Interaction of macrophage migration inhibitory factor with ceruloplasmin: Role of labile copper ions

Macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, is a target for pharmacological treatment of sepsis and malignant tumors. Inhibition of tautomerase activity of MIF in reaction with p-hydroxyphenylpyruvate (HPP) was observed in the presence of ceruloplasmin (CP), a copper-containing plasma protein. Binding labile copper ions to CP (CP+Cu(II)) is a prerequisite for MIF inhibiting. CP+Cu(II) is shown to be an uncompetitive inhibitor of MIF (K ~ 37 nM), which suggests formation of a complex 'MIF-HPP-CP-Cu(II)'. Filtration of CP+Cu(II) on a column with Chelex-100, otherwise the presence of high concentrations of histidine, cysteine or methionine abrogated the inhibitory effect of CP. Adding salts of Co(II) and Ni(II) that replace copper ions in the labile sites prevented the inhibitory effect of CP+Cu(II). Limited proteolysis of CP by thrombin diminished its oxidase activity in reaction with p-phenylenediamine, but endowed it with the capacity of inhibiting MIF. Covalent modification of MIF by phenylmethylsulfonyl fluoride (PMSF) resulted in binding of MIF-PMSF to CP immobilized on CM5 chip, the dissociation constant being 4.2 μM. In d-galactosamine-sensitized mice CP+Cu(II) increased the LPS-induced lethality from 54 to 100 %, while administration of antibodies against MIF prevented the lethal effect. The enhancement by CP+Cu(II) of the pro-inflammatory signal of MIF is discussed.