Dissection of the enzymatic and immunologic functions of macrophage migration inhibitory factor - Full immunologic activity of N-terminally truncatedmutants
R. Kleemann et al., Dissection of the enzymatic and immunologic functions of macrophage migration inhibitory factor - Full immunologic activity of N-terminally truncatedmutants, EUR J BIOCH, 267(24), 2000, pp. 7183-7192
Macrophage migration inhibitory factor (MIF) is a cytokine with broad regul
atory functions in innate immunity. MIF belongs to the few cytokines displa
ying 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. Her
e we report on the N-terminal mutational analysis and minimization of MIF a
nd on a dissection of the two catalytic activities by comparing mutants P2A
MIF, Delta 4MIF, Delta 5MIF Delta 6MIF, Delta 7MIF Delta 8MIF, and Delta 10
MIF with the cysteine mutants of MIF. As N-terminal deletion was predicted
to interfere with protein structure due to disruption of the central beta s
heet, it was surprising that deletion of up to six N-terminal residues resu
lted in normally expressed proteins with wild-type conformation. Strikingly
, such mutants exhibited full MIF-specific immunologic activity. While muta
tion of Pro2 eliminated tautomerase activity, the CALC cysteine residues ha
d no influence on this activity. However, mutant C81SMIF, which otherwise h
as full biologic activity, only had 32% tautomerase activity. Deletion of f
our 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 Delta 2MIF exhibiting 40% acti
vity, and Delta 4MIF completely failing to reduce HED. This study constitut
es the first comparison of the two catalytic activities of MIF and should a
ssist in understanding the molecular links between the catalytic and immuno
logic activities of this cytokine and in providing guidelines for N-termina
l protein minimization.