Complex of pregnancy-associated plasma protein-A and the proform of eosinophil major basic protein. Disulfide structure and carbohydrate attachment

J Biol Chem. 2003 Jan 24;278(4):2106-17. doi: 10.1074/jbc.M208777200. Epub 2002 Nov 5.


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 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 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 complex, as well as the inhibitory mechanism of pro-MBP.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acids / chemistry
  • Animals
  • Blood Proteins / chemistry*
  • Blood Proteins / metabolism
  • Blotting, Western
  • Carbohydrates / chemistry
  • Cations
  • Chromatography, Gel
  • Chromatography, High Pressure Liquid
  • Chromatography, Ion Exchange
  • Cyanogen Bromide / pharmacology
  • Cysteine / chemistry
  • DNA, Complementary / metabolism
  • Disulfides / chemistry
  • Electrophoresis, Polyacrylamide Gel
  • Eosinophil Granule Proteins
  • Humans
  • Mass Spectrometry
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Peptides / chemistry
  • Pregnancy-Associated Plasma Protein-A / chemistry*
  • Pregnancy-Associated Plasma Protein-A / metabolism
  • Protein Binding
  • Protein Folding
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Proteins / chemistry
  • Ribonucleases*
  • Sequence Homology, Amino Acid
  • Transfection


  • Amino Acids
  • Blood Proteins
  • Carbohydrates
  • Cations
  • DNA, Complementary
  • Disulfides
  • Eosinophil Granule Proteins
  • Peptides
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • Ribonucleases
  • Pregnancy-Associated Plasma Protein-A
  • Cysteine
  • Cyanogen Bromide