Apolipoprotein M – Studies of Structure and Function

Abstract: Apolipoprotein M (apoM), a 25 kDa plasma protein, is found in all major lipoprotein classes, although the majority is found in high density lipoproteins (HDL). ApoM has been suggested to be involved in the formation of and adding to the antiatherogenic functions of HDL, but its function is still not completely known. ApoM has been suggested to be a lipokalin. By studies in vitro using recombinant apoM we were able to show that apoM shares the ligand-binding abilities of lipokalins by being able to bind retinol and its two metabolites, all-trans retinoic acid and 9-cis retinoic acid. After successful crystallization trials we managed to determine the 3D structure of recombinant apoM expressed in E.coli. ApoM displays the typical lipocalin fold that was predicted, i.e. characterized by an 8-stranded antiparallel β-barrel enclosing an internal ligand-binding pocket, flanked by a α-helix. The ligand-binding pocket in apoM can be subdivided into a hydrophilic upper part, surrounded by several positively and negatively charged residues, and a hydrophobic lower part lined by numerous hydrophobic residues. ApoM was crystallized as a complex with either myristic acid or glycerol-1-myristate in the hydrophobic pocket showing that apoM can work as a fatty acid binding protein. We were also able to detect a binding of D-sphingosine and sphingosine-1-phosphate in ligand-binding studies. The physiological importance of the binding is not known. Mature apoM is circulating with a retained signal peptide. By constructing apoM with a cleavable signal peptide we were able to show that the signal peptide is necessary for the protein’s ability to associate to lipoproteins. We were also able to show that, in contrast to apoM with a cleavable signal peptide, full-length apoM was accumulated in stably transfected HEK293 cells, expressing apoM, unless serum was present. The addition of extra cellular HDL or co-expression of HDL was enough to completely restore the apoM expression. We have in a previous study found a marked positive correlation between plasma apoM and total cholesterol levels in healthy individuals. To investigate whether plasma apoM levels predict the risk of coronary heart disease, apoM was measured in plasma from subject later developing CHD and healthy controls from two prospective case-control studies, FINRISK ´92 and Copenhagen City Heart Study. In conditional logistic regression analyses, apoM was not a predictor of CHD events. We found positive correlations for apoM with both apoA-I and apoB.