cDNAs were obtained for macrophage migration-inhibitory factor (MIF)/l-dopachrome methyl ester tautomerase homologues from the parasitic nematodes Trichinella spiralis (TsMIF) and Trichuris trichiura (TtMIF). The translated sequences, which were partly confirmed by sequencing of proteolytic fragments, show 42 and 44% identity respectively with human or mouse MIF, and are shorter by one C-terminal residue. Unlike vertebrate MIF and MIF homologues of filarial nematodes, neither TsMIF nor TtMIF contain cysteine residues. Soluble recombinant TsMIF, expressed in Escherichia coli showed secondary structure (by CD spectroscopy) and quaternary structure (by light-scattering and gel filtration) similar to that of the trimeric mammalian MIFs and d-dopachrome tautomerase. The catalytic specificity of recombinant TsMIF in the ketonization of phenylpyruvate (1.4×106M−1·s−1) was comparable with that of human MIF, while that of p-hydroxyphenylpyruvate (9.1×104M−1·s−1) was 71-fold lower. TsMIF showed high specificity in tautomerization of the methyl ester of l-dopachrome compared with non-esterified l-dopachrome (>87000-fold) and a high kcat (≈ 4×104s−1). The crystal structure, determined to 1.65 Å (1 Å = 0.1nm), was generally similar to that of human MIF, but differed in the boundaries of the putative active-site pocket, which can explain the low activity towards p-hydroxyphenylpyruvate. The central pore was blocked, but was continuous, with the three putative tautomerase sites. Recombinant TsMIF (5ng/ml–5pg/ml) inhibited migration of human peripheral-blood mononuclear cells in a manner similar to that shown by human MIF, but had no effect from 5 to 500ng/ml on anti-CD3-stimulated murine T-cell proliferation. TsMIF was detected in supernatants of T. spiralis larvae cultured in vitro at 6ng/ml (55ng/mg total secreted protein). In conclusion TsMIF has structural, catalytic and cell-migration-inhibitory properties which indicate that it is partially orthologous to mammalian MIF.
↵1 Present address: Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, U.K.
The nucleotide sequence data discussed are available from the DDBJ, EMBL, GenBank® and GSDB Nucleotide Sequence Databases under the accession numbers AJ012740 (Trichinella spiralis MIF), AJ237770 (Trichuris trichiura MIF) and AJ237610 (for partial T. trichiura AMSH); the crystal structure co-ordinates of T. spiralis MIF are in the PDB (file 1HFO).
Abbreviations used: AMSH, a signal-transducing adaptor protein involved in interleukin-2- and granulocyte–macrophage-colony-stimulating-factor-stimulated cell growth; MIF, macrophage migration-inhibitory factor; (r)TsMIF, (recombinant) Trichinella spiralis MIF; TtMIF, Trichuris trichiura MIF; HsMIF, Homo sapiens (human) MIF; PDB, Protein Databank (Brookhaven, CT, U.S.A.); CC, correlation coefficient; IL, interleukin; IFN-γ, interferon-γ; CNS, Crystallography and NMR System; NCS, non-crystallographic symmetry.
- The Biochemical Society, London ©2001