Cytological analysis of pregnancy-associated plasma protein-A expression in porcine neonatal testis

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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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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Maternal Diabetes, Large-for-Gestational-Age Births, and First Trimester Pregnancy–Associated Plasma Protein-A

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2014-4103 ◽

2015 ◽

Vol 100 (6) ◽

pp. 2372-2379 ◽

Cited By ~ 18

Author(s):

George Wells ◽

Kerrin Bleicher ◽

Xuguang Han ◽

Monika McShane ◽

Yuk Fun Chan ◽

...

Keyword(s):

Plasma Protein ◽

Gestational Age ◽

Protein A ◽

Maternal Diabetes ◽

First Trimester ◽

Large For Gestational Age ◽

First Trimester Pregnancy ◽

Trimester Pregnancy

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OC23.02: Pregnancy-associated plasma protein A levels and neonatal complications in post-term pregnancies

Ultrasound in Obstetrics and Gynecology ◽

10.1002/uog.11372 ◽

2012 ◽

Vol 40 (S1) ◽

pp. 48-48

Author(s):

A. R. Zizzo ◽

I. Kirkegaard ◽

T. B. Heniksen ◽

N. Uldbjerg

Keyword(s):

Plasma Protein ◽

Protein A ◽

Neonatal Complications

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Insulin-Like Growth Factor Bioactivity, Stanniocalcin-2, Pregnancy-Associated Plasma Protein-A, and IGF-Binding Protein-4 in Pleural Fluid and Serum From Patients With Pulmonary Disease

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2017-00033 ◽

2017 ◽

Vol 102 (9) ◽

pp. 3526-3534 ◽

Cited By ~ 16

Author(s):

Ulrick Skipper Espelund ◽

Mette Bjerre ◽

Rikke Hjortebjerg ◽

Torben Riis Rasmussen ◽

Anders Lundby ◽

...

Keyword(s):

Growth Factor ◽

Pleural Fluid ◽

Plasma Protein ◽

Binding Protein ◽

Protein A ◽

Insulin Like Growth Factor ◽

Igf System ◽

Igf Binding ◽

Stanniocalcin 2 ◽

Igf Binding Protein

Abstract Context Members of the insulin-like growth factor (IGF) system are primarily produced in the liver and secreted into the circulation, but they are also produced, recruited, and activated locally in tissues. Objective To compare activity and concentrations of IGF system components in pleural fluid and blood. Design Pathological pleural fluid, secondary to lung cancer or nonmalignant disease, and matching blood samples were collected from 24 patients ages 66.7 to 81.9 years. Methods IGF-related proteins and cytokine levels were measured by immunoassays or immunoblotting. Bioactive IGF was measured by an IGF-1 receptor phosphorylation assay. Results Total IGF-1 concentration did not differ between the compartments, but concentrations of free IGF-1 and bioactive IGF were more than threefold higher in pleural fluid than in corresponding serum samples (P = 0.0004), regardless of etiology. Median pregnancy-associated plasma protein-A (PAPP-A) and interleukin (IL)-6 levels were increased 47-fold and 143-fold, respectively, in pleural fluid compared with plasma (P < 0.0001). PAPP-A and IL-6 concentrations correlated positively (r = 0.46; P = 0.02). In pleural fluid, levels of PAPP-A–generated IGF binding protein-4 fragments correlated inversely with that of stanniocalcin-2 (r ≤ −0.42; P ≤ 0.05), a PAPP-A inhibitor; such correlations were absent in plasma. Conclusion Pathological pleural fluid is characterized by increased in vitro IGF bioactivity and elevated concentrations of PAPP-A, an IGF-activating proteinase. Thus, the tissue activity of the IGF system may differ substantially from that of the circulating IGF system. The correlation between IL-6 and PAPP-A indicates that inflammation plays a role in promoting local tissue IGF activity.

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