
authors.initials: L
authors.lastname: Kristensen
authors.forename: Lars
authors.initials: L
authors.lastname: Sottrup-Jensen
authors.forename: Claus
authors.initials: C
authors.lastname: Oxvig
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry* / Q000378:metabolism; D015153:Blotting, Western; D002241:Carbohydrates / Q000737:chemistry; D002412:Cations; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003488:Cyanogen Bromide / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D047091:Eosinophil Granule Proteins; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D011266:Pregnancy-Associated Plasma Protein-A / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012260:Ribonucleases; D017386:Sequence Homology, Amino Acid; D014162:Transfection
chemical_list: D000596:Amino Acids; D001798:Blood Proteins; D002241:Carbohydrates; D002412:Cations; D018076:DNA, Complementary; D004220:Disulfides; D047091:Eosinophil Granule Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012260:Ribonucleases; D011266:Pregnancy-Associated Plasma Protein-A; D003545:Cysteine; D003488:Cyanogen Bromide