Inhibition of proteolysis by the proform of eosinophil major basic protein (proMBP) requires covalent binding to its target proteinase

FEBS Letters

Giudice, Linda C²  Sottrup-Jensen, Lars³  Glerup, Simon³  Olsen, Inger M³  Christiansen, Michael¹  Boldt, Henning B³  Rodacker, Vivien³  Oxvig, Claus³  Overgaard, Michael T³ 

[1] Department of Clinical Biochemistry, Statens Serum Institut, DK-2300 Copenhagen S, Denmark;Department of Gynecology and Obstetrics, Stanford University, Stanford, CA 94305, USA;Department of Molecular Biology, Science Park, University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark

关键词: Proform of eosinophil major basic protein;    Pregnancy-associated plasma protein-A;    Insulin-like growth factor binding protein;    Proteolysis;    Proteinase inhibition;    PAPP-A;    pregnancy-associated plasma protein-A;    proMBP;    proform of eosinophil major basic protein;    IGF;    insulin-like growth factor;    IGFBP;    IGF binding protein;   

DOI  :  10.1016/S0014-5793(04)00095-X

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown