ApoE of the HepG2 cell surface includes a major pool associated with chondroitin sulfate proteoglycans.
We have investigated the association of apolipoprotein E (apoE) with the HepG2 cell surface (i.e. plasma membrane and extracellular matrix) using domain specific monoclonal antibodies against apoE. Growth in beta-D-xyloside decreased the incorporation of 35S into glycosaminoglycans by 31% and cell surface apoE by 45% with a concomitant increase in apoE secretion (4.3-fold), underlining the importance of glycosaminoglycan association of apoE. Heparinase (3-10 U/mL) or heparin (1 mg/mL) decreased apoE by 25 and 30.5%, respectively, which suggests that some apoE is associated with cell surface heparan sulfate proteoglycans. Chondroitinase ABC (1.5 U/mL) reduced cell surface apoE by 40%, indicating that a major pool of apoE is associated with chondroitin sulfate proteoglycans. Further enzymatic and displacement analysis suggested that cell surface apoE associates specifically with GAGs containing chondroitin-4-sulfates. 3H1, a monoclonal antibody that recognizes an epitope within the lipid-binding C-terminal domain of apoE, decreased binding of apoE to chondroitin sulfate proteoglycans in solid-phase assays by 77% and to heparan sulfate proteoglycans by 46%, suggesting that this region is of increased importance for binding to chondroitin sulfate proteoglycans. Previous studies with 3H1 demonstrated that apoE of the extracellular matrix is lipid-poor (Burgess, J. W., Gould, D. R., and Marcel, Y. L. (1998) J. Biol. Chem. 273, 5645-5654), but we show here that apoE on the remaining cell surface is lipid-associated. In summary, lipidated apoE associates with the HepG2 plasma membrane through interactions with chondroitin-4-sulfate containing GAGs and, to a lesser extent, HSPG.
Burgess
J W
JW
Lipoprotein and Atherosclerosis Group, University of Ottawa Heart Institute, Ontario, Canada.
Liang
P
P
Vaidyanath
C
C
Marcel
Y L
YL
D001057:Apolipoproteins E / Q000737:chemistry* / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000737:chemistry* / Q000201:enzymology / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D011508:Chondroitin Sulfate Proteoglycans / Q000737:chemistry* / Q000494:pharmacology; D006025:Glycosaminoglycans / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D006923:Hymecromone / Q000031:analogs & derivatives / Q000494:pharmacology; D050356:Lipid Metabolism; D017434:Protein Structure, Tertiary; D013431:Sulfates / Q000378:metabolism; D013498:Suramin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D001057:Apolipoproteins E; D011508:Chondroitin Sulfate Proteoglycans; D006025:Glycosaminoglycans; D013431:Sulfates; D006923:Hymecromone; D013498:Suramin; C014111:methylumbelliferyl-beta-D-xyloside; D006493:Heparin