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 superfam ily metalloproteinase involved in normal and pathological insulin-like grow th factor (IGF) physiology. PAPP-A specifically cleaves IGF-binding protein (LGFBP)-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 cl eavage by PAPP-A uniquely depends on the presence of IGF. We here report ma mmalian expression and purification of recombinant 1547-residue PAPP-A (rPA PP-A). The recombinant protein is secreted as a homodimer of about 400 kDa composed of two 200-kDa disulfide-bound subunits. Antigenically and functio nally, 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 pro form of eosinophil major basic protein (proMBP), PAPP-A/proMBP. A compariso n between rPAPP-A and pregnancy serum PAPP-A/proMBP complex surprisingly re veals a difference greater than 100-fold in proteolytic activity, showing t hat proMBP functions as a proteinase inhibitor in vivo. We find that polycl onal antibodies against PAPP-A abrogate all detectable IGFBP-4 proteolytic activity in pregnancy serum, pointing at PAPP-A as the dominating, if not t he only, IGFBP-4 proteinase present in the circulation. We further show tha t pregnancy serum and plasma contain traces (<1%) of uncomplexed PAPP-A wit h a much higher specific activity than the PAPP-A/proMBP complex. The measu rable activity of the PAPP-A/proMBP complex probably results from the prese nce 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 in hibitory mechanism with the enzyme irreversibly bound to its inhibitor by d isulfide bonds.