Abstract
Our previous studies identified the lysosome as the compartment for degradation of newly synthesized apoE in primary macrophages. Lysosomal degradation of newly synthesized apoE is extensive and rapid (> 50% in 60 min). In the present study we tested the hypothesis that the macrophage cell surface is part of the itinerary of apoE in its path to the lysosomes. We therefore examined the existence and size of the apoE pool associated with the macrophage cell surface. Such a pool may not only provide a mechanism of targeting apoE for lysosomal degradation, by endocytosis, but also have important implications for the metabolism of lipoproteins by macrophages. Treatment of macrophages with heparin (10 micrograms/ml and 5 mg/ml) and heparinase I (1 U/ml), which releases substantial amounts of apoE from HepG2 cells, results in no additional release of apoE from macrophages. Treatment of macrophages with xyloside (1 mM) or GRGDTP (500 micrograms/ml) does not decrease the extent of cell-associated apoE. Both immunogold labeling, followed by electron microscopy, and immunofluorescent labeling and light microscopy further confirm the lack of significant amounts of cell surface-associated apoE in macrophages. In contrast, immunolabeled apoE is readily observed in permeabilized cells. Taken together, these data indicate the absence of significant apoE-glycosaminoglycan interaction at the macrophage cell surface. The lack of such an interaction is likely due to paucity of heparan sulfate proteoglycans on the macrophage cell surface, when compared to hepatocytes. Along with our previous observations. (Deng. J., V. Rudick, and L. Dory, 1995. J. Lipid Res. 36:2129-2140), these results suggest direct targeting of a portion of newly synthesized apoE from trans-Golgi network to lysosomes for degradation, without involving the plasma membrane and endocytosis.
Highlights
Our previous studies identified the lysosome as the compartment for degradation of newly synthesized Apolipoprotein E (apoE) in primary macrophages
The membrane-associated apoE o n these cells can be released by low concentrations of heparin, heparinase, and by xyloside treatment ( 7 ),suggesting that it is associated with the heparan sulfate moiety of cell surfaceassociated heparan sulfate proteoglycans (HSPG)
This is the most likely binding site, as a heparin or heparinase-releasable pool of apoE has been reported in HepG2 cells ( 7 ) .By adding exogenous heparin, the non-covalent interaction allows the displacement of apoE from the heparan sulfate moiety of the HSPG [19, 20]
Summary
0.05% tiypsin in 1 X EDTA, as described ( 7 ,9 ) .Briefly, 10 nil of trypsin solution was added to each T’75 parelit. Intracellular and surface-associated apoE were labeled by incubating permeabilized and non-permeabilized cells, respectively, with purified goat anti-rat apoE IgG for 60 min. Coverslips were washed over 5 min in three changes of either 1% Triton-X 100 in DPBS, for permeabilized cells, or DPBS, for non-permeabilized macrophages, before being exposed to secondary antibody. Another series of coverslips containing fixed non-permeabilized cells was incubated with rat anti-mouse Mac-2, an antibody specific for thioglycolate-elicited macrophage cell surface antigen. Grids containing the sections were washed with PBS; the aldehydes were quenched with 0.02 M glycine, blocked with 5% BSA in PBS, and exposed to various dilutions of either goat anti-apoE IgG or goat non-immune IgG. After incubation for 1 h at room temperature, the grids were washed and exposed to an anti-goat IgG 10 nm gold probe for 1 h. Sections were observed with a 910 Zeiss transmission electron microscope (ZeissInstruments, Thornwood, NY) and pictures were taken using Kodak SO-163 negative film
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