Visit our Expo - Redox and Inflammation signaling 2012

Session I : protein domains Poster I, 3
Preparation of leptin antagonists by site-directed mutagenesis of human, ovine, rat and
mouse leptin's site III: implications on blocking undesired leptin action in vivo.
Gili Salomon1, Leonora Niv-Spector1, Dana Berger1, Isabelle Callebaut2, Jean Djiane3
and Arieh Gertler1*
1Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew
University of Jerusalem, 2LMCP, CNRS UMR7590, Universites Paris 6 & Paris 7,
France, 3Institut National de la Recherche Agronomique, NOPA, France.
As no structural information of the 3D structure of leptin receptor (LEPR) is available the
model of interleukin 6 (IL6) was applied. In that model, a hexameric complex is formed
gradually, first by IL6 molecule which interacts with the IL6R-alpha, then with gp130
forming an inactive trimer which subsequently dimerizes forming an active hexamer, whose
formation is achieved due to interaction of IL6 bound in one trimer (through its site III) with
the immunoglobulin domain (IGD) of gp130 of the other trimer. We have identified the
putative leptin's binding site III by modeling LEPR, on the basis of its alignment with gp130,
and fitting leptin on IL6 in the IL6/gp130 complex as leptin's amino acids 39-42 (LDFI),
which are preserved in all leptin species. To verify this hypothesis and to test its generality we
have prepared and purified to homogeneity human, ovine, rat and mouse triple
(L39A/D40A/F41A) and quadruple (L39A/D40A/F41A/I42A, human and ovine only) leptin
muteins. All six muteins had typical cytokine secondary structure, acted as true antagonists,
namely they interacted with LEPR with affinity similar to the wild type hormone (as
evidenced by SRP and RRA), were devoid of biological activity in several leptin-responsive
bioassays and specifically inhibited leptin action. Those muteins can be prepared in gram
amounts and thus serve as a novel tool of studying leptin function in vitro and in vivo. To
prolong their life in circulation some muteins were pegylated using 40 kDa polyethylene
glycol. Although pegylation decreases the affinity, increasing circulation half-life can
recompensate this deficit so pegylated antagonists are expected to be more potent in vivo.
Antagonizing leptin has been suggested as a possible therapy in auto-immune diseases and
heart failure. Thus leptin antagonists not only offer a novel tool to elucidate the role of leptin
in mammalian physiology and pathology but have a potential of becoming a drug.
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