Transport and processing of endogenously synthesized ApoE on the macrophage cell surface

We have previously established the presence of a pool of apoE sequestered o n 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 sur face biotinylation protocol to directly quantitate apoE on the macrophage c ell 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, ap proximately 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 apo E. The half-life of apoE on the macrophage cell surface was calculated to b e approximately 12 min. On SDS-polyacrylamide gel electrophoresis, the apoE isolated from the surface fraction of cells labeled to equilibrium migrate d in an isoform pattern distinct from that observed from the intracellular fraction, with the surface fraction migrating predominantly in a higher mol ecular weight isoform, Pulse labeling experiments demonstrated that newly s ynthesized 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 ap oE, which accumulated in the medium, even from pulse labeled cells, was pre dominantly in the high molecular weight isoform. Additional experiments dem onstrated that low molecular weight apoE present on the cell surface was mo dified to higher molecular weight apoE by the addition of sialic acid resid ues 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 re main to be identified and investigated.