BACKGROUND: Maternal serum concentrations of pregnancy-associated plasma protein-A (PAPP-A, pappalysin-1, EC 3.4.24.79) are used to predict the occurrence of Down syndrome. In pregnancy, PAPP-A primarily circulates as a covalent 2:2 complex with the proform of eosinophil major basic protein (proMBP), which inhibits the proteolytic activity of PAPP-A. At term, however, approximately 1% of PAPP-A exists as an active, uncomplexed dimer with proteolytic activity directed specifically toward insulin-like growth factor binding protein (IGFBP)-4 and IGFBP-5. No assays have been developed that allow quantification of PAPP-A proteolytic activity. METHODS: We developed a sensitive and specific immunocapture assay for PAPP-A activity based on intramolecular quenched fluorescence. We used a 26-residue synthetic peptide derived from IGFBP-4 in which specific positions on each side of the PAPP-A cleavage site were substituted with 3-nitrotyrosine and o-aminobenzoic acid. RESULTS: The assay detected the activity of recombinant PAPP-A as well as PAPP-A in serum samples from pregnant women. The limit of detection (mean signal of blank plus 3 SD) of the active PAPP-A subunit was 13 pmol/L, and the intra- and interassay CVs were <10% and <15%, respectively. Interestingly, the fraction of active PAPP-A decreased gradually from week 7 to week 19 of pregnancy. CONCLUSIONS: This method allows the measurement of PAPP-A enzymatic activity and because of its specificity it is relevant to the study of the biological function of PAPP-A. The method may also be useful in the diagnosis of pregnancy disorders.
BACKGROUND: Maternal serum concentrations of pregnancy-associated plasma protein-A (PAPP-A, pappalysin-1, EC 3.4.24.79) are used to predict the occurrence of Down syndrome. In pregnancy, PAPP-A primarily circulates as a covalent 2:2 complex with the proform of eosinophil major basic protein (proMBP), which inhibits the proteolytic activity of PAPP-A. At term, however, approximately 1% of PAPP-A exists as an active, uncomplexed dimer with proteolytic activity directed specifically toward insulin-like growth factor binding protein (IGFBP)-4 and IGFBP-5. No assays have been developed that allow quantification of PAPP-A proteolytic activity. METHODS: We developed a sensitive and specific immunocapture assay for PAPP-A activity based on intramolecular quenched fluorescence. We used a 26-residue synthetic peptide derived from IGFBP-4 in which specific positions on each side of the PAPP-A cleavage site were substituted with 3-nitrotyrosine and o-aminobenzoic acid. RESULTS: The assay detected the activity of recombinant PAPP-A as well as PAPP-A in serum samples from pregnant women. The limit of detection (mean signal of blank plus 3 SD) of the active PAPP-A subunit was 13 pmol/L, and the intra- and interassay CVs were <10% and <15%, respectively. Interestingly, the fraction of active PAPP-A decreased gradually from week 7 to week 19 of pregnancy. CONCLUSIONS: This method allows the measurement of PAPP-A enzymatic activity and because of its specificity it is relevant to the study of the biological function of PAPP-A. The method may also be useful in the diagnosis of pregnancy disorders.
Authors: Søren Kløverpris; Jakob H Mikkelsen; Josefine H Pedersen; Malene R Jepsen; Lisbeth S Laursen; Steen V Petersen; Claus Oxvig Journal: J Biol Chem Date: 2015-07-20 Impact factor: 5.157
Authors: Sara Dam Kobberø; Michael Gajhede; Osman Asghar Mirza; Søren Kløverpris; Troels Rønn Kjær; Jakob Hauge Mikkelsen; Thomas Boesen; Claus Oxvig Journal: Nat Commun Date: 2022-10-18 Impact factor: 17.694
Authors: Raziel Rojas-Rodriguez; Rachel Ziegler; Tiffany DeSouza; Sana Majid; Aylin S Madore; Nili Amir; Veronica A Pace; Daniel Nachreiner; David Alfego; Jomol Mathew; Katherine Leung; Tiffany A Moore Simas; Silvia Corvera Journal: Sci Transl Med Date: 2020-11-25 Impact factor: 17.956
Authors: Clive J Petry; Ken K Ong; Ieuan A Hughes; Carlo L Acerini; Jan Frystyk; David B Dunger Journal: J Clin Endocrinol Metab Date: 2017-06-01 Impact factor: 5.958