Dissection of the enzymatic and immunologic functions of macrophage migration inhibitory factor. Full immunologic activity of N-terminally truncated mutants.
Macrophage migration inhibitory factor (MIF) is a cytokine with broad regulatory functions in innate immunity. MIF belongs to the few cytokines displaying catalytic activities, i.e. MIF has a Pro2-dependent tautomerase and a Cys-Ala-Leu-Cys (CALC) cysteine-based thiol-protein oxidoreductase activity. Previous studies have addressed the roles of the catalytic site residues and the C-terminus. The two activities have not been directly compared. Here we report on the N-terminal mutational analysis and minimization of MIF and on a dissection of the two catalytic activities by comparing mutants P2AMIF, Delta4MIF, Delta5MIF, Delta6MIF, Delta7MIF, Delta8MIF, and Delta10MIF with the cysteine mutants of MIF. As N-terminal deletion was predicted to interfere with protein structure due to disruption of the central beta sheet, it was surprising that deletion of up to six N-terminal residues resulted in normally expressed proteins with wild-type conformation. Strikingly, such mutants exhibited full MIF-specific immunologic activity. While mutation of Pro2 eliminated tautomerase activity, the CALC cysteine residues had no influence on this activity. However, mutant C81SMIF, which otherwise has full biologic activity, only had 32% tautomerase activity. Deletion of four N-terminal residues did not interfere with insulin reduction by MIF. By contrast, reduction of 2-hydroxyethyldisulfide (HED) was markedly affected by N-terminal manipulation, with P2AMIF and Delta2MIF exhibiting 40% activity, and Delta4MIF completely failing to reduce HED. This study constitutes the first comparison of the two catalytic activities of MIF and should assist in understanding the molecular links between the catalytic and immunologic activities of this cytokine and in providing guidelines for N-terminal protein minimization.
Kleemann
R
R
Laboratory of Biochemistry, Institute for Interfacial Engineering, University of Stuttgart, Germany.
Rorsman
H
H
Rosengren
E
E
Mischke
R
R
Mai
N T
NT
Bernhagen
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers; D008263:Macrophage Migration-Inhibitory Factors / Q000276:immunology* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet
D017931:DNA Primers; D008263:Macrophage Migration-Inhibitory Factors