The HepG2 extracellular matrix contains separate heparinase- and lipid-releasable pools of ApoE. Implications for hepatic lipoprotein metabolism.
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.
Burgess
J W
JW
Lipoprotein and Atherosclerosis Group, University of Ottawa Heart Institute, Department of Biochemistry, University of Ottawa, Ottawa, Ontario K1Y 4E9, Canada.
Gould
D R
DR
Marcel
Y L
YL
D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D001057:Apolipoproteins E / Q000032:analysis / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular / Q000737:chemistry; D004533:Egtazic Acid / Q000494:pharmacology; D004655:Emulsions; D005109:Extracellular Matrix / Q000737:chemistry*; D005217:Fat Emulsions, Intravenous / Q000494:pharmacology; D019763:Heparin Lyase / Q000378:metabolism*; D006497:Heparan Sulfate / Q000378:metabolism; D006801:Humans; D008055:Lipids / Q000494:pharmacology*; D008074:Lipoproteins / Q000378:metabolism; D010713:Phosphatidylcholines / Q000494:pharmacology; D010743:Phospholipids; D011485:Protein Binding / Q000502:physiology; D012450:Safflower Oil; D013024:Soybean Oil; D013498:Suramin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D001057:Apolipoproteins E; D004533:Egtazic Acid; D004655:Emulsions; D005217:Fat Emulsions, Intravenous; D019763:Heparin Lyase; D006497:Heparan Sulfate; D008055:Lipids; D008074:Lipoproteins; D010713:Phosphatidylcholines; D010743:Phospholipids; D012450:Safflower Oil; D013024:Soybean Oil; D013498:Suramin; C063336:safflower oil, soybean oil, phospholipid emulsion