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J Biol Chem, Vol. 275, Issue 7, 4759-4765, February 18, 2000

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

Yuwei Zhao and Theodore Mazzone

From the Departments of Medicine and Biochemistry, Rush Medical College, Chicago, Illinois 60612

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 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.

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^* This work was supported by National Institutes of Health Grants HL 39653 and HL 57489.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Rush Medical College, 1653 W. Congress Pkwy., Chicago, IL 60612. Tel.: 312-942-8231; Fax: 312-942-8233; E-mail: tmazzone@rush.edu.

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Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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