Abstract
This study was undertaken to determine the mechanism by which proform of eosinophil major basic protein (proMBP) inhibits the IGFBP-4 proteolytic activity of pregnancy-associated plasma protein (PAPP)-A. Co-overexpression of PAPP-A with proMBP in 293T cells, or co-incubation of 293T cells, respectively, overexpressing proMBP and PAPP-A resulted in the formation of a covalent proMBP-PAPP-A complex and inhibition of IGFBP-4 proteolysis. Similar results were obtained when recombinant proMBP and PAPP-A were incubated in the presence of U2 osteosarcoma cells or when recombinant proMBP was added to the U2 cells overexpressing PAPP-A. In contrast, no formation of covalent proMBP-PAPP-A complex or inhibition of IGFBP-4 proteolysis was observed when recombinant proMBP and PAPP-A were incubated under cell-free conditions, although proMBP was able to interact with PAPP-A in a non-covalent manner. These new findings suggest that formation of covalent proMBP-PAPP-A complex is a cell-mediated event and is required for proMBP to inhibit the catalytic activity of PAPP-A.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Blood Proteins / chemistry*
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Blood Proteins / genetics
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Blood Proteins / metabolism*
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Cell Line
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Enzyme Inhibitors / chemistry*
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Enzyme Inhibitors / metabolism
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Enzyme Inhibitors / pharmacology
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Eosinophil Granule Proteins
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Gene Expression
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Humans
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Immunoblotting
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Insulin-Like Growth Factor Binding Protein 4 / metabolism
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Pregnancy-Associated Plasma Protein-A / antagonists & inhibitors
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Pregnancy-Associated Plasma Protein-A / chemistry*
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Pregnancy-Associated Plasma Protein-A / genetics
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Pregnancy-Associated Plasma Protein-A / metabolism*
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Protein Precursors / chemistry*
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Protein Precursors / genetics
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Protein Precursors / physiology*
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Recombinant Proteins / pharmacology
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Ribonucleases / chemistry*
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Ribonucleases / genetics
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Ribonucleases / metabolism*
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Transfection
Substances
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Blood Proteins
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Enzyme Inhibitors
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Eosinophil Granule Proteins
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Insulin-Like Growth Factor Binding Protein 4
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Protein Precursors
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Recombinant Proteins
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Ribonucleases
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Pregnancy-Associated Plasma Protein-A