Abstract
Apolipoprotein M (apoM) is a plasma protein associated mainly with HDL. ApoM is suggested to be important for the formation of prebeta-HDL, but its mechanism of action is unknown. Homology modeling has suggested apoM to be a lipocalin. Lipocalins share a structurally conserved beta-barrel, which in many lipocalins bind hydrophobic ligands. The aim of this study was to test the ability of apoM to bind different hydrophobic substances. ApoM was produced both in Escherichia 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 (dissociation constant = 2-3 microM), 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 wild-type 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 has yet to be elucidated.
Highlights
IntroductionApolipoprotein M (apoM) is a plasma protein associated mainly with HDL. ApoM is suggested to be important for the formation of preb-HDL, but its mechanism of action is unknown
Apolipoprotein M is a plasma protein associated mainly with HDL
The lipocalin domain of human Apolipoprotein M (apoM) was expressed both in prokaryotic (E. coli) and eukaryotic (HEK 293) cells
Summary
Apolipoprotein M (apoM) is a plasma protein associated mainly with HDL. ApoM is suggested to be important for the formation of preb-HDL, but its mechanism of action is unknown. A noteworthy feature of the apoM amino acid sequence is the lack of a signal peptidase cleavage site, explaining why circulating apoM contains its signal peptide [1]. This unusual property is found in two other HDL-associated proteins: paraoxonase-1 and the haptoglobin-related protein [5]. The lipocalin family contains a highly conserved tryptophan (position 24 in RBP and position 47 in apoM) that points toward the inside of the hydrophobic pocket [9]. ApoM contains a second tryptophan at position 100 (Trp100) facing the hydrophobic pocket close to its opening (Fig. 1) These tryptophans can be used for assessing the binding
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