Transport and Processing of Endogenously Synthesized ApoE on the Macrophage Cell Surface

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°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 monocytederived 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.

Oleic Acid Modulates the Post-translational Glycosylation of Macrophage ApoE to Increase Its Secretion

“…We evaluated this possibility by examining the time course of the oleic acid induction of apoE secretion. As shown in Table II, a substantial induction of apoE secretion by fatty acid required at least 6 h. This result argues against simple displacement of a cell surface pool of apoE by fatty acid as a mechanism for increased apoE secretion as we have previously shown that the half-life of apoE on the cell surface of macrophages is ϳ12 min (32). Further, the 6-h lag suggests that some homeostatic alteration in cells is necessary before apoE secretion is increased.…”

“…Therefore, it is unlikely that the effect of oleic acid on apoE secretion is mediated via changes in cellular sterol trafficking. We have also previously reported that apoE can be sequestered on the macrophage cell surface prior to secretion and may, in fact, undergo recycling through intracellular compartments prior to secretion (31,32). We therefore considered the possibility that exogenous fatty acids could displace apoE from the cell surface and thus produce the simultaneous increase in medium apoE and decrease in cellular apoE that we observed in pulse-chase experiments.…”

“…after exhaustive digestion of labeled cellular and secreted apoE with neuraminidase, the multiple bands shown in Fig. 5A collapse into the lowest band, which represents asialo apoE (32,34,36). In Fig.…”

“…We have also previously shown that apoE can be sequestered in a macrophage cell surface compartment prior to its secretion (31,32). We therefore next evaluated the possibility that oleic acid increased the secreted fraction and reduced the cellular fraction of apoE by facilitating the release of apoE from a cell surface pool.…”

Divergent Effects of Peroxisome Proliferator-activated Receptor γ Agonists and Tumor Necrosis Factor α on Adipocyte ApoE Expression

“…Twenty g of cell lysate or 25 l of medium were resolved by 10% SDS-PAGE and electroblotted onto nitrocellulose membranes. The membranes were blocked in 5% nonfat milk, 1% bovine serum albumin, and apoE was detected by ECL kit (Amersham Biosciences) as previously described (24,25). Signals on immunoblot were quantitated using Zero DScan software (Scanalytics Inc., Fairfax, VA).…”