Abstract
Apolipoprotein M (apoM) is a novel apolipoprotein that is reportedly necessary for pre beta HDL formation; however, its detailed function remains unknown. We investigated the biogenesis and properties of apoM and its effects on the initial steps of nascent pre beta HDL assembly by ABCA1 in HEK293 cells. Transiently transfected apoM was localized primarily in the endomembrane compartment. Pulse-chase analyses demonstrated that apoM is inefficiently secreted, relative to human serum albumin, and that approximately 50% remains membrane-associated after extraction with sodium carbonate, pH 11.5. To investigate the role of apoM in nascent pre beta HDL formation, ABCA1-expressing or control cells, transfected with empty vector, apoM, or C-terminal epitope-tagged apoM (apoM-C-FLAG), were incubated with (125)I-apoA-I for 24 h. Conditioned media were harvested and fractionated by fast-protein liquid chromatography (FPLC) to monitor HDL particle size. Pre beta HDL particles were formed effectively in the absence of apoM expression; however, increased apoM expression stimulated the formation of larger-sized nascent pre beta HDLs. Immunoprecipitation with anti-apoA-I antibody followed by apoM Western blot analysis revealed that little secreted apoM was physically associated with pre beta HDL. Our results suggest that apoM is an atypical secretory protein that is not necessary for ABCA1-dependent pre beta HDL formation but does stimulate the formation of larger-sized pre beta HDL. We propose that apoM may function catalytically at an intracellular site to transfer lipid onto pre beta HDL during or after their formation by ABCA1.
Highlights
Apolipoprotein M is a novel apolipoprotein that is reportedly necessary for pre HDL formation; its detailed function remains unknown
ApoM expression was investigated by Western blot analysis using control and stably transfected ABCA1-expressing cells transiently transfected with empty vector, Apolipoprotein M (apoM)-wild type (WT), or apoM-C-FLAG cDNA
Control and ABCA1-expressing cells were transfected with pcDNA3 or apoM-C-FLAG, and immunofluorescence was detected by incubating fixed cells with monoclonal antibody to the FLAG epitope, followed by goat anti-mouse IgG FITC-labeled secondary antibody
Summary
Apolipoprotein M (apoM) is a novel apolipoprotein that is reportedly necessary for pre HDL formation; its detailed function remains unknown. To investigate the role of apoM in nascent pre HDL formation, ABCA1-expressing or control cells, transfected with empty vector, apoM, or C-terminal epitope-tagged apoM (apoM-C-FLAG), were incubated with 125I-apoA-I for 24 h. Pre HDL particles were formed effectively in the absence of apoM expression; increased apoM expression stimulated the formation of larger-sized nascent pre HDLs. Immunoprecipitation with anti-apoA-I antibody followed by apoM Western blot analysis revealed that little secreted apoM was physically associated with pre HDL. We propose that apoM may function catalytically at an intracellular site to transfer lipid onto pre HDL during or after their formation by ABCA1.—Mulya, A., J. Apolipoprotein M expression increases the size of nascent pre HDL formed by ATP binding cassette transporter A1.
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