Inhibition of proteolysis by the proform of eosinophil major basic protein (proMBP) requires covalent binding to its target proteinase.
By proteolytic cleavage of insulin-like growth factor binding proteins, the metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) is able to control the biological activity of insulin-like growth factors. PAPP-A circulates in pregnancy as a proteolytically inactive complex, disulfide bound to the proform of eosinophil major basic protein (proMBP). We here demonstrate that co-transfection of mammalian cells with PAPP-A and proMBP cDNA results in the formation of a covalent PAPP-A/proMBP complex in which PAPP-A is inhibited. Formation of the complex also occurs when PAPP-A and proMBP synthesized separately are incubated. Complex formation was monitored by Western blotting, and by using an immunoassay specific for the complex. Using mutagenesis, we further demonstrate that the complex forms in a specific manner and depends on the presence of two proMBP cysteine residues. Mutated proMBP, in which Cys-51 and -169 are replaced by serine, is unable to form the covalent complex with PAPP-A. Of particular interest, such mutated proMBP further lacks the ability to inhibit PAPP-A. For the first time, this conclusively demonstrates that proMBP is a proteinase inhibitor. We further conclude that proMBP inhibits PAPP-A in an unusual manner, not paralleled by other proteinase inhibitors of our knowledge, which requires proMBP to be covalently bound to PAPP-A by disulfide bonds. ProMBP binding to PAPP-A most likely either abrogates substrate access to the active site of PAPP-A or induces a conformational change in the structure of PAPP-A, as we, by further mutagenesis, were able to exclude that the inhibitory mechanism of proMBP is based on a cysteine switch-like mechanism.
Department of Molecular Biology, Science Park, University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark.
Michael T
MT
Overgaard
Simon
S
Glerup
Henning B
HB
Boldt
Vivien
V
Rodacker
Inger M
IM
Olsen
Michael
M
Christiansen
Lars
L
Sottrup-Jensen
Linda C
LC
Giudice
Claus
C
Oxvig
D019943:Amino Acid Substitution; D001798:Blood Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D004220:Disulfides / Q000378:metabolism; D047091:Eosinophil Granule Proteins; D004804:Eosinophils / Q000737:chemistry / Q000378:metabolism; D005260:Female; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology; D007700:Kinetics; D010957:Plasmids; D011247:Pregnancy; D011266:Pregnancy-Associated Plasma Protein-A / Q000737:chemistry / Q000378:metabolism*; D011480:Protease Inhibitors / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012260:Ribonucleases / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D014162:Transfection
D001798:Blood Proteins; D018076:DNA, Complementary; D004220:Disulfides; D047091:Eosinophil Granule Proteins; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D012694:Serine; D012260:Ribonucleases; D011266:Pregnancy-Associated Plasma Protein-A