Expression of recombinant human pregnancy-associated plasma protein-A and identification of the proform of eosinophil major basic protein as its physiological inhibitor

Michael T. Overgaard, Jesper Haaning, Henning B. Boldt, Inger M. Olsen, Lisbeth S. Laursen, Michael Christiansen, Gerald J. Gleich, Lars Sottrup-Jensen, Cheryl A Conover, Claus Oxvig

Research output: Contribution to journalArticle

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Abstract

Pregnancy-associated plasma protein-A (PAPP-A), originally known from human pregnancy serum, has recently been demonstrated to be a metzincin superfamily metalloproteinase involved in normal and pathological insulin-like growth factor (IGF) physiology. PAPP-A specifically cleaves IGF-binding protein (IGFBP)-4, one of six antagonists of IGF action, which results in release of IGF bound to IGFBP-4. IGFBP-4 is the only known PAPP-A substrate. Its cleavage by PAPP-A uniquely depends on the presence of IGF. We here report mammalian expression and purification of recombinant 1547-residue PAPP-A (rPAPP-A). The recombinant protein is secreted as a homodimer of about 409 kDa composed of two 20O-kDa disulfide-bound subunits. Antigenically and functionally, rPAPP-A behaves like the native protein. In human pregnancy, PAPP-A is known to circulate as a 500-kDa disulfide-bound 2:2 complex with the proform of eosinophil major basic protein (proMBP), PAPP-A/proMBP. A comparison between rPAPP-A and pregnancy serum PAPP-A/proMBP complex surprisingly reveals a difference greater than 100-fold in proteolytic activity, showing that proMBP functions as a proteinase inhibitor in vivo. We find that polyclonal antibodies against PAPP-A abrogate all detectable IGFBP-4 proteolytic activity in pregnancy serum, pointing at PAPP-A as the dominating, if not the only, IGFBP-4 proteinase present in the circulation. We further show that pregnancy serum and plasma contain traces (<1%) of uncomplexed PAPP-A with a much higher specific activity than the PAPP-A/proMBP complex. The measurable activity of the PAPP-A/proMBP complex probably results from the presence of a minor subpopulation of partly inhibited PAPP-A that exists in a 2:1 complex with proMBP. Inhibition of PAPP-A by proMBP represents a novel inhibitory mechanism with the enzyme irreversibly bound to its inhibitor by disulfide bonds.

Original languageEnglish (US)
Pages (from-to)31128-31133
Number of pages6
JournalJournal of Biological Chemistry
Volume275
Issue number40
StatePublished - Oct 6 2000

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Eosinophil Major Basic Protein
Pregnancy-Associated Plasma Protein-A
Insulin-Like Growth Factor Binding Protein 4
Somatomedins
Pregnancy
Disulfides
Serum
Peptide Hydrolases

ASJC Scopus subject areas

  • Biochemistry

Cite this

Overgaard, M. T., Haaning, J., Boldt, H. B., Olsen, I. M., Laursen, L. S., Christiansen, M., ... Oxvig, C. (2000). Expression of recombinant human pregnancy-associated plasma protein-A and identification of the proform of eosinophil major basic protein as its physiological inhibitor. Journal of Biological Chemistry, 275(40), 31128-31133.

Expression of recombinant human pregnancy-associated plasma protein-A and identification of the proform of eosinophil major basic protein as its physiological inhibitor. / Overgaard, Michael T.; Haaning, Jesper; Boldt, Henning B.; Olsen, Inger M.; Laursen, Lisbeth S.; Christiansen, Michael; Gleich, Gerald J.; Sottrup-Jensen, Lars; Conover, Cheryl A; Oxvig, Claus.

In: Journal of Biological Chemistry, Vol. 275, No. 40, 06.10.2000, p. 31128-31133.

Research output: Contribution to journalArticle

Overgaard, MT, Haaning, J, Boldt, HB, Olsen, IM, Laursen, LS, Christiansen, M, Gleich, GJ, Sottrup-Jensen, L, Conover, CA & Oxvig, C 2000, 'Expression of recombinant human pregnancy-associated plasma protein-A and identification of the proform of eosinophil major basic protein as its physiological inhibitor', Journal of Biological Chemistry, vol. 275, no. 40, pp. 31128-31133.
Overgaard, Michael T. ; Haaning, Jesper ; Boldt, Henning B. ; Olsen, Inger M. ; Laursen, Lisbeth S. ; Christiansen, Michael ; Gleich, Gerald J. ; Sottrup-Jensen, Lars ; Conover, Cheryl A ; Oxvig, Claus. / Expression of recombinant human pregnancy-associated plasma protein-A and identification of the proform of eosinophil major basic protein as its physiological inhibitor. In: Journal of Biological Chemistry. 2000 ; Vol. 275, No. 40. pp. 31128-31133.
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abstract = "Pregnancy-associated plasma protein-A (PAPP-A), originally known from human pregnancy serum, has recently been demonstrated to be a metzincin superfamily metalloproteinase involved in normal and pathological insulin-like growth factor (IGF) physiology. PAPP-A specifically cleaves IGF-binding protein (IGFBP)-4, one of six antagonists of IGF action, which results in release of IGF bound to IGFBP-4. IGFBP-4 is the only known PAPP-A substrate. Its cleavage by PAPP-A uniquely depends on the presence of IGF. We here report mammalian expression and purification of recombinant 1547-residue PAPP-A (rPAPP-A). The recombinant protein is secreted as a homodimer of about 409 kDa composed of two 20O-kDa disulfide-bound subunits. Antigenically and functionally, rPAPP-A behaves like the native protein. In human pregnancy, PAPP-A is known to circulate as a 500-kDa disulfide-bound 2:2 complex with the proform of eosinophil major basic protein (proMBP), PAPP-A/proMBP. A comparison between rPAPP-A and pregnancy serum PAPP-A/proMBP complex surprisingly reveals a difference greater than 100-fold in proteolytic activity, showing that proMBP functions as a proteinase inhibitor in vivo. We find that polyclonal antibodies against PAPP-A abrogate all detectable IGFBP-4 proteolytic activity in pregnancy serum, pointing at PAPP-A as the dominating, if not the only, IGFBP-4 proteinase present in the circulation. We further show that pregnancy serum and plasma contain traces (<1{\%}) of uncomplexed PAPP-A with a much higher specific activity than the PAPP-A/proMBP complex. The measurable activity of the PAPP-A/proMBP complex probably results from the presence of a minor subpopulation of partly inhibited PAPP-A that exists in a 2:1 complex with proMBP. Inhibition of PAPP-A by proMBP represents a novel inhibitory mechanism with the enzyme irreversibly bound to its inhibitor by disulfide bonds.",
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AU - Overgaard, Michael T.

AU - Haaning, Jesper

AU - Boldt, Henning B.

AU - Olsen, Inger M.

AU - Laursen, Lisbeth S.

AU - Christiansen, Michael

AU - Gleich, Gerald J.

AU - Sottrup-Jensen, Lars

AU - Conover, Cheryl A

AU - Oxvig, Claus

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N2 - Pregnancy-associated plasma protein-A (PAPP-A), originally known from human pregnancy serum, has recently been demonstrated to be a metzincin superfamily metalloproteinase involved in normal and pathological insulin-like growth factor (IGF) physiology. PAPP-A specifically cleaves IGF-binding protein (IGFBP)-4, one of six antagonists of IGF action, which results in release of IGF bound to IGFBP-4. IGFBP-4 is the only known PAPP-A substrate. Its cleavage by PAPP-A uniquely depends on the presence of IGF. We here report mammalian expression and purification of recombinant 1547-residue PAPP-A (rPAPP-A). The recombinant protein is secreted as a homodimer of about 409 kDa composed of two 20O-kDa disulfide-bound subunits. Antigenically and functionally, rPAPP-A behaves like the native protein. In human pregnancy, PAPP-A is known to circulate as a 500-kDa disulfide-bound 2:2 complex with the proform of eosinophil major basic protein (proMBP), PAPP-A/proMBP. A comparison between rPAPP-A and pregnancy serum PAPP-A/proMBP complex surprisingly reveals a difference greater than 100-fold in proteolytic activity, showing that proMBP functions as a proteinase inhibitor in vivo. We find that polyclonal antibodies against PAPP-A abrogate all detectable IGFBP-4 proteolytic activity in pregnancy serum, pointing at PAPP-A as the dominating, if not the only, IGFBP-4 proteinase present in the circulation. We further show that pregnancy serum and plasma contain traces (<1%) of uncomplexed PAPP-A with a much higher specific activity than the PAPP-A/proMBP complex. The measurable activity of the PAPP-A/proMBP complex probably results from the presence of a minor subpopulation of partly inhibited PAPP-A that exists in a 2:1 complex with proMBP. Inhibition of PAPP-A by proMBP represents a novel inhibitory mechanism with the enzyme irreversibly bound to its inhibitor by disulfide bonds.

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