The Immunoregulatory Mediator Macrophage Migration Inhibitory Factor (MIF) Catalyzes a Tautomerization Reaction

Abstract

Background: Recent studies of melanin biosynthesis have uncovered an unusual enzymatic activity which converts the non-naturally occurring D-isomer of 2-carboxy-2, 3-dihydroindole-5,6-quinone (dopachrome) into 5,6-dihydroxyindole-2-carboxylic acid (DHICA). The aim of the present investigation was to isolate and characterize the enzyme catalyzing this tautomerization reaction. Materials and Methods: After we performed a tissue survey of D-dopachrome tautomerase activity, 10 bovine lenses were homogenized and used as a source of enzyme. A soluble fraction was obtained by high-speed centrifugation and subjected to successive FPLC chromatography on Phenyl-sepharose, Mono S cation-exchange, and Superdex gel-filtration. The isolated enzyme was electrophoresed, blotted onto PVDF membrane, and the N terminus analyzed by gas phase micro-sequencing. Results: The protein catalyzing the conversion of Ddopachrome to DHICA was purified to homogeneity in 14% yield and showed a molecular weight of 12 kD when analyzed by SDS-PAGE. The first 27 amino acid residues of this protein were sequenced and found to be identical with those of bovine macrophage migration inhibitory factor (MIF). The catalytic activity of native MIX was confirmed by studies of purified recombinant human MIW, which showed the same tautomerase activity. While L-dopachrome was not a substrate for this reaction, the methyl esters of the Land D-isomers were found to be better substrates for MIF than D-dopachrome. Conclusions: MIW has been described recently to be an anterior pituitary hormone and to be released from immune cells stimulated by low concentrations of glucocorticoids. Once secreted, MIF acts to control, or counter-regulate, the immunosuppressive effects of glucocorticoids on the immune system. Although the tested substrate, D-dopachrome, does not occur naturally, the observation that MIF has tautomerase activity suggests that MIF may mediate its biological effects by an enzymatic reaction. These data also offer a potential approach for the design of small molecule pharmacological inhibitors of MIF that may modulate its potent immunoregulatory effects in vivo.

Macrophage Migration Inhibitory Factor: a Potential Marker for Cancer Diagnosis and Therapy

“…The crystal structure shows that the active form is a 37.5 kDa homotrimer with new protein folds (Sun et al, 1996b). MIF has Pro-Pro dependent tautomerase activity and CALC dependent oxidoreductase activity (Rosengren et al, 1996;Thiele & Bernhagen, 2005). The tautomerase activity allows the MIF to catalyze D-dopachrome or L-dopachrome to its indole derivatives (Rosengren et al, 1996).…”

Molecular Interaction and Enzymatic Activity of Macrophage Migration Inhibitory Factor with Immunorelevant Peptides

“…MIF tautomerase/isomerase activity relies on Pro-1 that promotes enolization and ketonization (10,43). Because its natural ligand is unknown, MIF enzymatic properties have been described in detail using molecular models as substrates (44).…”

“…First, MIF can function as a pro-inflammatory protein, as assessed by its inhibitory effect on dexamethasone-mediated TNF-␣ production (2) and its ability to sustain CD3-mediated T cell proliferation (8) and pro-inflammatory cytokine secretion (9). MIF also possesses enzymatic activities as a tautomerase/isomerase (10) and thiol oxidoreductase (11). However, the natural substrates for MIF catalytic activity are unknown even though its enzymatic activity has been characterized in detail using insulin and L-DOPA as models (11,12).…”

Crystal structure of macrophage migration inhibitory factor from human Å lymphocyte at 2.1 Å resolution

“…On the other hand, although MIF was recently reported to catalyze the tautomerization of 2-carboxy-2,3-dihydroindole-5,6-quinone (o-dopachrome) to 5,6-dihidroxyindole-2-carboxylic acid (DHICA) [8], nothing is known about its active site. MIF does have a pocket in a similar region close to the N-terminal Pro residue.…”

“…MIF exists not only in immune ccIls, but also in other cell types such as chicken differentiatin: cells of the eye lens, mouse 3T3 fibroblastic cells and rat Ii\ er cells . A recent report showed that MIF from the bl $vine lens has enzyme activity to catalyze the tautomerizatit m of 2-carboxy-2,3-dihydroindole-5,6-quinone (D-dopacl rome) to 5,6-dihidroxyindole-2-carboxylic acid (DHICA) [8 . Moreover, the three-dimensional structure of MIF from tb,?…”