Cross‐linking and mutational analysis of the oligomerization state of the cytokine macrophage migration inhibitory factor (MIF)

Abstract

The structure of the cytokine MIF has been investigated by X-ray crystallography, NMR, and biochemical methods with conflicting results regarding the structural and functional oligomerization state of this protein. Determination of the oligomeric state(s) is important for understanding more precisely the molecular mechanism of MIF action. To address this issue, we performed cross-linking of human and mouse MIF and selected mutants by various methods and analyzed the oligomerization by SDS-PAGE and gel filtration. MIF was found to form a mixture of monomeric, dimeric, and trimeric states at physiological concentrations, with the monomer and dimer representing the major species. Similar results 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 critical for trimer stabilization. Cross-linking analysis of the isosteric CysCSer mutants C56S and C80S of human MIF resulted in a similar oligomer distribution, whereas substitution of Cys SW led to a significant reduction in the dimeric and trimeric forms, indicating that the hydrophobic region around Cys SW is important for the oligomerization of MIF. Together, our data argue that physiological MIF solutions contain a mixture of monomers, dimers, and trimers.z 1998 Federation of European Biochemical Societies.

Functional Characterization of thePlasmodium falciparumandP. bergheiHomologues of Macrophage Migration Inhibitory Factor

“…Recombinant His 6 -tagged PMIF and huMIF elute at apparent molecular masses of 30 and 22 kDa in size exclusion chromatography, and these sizes are both consistent with a dimeric form in solution. Until (37), the His 6 tag does not appear to affect oligomerization. However, a more in-depth study is required to determine the exact oligomerization status of PMIF in solution.…”

Amyloid fibril formation by macrophage migration inhibitory factor

“…X-ray crystallography studies indicate that MIF exists predominantly in a trimeric form [36,37], whereas NMR [21], sedimentation velocity [24], size exclusion chromatography [22], and solution crosslinking studies [23] indicate that MIF also may exist in dimeric and monomeric forms. However, recent studies reexamining the molecular size and hydrodynamic properties of MIF in solution suggest that it exists in solution predominantly (>95%) as a trimer, which sediments with a sedimentation coefficient of 3.1S [38].…”

“…1B). Although MIF crystallizes as a trimer [19], experimental studies employing NMR spectroscopy [21], size-exclusion chromatography [22], chemical cross-linking [23,24], and analytical ultracentrifugation support the existence of dimeric and monomeric forms in solution [24].…”

Macrophage migration inhibitory factor: molecular, cellular and genetic aspects of a key neuroendocrine molecule

“…However, it is currently not known whether this represents the true, physiological state of MIF protein, as studies have suggested bioactive MIF monomers or dimers (Mischke et al 1998). Each monomer of MIF is known to be composed of the following: two antiparallel -helices ( 1 and 2) and six strands ( 1-6).…”