CROSS-LINKING AND MUTATIONAL ANALYSIS OF THE OLIGOMERIZATION STATE OFTHE CYTOKINE MACROPHAGE-MIGRATION INHIBITORY FACTOR (MIF)

The structure of the cytokine MIF has been investigated by X-ray cryst allography, NMR, and biochemical methods with conflicting results rega rding the structural and functional oligomerization state of this prot ein, Determination of the oligomeric state(s) is important for underst anding more precisely the molecular mechanism of MIF action. To addres s this issue, we performed cross-linking of human and mouse MIF and se lected mutants by various methods and analyzed the oligomerization by SDS-PAGE and gel filtration. MIF was found to form a mixture of monome ric, dimeric, and trimeric states at physiological concentrations, wit h the monomer and dimer representing the major species. Similar result s were obtained when the carboxy-truncated mutants MIF(1-104) and MIF( 1-109) were examined, indicating that the C-terminus of MIF is not cri tical for trimer stabilization. Cross-linking analysis of the isosteri c Cys --> Ser mutants C56S and C80S of human MIF resulted in a similar oligomer distribution, whereas substitution of Cys(59) led to a signi ficant reduction in the dimeric and trimeric forms, indicating that th e hydrophobic region around Cys(59) is important for the oligomerizati on of MIF, Together, our data argue that physiological MIP solutions c ontain a mixture of monomers, dimers, and trimers, (C) 1998 Federation of European Biochemical Societies.