Expression of recombinant human pregnancy-associated plasma protein-A and identification of the proform of eosinophil major basic protein as its physiological inhibitor.
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 400 kDa composed of two 200-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.
Department of Molecular and Structural Biology, Science Park, University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark.
M T
MT
Overgaard
J
J
Haaning
H B
HB
Boldt
I M
IM
Olsen
L S
LS
Laursen
M
M
Christiansen
G J
GJ
Gleich
L
L
Sottrup-Jensen
C A
CA
Conover
C
C
Oxvig
D001798:Blood Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D018076:DNA, Complementary / Q000378:metabolism; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors; D004797:Enzyme-Linked Immunosorbent Assay; D047091:Eosinophil Granule Proteins; D004804:Eosinophils / Q000737:chemistry; D005260:Female; D006801:Humans; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000097:blood; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011247:Pregnancy; D011266:Pregnancy-Associated Plasma Protein-A / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism*; D012260:Ribonucleases; D013002:Somatomedins / Q000378:metabolism; D014162:Transfection
D001798:Blood Proteins; D018076:DNA, Complementary; D004220:Disulfides; D004792:Enzyme Precursors; D047091:Eosinophil Granule Proteins; D018974:Insulin-Like Growth Factor Binding Protein 4; D011994:Recombinant Proteins; D013002:Somatomedins; D012260:Ribonucleases; D008666:Metalloendopeptidases; D011266:Pregnancy-Associated Plasma Protein-A