Abstract
We have previously established the presence of a pool of apoE sequestered on the macrophage cell surface by demonstrating its displacement from a cell monolayer at 4 degrees C. In this series of experiments, we use a cell surface biotinylation protocol to directly quantitate apoE on the macrophage cell surface and evaluate its transport to and from this cell surface pool. In human monocyte-derived macrophages labeled to equilibrium and in a mouse macrophage cell line transfected to constitutively express human apoE3, approximately 8% of total cellular apoE was present on the surface, but only a portion of this surface pool served as a direct precursor to secreted apoE. The half-life of apoE on the macrophage cell surface was calculated to be approximately 12 min. On SDS-polyacrylamide gel electrophoresis, the apoE isolated from the surface fraction of cells labeled to equilibrium migrated in an isoform pattern distinct from that observed from the intracellular fraction, with the surface fraction migrating predominantly in a higher molecular weight isoform. Pulse labeling experiments demonstrated that newly synthesized apoE reached the cell surface by 10 min but was predominantly in a low molecular weight isoform. There was also a lag between appearance of apoE on the cell surface and its appearance in the medium. Biotinylated apoE, which accumulated in the medium, even from pulse labeled cells, was predominantly in the high molecular weight isoform. Additional experiments demonstrated that low molecular weight apoE present on the cell surface was modified to higher molecular weight apoE by the addition of sialic acid residues prior to secretion and that this conversion was inhibited by brefeldin A. These results demonstrate an unexpected complexity in the transport and cellular processing of macrophage cell surface apoE. Factors that modulate the size and turnover of the cell surface pool of apoE in the macrophage remain to be identified and investigated.
Highlights
Several potential fates for cell surface apoE in the macrophage can be considered
Assessment of the Steady State Distribution of apoE on the Macrophage Cell Surface—Biotinylation of cell surface proteins followed by immunoisolation of cellular apoE and separation of biotinylated from unbiotinylated apoE was conducted in human monocytes/macrophages and J774-E cells labeled to equilibrium
The total cellular fraction (IC ϩ S) present in the streptavidin supernatant from unbiotinylated cells, displayed a banding pattern that was similar to a combination of the isoform pattern from the surface and intracellular fractions obtained from biotinylated cells, i.e. prominent high and low molecular weight apoE bands
Summary
Several potential fates for cell surface apoE in the macrophage can be considered. The first would be that this pool serves as the immediate precursor for secreted apoE. The total cellular fraction (IC ϩ S) present in the streptavidin supernatant from unbiotinylated cells (lanes 7 and 8), displayed a banding pattern that was similar to a combination of the isoform pattern from the surface and intracellular fractions obtained from biotinylated cells, i.e. prominent high and low molecular weight apoE bands.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
