Structural Characterisation of the Leishmania Major Orthologues of Macrophage Migration Inhibitory Factor (mif)

Leishmania major, an intracellular parasitic protozoon that infects, differentiates and replicates within macrophages, encodes two closely related MIF-like proteins, which have only ~20% amino acid identity with mammalian MIF. Recombinant L. major MIF1 and MIF2 have been expressed and the structures, resolved by X-ray crystallography, show a trimeric ring architecture similar to mammalian MIF but with some structurally distinct features. LmjMIF1, but not LmjMIF2, has tautomerase activity, indicating that the LmjMIFs have evolved potentially different biological roles. This is further demonstrated by the differential life cycle expression of the proteins. LmjMIF2 is found in all life cycle stages whereas LmjMIF1 is found exclusively in amastigotes, the intracellular stage responsible for mammalian disease. The findings are consistent with parasite MIFs modulating or circumventing the host macrophage response and thereby promoting parasite survival, however analysis of the L. braziliensis genome showed that this species lacks intact MIF genes highlighting that MIF is not a virulence factor in all species of Leishmania. Parasites have adopted many strategies to avoid or subvert their hosts’ immune responses including antigenic variation (1), signalling subversion (2) and immune evasion genes (3). Leishmania parasites are obligate intracellular pathogens and the causative agents of leishmaniases. The form and severity of the disease ranges from relatively mild dermal lesions to often fatal visceral infection depending on the infecting Leishmania species and the immune status of the host (4). Leishmania enter macrophages by receptor-mediated endocytosis (5) where they differentiate into amastigotes. A number of strategies are employed by the amistagotes to survive within parasitophorous vacuoles in host macrophages, including inhibition of NO production, hydrolytic enzymes and calcium chelation (6). During the annotation of the L. major genome (7), two genes in tandem (LmjMIF1 and LmjMIF2) were identified that encode proteins related to mammalian Macrophage Migration Inhibitory Factor (MIF), which is a major mediator of inflammatory processes (8). Increased serum levels of MIF contribute to a plethora of auto-immune diseases Address correspondence to: JCM and MDW. 3Current address: Department of Biology (area 11), University of York, York, YO10 4YW, UK 4Current address: Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, The John Arbuthnott Building, 27 Taylor Street, Glasgow G4 0NR, Scotland Europe PMC Funders Group Author Manuscript Biochem Biophys Res Commun. Author manuscript; available in PMC 2011 December 18. Published in final edited form as: Biochem Biophys Res Commun. 2009 March 13; 380(3): 442–448. doi:10.1016/j.bbrc.2009.01.030. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts including rheumatoid arthritis (9), type 2 diabetes (10) and colitis (11). Conversely, knockout experiments in mice have shown that normal levels of MIF are required for the resolution of infections by Salmonella typhimurium (12), Mycobacterium tuberculosis (13), the helminth parasite Taenia crassiceps (14) and L. major (15). Macrophage activation by IFN-γ is prevented in MIF knockout mice, so that mice normally resistant to L. major develop non-healing leishmaniasis (15). Homologues of MIF are found in all mammalian genomes and some helminth parasites, such as Trichinella spiralis (16), Ancylostoma ceylanicum (17) and Brugia malayi (18). Helminth MIF-like proteins are postulated to modify host immune responses thus promoting parasite survival (19). Protozoan parasites including Plasmodium falciparum, P. berghei (20), and Eimeria species (21) also have characterised MIF orthologues that are released by the parasites to modulate the host immune system (22). MIF-like proteins are notably absent from African trypanosomes, which are closely related to Leishmania but do not infect macrophages. There are now over 30 X-ray structures of MIF available from 8 species including, human (23), rat (24), mouse (25), frog (26) and the parasites Brugia malayi (27) and L. major (28) and this study). The trimeric architecture is maintained in all structures. Sequence identities among mammalian MIF sequences are high (above 85%) however this drops to 20-27% identity between the human and protozoan Leishmania and Plasmodium sequences. All of the structurally characterised MIF sequences show keto-enol tautomerase activity with model substrates like p-hydroxyphenylpyruvate and D-dopachrome, though the biological relevance of this well characterised activity is still unknown. Small molecule tautomerase inhibitors of mammalian MIF (29) are being developed for treatment of inflammatory diseases such as rheumatoid arthritis (30). Mammalian MIFs also have an oxidoreductase activity that is dependant on a CxxC thioredoxin-like motif. This motif is also implicated in the intracellular interaction of mammalian MIF with Jab-1, the catalytic subunit of the COP9 signalosome (31). In this work we describe the X-ray structures of LmjMIF1 and LmjMIF2 and compare these structures with the known mammalian MIF structures. The possible biological roles of the two isoforms are discussed in relation to their structures and to their different enzyme activities. EXPERIMENTAL PROCEDURES