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J Biol Chem, Vol. 273, Issue 10, 5645-5654, March 6, 1998
From the Lipoprotein and Atherosclerosis Group, University of
Ottawa Heart Institute, Departments of Biochemistry, and Pathology and
Laboratory Medicine, University of Ottawa,
Ottawa, Ontario K1Y 4E9, Canada
We have examined the association of apoE with the
extracellular matrix (ECM) of HepG2 cells. Comparison of ECM
prepared by previously published methods demonstrated that
cytochalasin B-prepared material yielded the highest endogenous apoE,
representing 23.6% of that in cell monolayers. ECM prepared with EDTA
or Triton X-100 exhibited decreased levels of apoE, 3 and 6%,
respectively. ECM bound very low density lipoprotein poorly (5-6% of
the monolayer capacity); however, these incubations dramatically
increased the apoE content of the ECM. Heparinase or suramin decreased
apoE of the ECM by 19.6 and 37.3%, respectively, suggesting
association with heparin sulfate proteoglycans. EDTA or EGTA also
displaced 35% of the apoE, suggesting a
Ca2+-dependent association. Incubation
with phosphatidylcholine vesicles (PCV) displaced 30% of the apoE,
suggesting that lipid content affects association of apoE with the ECM.
Data derived from sequential incubations with combinations of suramin,
EGTA, and PCV were consistent with the presence of two distinct pools
of apoE on the HepG2 ECM, one releasable with suramin and EGTA and the
other releasable with lipids. Exogenously applied lipid-free apoE
readily bound to the ECM; however, increasing the lipid content
decreased its association. Lipid-free apoE could be equally displaced
from the ECM with PCV or suramin. When lipid-free apoE adsorbed to
microtiter wells was incubated with a triglyceride emulsion or
palmitoyloleyl phosphatidylcholine micelles, the immunoreactivity of
3H1 (but not other antibodies), a monoclonal antibody against an
epitope in the C-terminal domain of apoE, increased about 4-fold. In a similar manner, incubation of ECM with lipid dramatically increased the
immunoreactivity of 3H1, indicating that apoE of the ECM exists in a
lipid-poor form. Scatchard analysis demonstrated that the increased
immunoreactivity was due to an increase in the number of antibody
binding sites. In conclusion, the ECM contains two pools of lipid-poor
apoE. One pool associates with the ECM through heparin sulfate
proteoglycans- and Ca2+-dependent interactions.
A second pool of apoE dissociates from the ECM upon lipidation. The
lipid-sensitive pool of apoE may participate in secretion or efflux of
lipids or in the capture of lipoproteins by providing the apoE
needed for receptor-mediated uptake.
The HepG2 Extracellular Matrix Contains Separate Heparinase- and
Lipid-releasable Pools of ApoE
IMPLICATIONS FOR HEPATIC LIPOPROTEIN METABOLISM
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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