Quantification of Proteolytically Active Pregnancy-Associated Plasma Protein-A with an Assay Based on Quenched Fluorescence

Abstract 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, ∼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.

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Immunofluorometric Point-of-Care Assays for the Detection of Acute Coronary Syndrome-Related Noncomplexed Pregnancy-Associated Plasma Protein A

Clinical Chemistry ◽

10.1373/clinchem.2006.070607 ◽

2006 ◽

Vol 52 (9) ◽

pp. 1794-1801 ◽

Cited By ~ 30

Author(s):

Saara Wittfooth ◽

Qiu-Ping Qin ◽

Juha Lund ◽

Ilkka Tierala ◽

Kari Pulkki ◽

...

Keyword(s):

Detection Limit ◽

Plasma Protein ◽

Specific Antibody ◽

Point Of Care ◽

Protein A ◽

Serum Samples ◽

Coronary Syndrome ◽

A Subunit ◽

The Difference ◽

Eosinophil Major Basic Protein

Abstract Background: We recently reported that the pregnancy-associated plasma protein A (PAPP-A) form specifically related to acute coronary syndromes (ACS) is not complexed with the proform of eosinophil major basic protein (proMBP). The aim of this study was to develop rapid point-of-care immunoassays for the measurement of the noncomplexed PAPP-A. Methods: We developed immunofluorometric noncompetitive dry-reagent assays for total PAPP-A with 2 PAPP-A subunit-specific monoclonal antibodies and for PAPP-A/proMBP complex with 1 PAPP-A subunit-specific antibody and 1 proMBP subunit-specific antibody. The concentration of noncomplexed PAPP-A was determined as the difference of the results obtained with the 2 assays. Results: The assays were linear from 0.5 to 300 mIU/L. The analytical detection limit and functional detection limit (CV <20%) were 0.18 mIU/L and 0.27 mIU/L for total PAPP-A assay and 0.23 mIU/L and 0.70 mIU/L for PAPP-A/proMBP assay, respectively. The total assay imprecisions were <10%, and recoveries were 88%–107% for both assays. The mean difference (95% limits of agreement) between the new total PAPP-A assay and a previously reported total PAPP-A assay was −3.2% (−45.7% to 39.3%; n = 546; P = 0.0019). In serum samples from 159 non-ACS individuals, median concentrations (interquartile range) were 2.42 (1.14) mIU/L for total PAPP-A, 2.20 (1.18) mIU/L for PAPP-A/proMBP, and 0.18 (0.63) mIU/L for noncomplexed PAPP-A. Total PAPP-A and PAPP-A/proMBP, but not noncomplexed PAPP-A, correlated with age (r = 0.290, P = 0.0002; r = 0.230, P = 0.0035; r = 0.075, P = 0.3483, respectively). Conclusions: The new assays described revealed that noncomplexed PAPP-A is found only in negligible amounts in non-ACS samples.

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Studies on regulation of IGF (insulin-like growth factor)-binding protein (IGFBP) 4 proteolysis by pregnancy-associated plasma protein-A (PAPP-A) in cells treated with phorbol ester

Biochemical Journal ◽

10.1042/bj20030937 ◽

2004 ◽

Vol 379 (1) ◽

pp. 57-64 ◽

Cited By ~ 5

Author(s):

Arun S. SIVANANDAM ◽

Subburaman MOHAN ◽

Hirohito KITA ◽

Sanjay KAPUR ◽

Shin-Tai CHEN ◽

...

Keyword(s):

Growth Factor ◽

Proteolytic Activity ◽

Plasma Protein ◽

Binding Protein ◽

Protein A ◽

Insulin Like Growth Factor ◽

Factor Binding ◽

Fold Reduction ◽

Sds Page ◽

Eosinophil Major Basic Protein

PAPP-A (pregnancy-associated plasma protein-A) is produced by hSFs (human skin fibroblasts) and hOBs (human osteoblasts) and enhances the mitogenic activity of IGFs (insulin-like growth factors) by degradation of IGFBP-4 (insulin-like growth factor-binding protein 4). PKC (protein kinase C) activation in these cells led to reduction in IGFBP-4 proteolysis. This study was undertaken to determine the mechanism by which activation of PKC suppresses IGFBP-4 proteolysis. Treatment of hSFs/hOBs with TPA (PMA; 100 nM) reduced IGFBP-4 proteolysis without significantly decreasing the PAPP-A level in the CM (conditioned medium). Immunodepletion of the proform of eosinophil major basic protein (proMBP), a known PAPP-A inhibitor, from CM of TPA-treated cells (TPA CM) failed to increase IGFBP-4 proteolytic activity. Transduction of hSFs with proMBP retrovirus increased the concentration of proMBP up to 30 ng/ml and led to a moderate reduction in IGFBP-4 proteolysis. In contrast, TPA treatment blocked IGFBP-4 proteolysis but failed to induce a detectable amount of proMBP in the CM. While proMBP overexpression led to the formation of a covalent proMBP–PAPP-A complex and reduced the migration of PAPP-A on SDS/PAGE, TPA treatment dose- and time-dependently increased the conversion of a ≈470 kDa PAPP-A form (PAPP-A470) to a ≈400 kDa PAPP-A form (PAPP-A400). Since unreduced PAPP-A400 co-migrated with the 400 kDa recombinant PAPP-A homodimer and since PAPP-A monomers from reduced PAPP-A470 and PAPP-A400 co-migrated on SDS/PAGE, conversion of PAPP-A470 to PAPP-A400 is unlikely to be caused by proteolytic cleavage of PAPP-A. Consistent with the data showing that the increase in the ratio of PAPP-A400/PAPP-A470 is correlated with the extent of reduction in IGFBP-4 proteolysis, partially purified PAPP-A400 exhibited a 4-fold reduction in IGFBP-4 proteolytic activity compared with PAPP-A470. These data suggest that a novel mechanism, namely conversion of PAPP-A470 to the less-active PAPP-A400, could account for the TPA-induced suppression of PAPP-A activity.

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The proform of eosinophil major basic protein: a new maternal serum marker for Down syndrome

Prenatal Diagnosis ◽

10.1002/(sici)1097-0223(199910)19:10<905::aid-pd658>3.0.co;2-q ◽

1999 ◽

Vol 19 (10) ◽

pp. 905-910 ◽

Cited By ~ 20

Author(s):

Michael Christiansen ◽

Claus Oxvig ◽

Jill M. Wagner ◽

Qiu-Ping Qin ◽

Tri H. Nguyen ◽

...

Keyword(s):

Down Syndrome ◽

Basic Protein ◽

Protein A ◽

Serum Marker ◽

Maternal Serum ◽

Major Basic Protein ◽

Eosinophil Major Basic Protein

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Molecular Distinction of Circulating Pregnancy-Associated Plasma Protein A in Myocardial Infarction and Pregnancy

Clinical Chemistry ◽

10.1373/clinchem.2004.036467 ◽

2005 ◽

Vol 51 (1) ◽

pp. 75-83 ◽

Cited By ~ 53

Author(s):

Qiu-Ping Qin ◽

Saara Kokkala ◽

Juha Lund ◽

Natalia Tamm ◽

Liisa-Maria Voipio-Pulkki ◽

...

Keyword(s):

Myocardial Infarction ◽

Pregnant Women ◽

Molecular Mass ◽

Plasma Protein ◽

Specific Antibody ◽

Protein A ◽

Gel Filtration ◽

Single Peak ◽

Serum Samples ◽

A Subunit

Abstract Background: In the blood of pregnant women, pregnancy-associated plasma protein A (PAPP-A) is present as a covalent complex with the proform of eosinophil major basic protein (proMBP). Recently, increased serum concentrations of PAPP-A have been found in acute coronary syndromes (ACS). The aim of this study was to investigate whether the circulating PAPP-A in ACS is the same as that in pregnancy. Methods: We developed two time-resolved immunofluorometric assays based on a relative epitope map constructed by the use of 17 monoclonal antibodies. One assay, which measured total PAPP-A, used two PAPP-A subunit-specific antibodies. The other assay, which measured PAPP-A/proMBP complex, used one proMBP subunit-specific antibody and one PAPP-A subunit-specific antibody. Serum samples from four patients with myocardial infarction (MI), three pregnant women in their first trimester, and one in her third trimester were fractionated by gel filtration on a Superose™ 6 precision column. The two assays were used to analyze fractions obtained by gel filtration as well as serum samples serially collected from four other MI patients. Results: Pregnancy-related PAPP-A was eluted as a single peak with a molecular mass of ∼700 kDa, whereas ACS-related PAPP-A was also eluted as a single peak but with a molecular mass of ∼530 kDa. Pregnancy-related PAPP-A was detected equally by the two assays, whereas increased ACS-related PAPP-A was detected only by the assay for total PAPP-A. Conclusions: Our results provide the first evidence that circulating ACS-related PAPP-A is different from circulating pregnancy-related PAPP-A in that it is not complexed with proMBP. These findings provide a solid foundation for the design of immunoassays to accurately measure atherosclerosis-associated plasma protein A in the circulation.

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PO19-583 LONG-TERM PROGNOSTIC IMPLICATIONS OF CIRCULATING PREGNANCY-ASSOCIATED PLASMA PROTEIN-A AND PROFORM-EOSINOPHIL MAJOR BASIC PROTEIN IN CHRONIC STABLE ANGINA

Atherosclerosis Supplements ◽

10.1016/s1567-5688(07)71593-3 ◽

2007 ◽

Vol 8 (1) ◽

pp. 160

Author(s):

L. Consuegra-Sanchez ◽

I. Petrovic ◽

J. Cosin-Sales ◽

D. Holt ◽

J.C. Kaski

Keyword(s):

Plasma Protein ◽

Stable Angina ◽

Basic Protein ◽

Protein A ◽

Chronic Stable Angina ◽

Major Basic Protein ◽

Prognostic Implications ◽

Eosinophil Major Basic Protein

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Messenger Ribonucleic Acid Levels of Pregnancy-Associated Plasma Protein-A and the Proform of Eosinophil Major Basic Protein: Expression in Human Reproductive and Nonreproductive Tissues1

Biology of Reproduction ◽

10.1095/biolreprod61.4.1083 ◽

1999 ◽

Vol 61 (4) ◽

pp. 1083-1089 ◽

Cited By ~ 75

Author(s):

Michael T. Overgaard ◽

Claus Oxvig ◽

Michael Christiansen ◽

James B. Lawrence ◽

Cheryl A. Conover ◽

...

Keyword(s):

Protein Expression ◽

Plasma Protein ◽

Ribonucleic Acid ◽

Basic Protein ◽

Protein A ◽

Major Basic Protein ◽

Messenger Ribonucleic Acid ◽

Human Reproductive ◽

Eosinophil Major Basic Protein

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The proform of eosinophil major basic protein: a new maternal serum marker for adverse pregnancy outcome

Prenatal Diagnosis ◽

10.1002/pd.2331 ◽

2009 ◽

Vol 29 (11) ◽

pp. 1013-1019 ◽

Cited By ~ 6

Author(s):

Kasper Pihl ◽

Torben Larsen ◽

Steen Rasmussen ◽

Lone Krebs ◽

Michael Christiansen

Keyword(s):

Pregnancy Outcome ◽

Basic Protein ◽

Adverse Pregnancy Outcome ◽

Protein A ◽

Serum Marker ◽

Maternal Serum ◽

Major Basic Protein ◽

Adverse Pregnancy ◽

Eosinophil Major Basic Protein

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Double-monoclonal immunofluorometric assays for pregnancy-associated plasma protein A/proeosinophil major basic protein (PAPP-A/proMBP) complex in first-trimester maternal serum screening for Down syndrome

Clinical Chemistry ◽

10.1093/clinchem/43.12.2323 ◽

1997 ◽

Vol 43 (12) ◽

pp. 2323-2332 ◽

Cited By ~ 56

Author(s):

Qiu-Ping Qin ◽

Michael Christiansen ◽

Claus Oxvig ◽

Kim Pettersson ◽

Lars Sottrup-Jensen ◽

...

Keyword(s):

Down Syndrome ◽

Plasma Protein ◽

Basic Protein ◽

Protein A ◽

First Trimester ◽

Maternal Serum ◽

Major Basic Protein ◽

Maternal Serum Screening ◽

Detection Rates ◽

Serum Screening

Abstract Four double-monoclonal time-resolved immunofluorometric assays (TrIFMAs) have been developed for the specific determination of pregnancy-associated plasma protein A/proeosinophil major basic protein (PAPP-A/proMBP) complex in first-trimester maternal serum samples. The assays have a functional sensitivity of <4 mIU/L and a working range from 4 to 1000 mIU/L. These 4 assays, together with a polyclonal sandwich TrIFMA, were compared for their ability to discriminate between normal pregnancies (n = 149) and pregnancies carrying a Down syndrome fetus (n = 36) in maternal serum screening samples from gestational weeks 4–13. In 26 Down syndrome pregnancies from gestational weeks 7–12, the median PAPP-A multiples of the median concentration in controls (MoMs) determined by monoclonal antibody combinations 234–3/234–2*, 234–4/234–2*, 234–4/234–5*, and 234–5/234–6* were 0.35, 0.37, 0.42, and 0.44, respectively, whereas the median MoM determined by the polyclonal assay was 0.56. ROC curve analysis also showed that better overall diagnostic accuracy and detection rates were achieved by the monoclonal TrIFMAs than by the polyclonal TrIFMA. This report is the first to describe assays that specifically measure PAPP-A/proMBP complex without possible interference from other proMBP-containing complexes.

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