Abstracts (complete list) - Wissenschaft Online

Sandra Kraemer, Regina Krohn, Hongqi Lue, Manfred Dewor, Christian Weber, Jürgen
Bernhagen
The pseudo-ELR motif of MIF is required for its functions as
CXC arrest chemokine
Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory cytokine that
plays a pivotal role in acute and chronic inflammatory diseases such as septic shock,
and atherosclerosis. We have recently demonstrated that MIF, contrary to its historic
and eponymous name, is a non-cognate ligand of the chemokine receptor CXCR2. MIF
binds to CXCR2 with nanomolar affinity and induces leukocyte arrest and chemotaxis
through this receptor (Bernhagen et al., Nat. Med. 13, 2007). CXCR2 signaling induced
by cognate ligands, such as IL-8, requires an N-terminal Glu-Leu-Arg (ELR) motif. The
MIF monomer not only displays a striking architectural homology to the IL-8 dimer, but
we also noticed that MIF has an ELR-like 3D motif, which we tentatively term pseudo-
ELR motif. It is formed by the two non-adjacent residues Arg11 (R11) and Asp44 (D44),
which reside in an ELR-like spacing in exposed neighboring loops, imitating the ELRmotif.
While it is tempting to speculate that the pseudo-ELR motif conveys binding and
activity specificity comparable to what is observed for classical ELR+ CXC chemokines,
no functional data has been available. We have thus generated MIF mutants with
residues R11 and D44 mutated to address the question whether the pseudo-ELR motif is
functionally required for an interaction with CXCR2 and for MIF’s chemokine-like
activities. We have cloned the mutants R11A-MIF, D44A-MIF, and the double mutant
R11A-D44A-MIF. Although mutagenesis involved alterations in charge and isoelectric
point, the mutants were expressed and purified by an almost identical procedure as that
established for wildtype MIF. Spectroscopic analysis by far-UV circular dichroism
confirmed that the mutants exhibited an overall structural integrity. However, R11A-MIF
showed an increase in β-sheet structure, indicating that the pseudo-ELR site constitutes
a sensitive conformational region of the molecule. We will report on the functional
relevance of this motif for the leukocyte recruitment activity of MIF. Intriguingly, initial
experiments suggest that mutating R11/D44 markedly interferes with CXCR2dependent
monocyte arrest on human aortic endothelial cells in flow induced by MIF.