Secretion-capture role for apolipoprotein E in remnant lipoprotein metabolism involving cell surface heparan sulfate proteoglycans.
To determine the impact of enhanced apolipoprotein (apo) E secretion on the mechanism of remnant lipoprotein uptake, rat hepatoma cells (McA-RH7777) were stably transfected with normal human apoE3 or receptor binding-defective apoE-Leiden. After a 2-h incubation, the human apoE secreted from the transfected hepatocytes was 10-12 times greater than the endogenous rat apoE. The apoE3-transfected cells bound and internalized rabbit beta-very low density lipoproteins (beta-VLDL) to a much greater degree than did apoE-Leiden-transfected cells and nontransfected cells. The apoE3-secreting cells displayed a 2-3.5-fold enhancement of cell-associated beta-VLDL compared to either the apoE-Leiden-transfected or nontransfected cells. Fluorescently labeled beta-VLDL were observed to concentrate within intracellular granules of the apoE3-transfected cells, presumably within endosomes and lysosomes. Furthermore, electron microscopy revealed that the apoE3-secreting cells displayed abundant beta-VLDL and chylomicron remnants on their cell surfaces and microvilli, in contrast to non-transfected or apoE-Leiden-secreting cells. Electron microscopy also revealed an abundance of chylomicron remnants within intracellular vesicles and multivesicular bodies of apoE3-transfected hepatocytes. Heparinase treatment (3 units/ml) completely abolished the increased association of beta-VLDL with apoE3-transfected cells but did not affect the limited association of beta-VLDL with apoE-Leiden-transfected or nontransfected cells. We established that the apoE3-enriched beta-VLDL were bound to cell surface heparan sulfate proteoglycans, as was the newly synthesized and secreted apoE3 (approximately 12% of the total secreted apoE3 was released by heparinase and suramin; 4% by heparin). In addition, reisolation of beta-VLDL by fast performance liquid chromatography after their incubation with exogenous apoE3, with medium from apoE3-secreting cells, or with the apoE3-secreting cells themselves revealed that the particles were enriched in apoE3 and displayed enhanced binding. These results suggest a secretion-capture role for apoE and indicate an important role for heparan sulfate proteoglycans on the cell surface for remnant lipoprotein metabolism.
Ji
Z S
ZS
Gladstone Institute of Cardiovascular Disease, University of California, San Francisco 94141-9100.
Fazio
S
S
Lee
Y L
YL
Mahley
R W
RW
D000818:Animals; D053318:Apolipoprotein E3; D001057:Apolipoproteins E / Q000302:isolation & purification / Q000378:metabolism*; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008079:Lipoproteins, VLDL / Q000378:metabolism*; D008113:Liver Neoplasms; D008114:Liver Neoplasms, Experimental; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008854:Microscopy, Electron; D008954:Models, Biological; D008961:Models, Structural; D011509:Proteoglycans / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D018110:Receptors, Lipoprotein / Q000096:biosynthesis / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D053318:Apolipoprotein E3; D001057:Apolipoproteins E; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D008079:Lipoproteins, VLDL; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D011509:Proteoglycans; D018110:Receptors, Lipoprotein; C052720:apolipoprotein E3 (Leidein)