Hydrophobic ligand-binding properties of the human lipocalin apolipoprotein M

Josefin Ahnstrom, Kirsten Faber, Olof Axler, and Bjorn Dahlback

Laboratory Medicine, Clinical Chemistry, Malmö University Hospital, Malmo 20502

Corresponding Author: bjorn.dahlback{at}med.lu.se

Apolipoprotein M (apoM) is a plasma protein mainly associated with HDL. ApoM is suggested to be important for the formation of pre-ß HDL, but its mechanism of action is unknown. Homology modeling has suggested apoM to be a lipocalin. Lipocalins share a structurally conserved ß-barrel, which in many lipocalins bind hydrophobic ligands. The aim of the present study was to test the ability of apoM to bind different hydrophobic substances. ApoM was produced both in E. coli and in HEK 293 cells. Characterization of both variants with electrophoretic and immunological methods suggested apoM from E.coli to be correctly folded. Intrinsic tryptophan fluorescence of both apoM variants revealed that retinol, all-trans retinoic acid and 9-cis retinoic acid bound (Kd 2-3 µM), whereas other tested substances e.g. cholesterol, vitamin K and arachidonic acid did not. The intrinsic fluorescence of two apoM mutants carrying single tryptophans was quenched by retinol and retinoic acid to the same extent as wildtype apoM, indicating that the environment of both tryptophans was affected by the binding. In conclusion, the binding of retinol and retinoic acid supports the hypothesis that apoM is a lipocalin. The physiological relevance of this binding is yet to be elucidated.

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