Dynamic and stable pools of apoE differ functionally at the HepG2 cell surface

Jim W Burgess,Yves L Marcel

doi:10.1016/s0022-2275(20)30273-x

Abstract

Synthesis of apolipoprotein (apo)E in hepatocytes leads to both secretion and retention in cell surface pools. Inclusion of Brefeldin A to HepG2 cells prompted a rapid decrease of cell surface apoE to about 37% of control values after a 3-h incubation. The t1/2 for this dynamic pool was estimated to be 15 min. In contrast, a stable fraction of apoE (t1/2 > 20 h) was found in association with the extracellular matrix (ECM). Increased content of apoE on the ECM correlated with decreased binding of VLDL. Decreased apoE on the cell surface correlated with increased binding of VLDL to cells. Collectively, this suggests that glycosaminoglycan-bound apoE can occlude binding sites for apoE-containing lipoproteins on glycosaminoglycans. In solid-phase assays, heparin, suramin, and chondroitin sulfates A and B efficiently inhibited the binding of apoE to heparan sulfate proteoglycans, but were unable to displace apoE from this glycosaminoglycan. Finally, decreasing cell surface apoE with suramin subsequently decreased the apoE content on secreted apoB-containing lipoproteins without affecting the overall secretion of apoE or apoB to the extracellular medium. In summary, cell surface apoE comprises both dynamic fractions, which can be donated to newly secreted lipoproteins, and stable fractions, which may act to minimize the unproductive binding of lipoproteins to the ECM. —Burgess, J. W., and Y. L. Marcel. Dynamic and stable pools of apoE differ functionally at the HepG2 cell surface.

Highlights

Synthesis of apolipoproteinE in hepatocytes leads to both secretion and retention in cell surface pools
The results presented here introduce novel findings regarding cell surface apolipoprotein E (apoE) including i) a dynamic pool of cell surface apoE exists on the HepG2 cell surface and can be transferred to extracellular lipoproteins, ii) apoE associated with glycosaminoglycans reduces the subsequent binding of apoE-enriched lipid particles, iii) a stable pool of apoE associated with the extracellular matrix (ECM) reduces unproductive binding of apoE-enriched particles to this surface, and iv) cell surface apoE can be transferred to newly secreted apolipoprotein B (apoB)-lipoproteins
Studies with Brefeldin A indicate that the HepG2 cell surface maintains both dynamic and stable pools of apoE

Summary

EXPERIMENTAL PROCEDURES

Na125I was obtained from Amersham (Oakville, ON). Iodobeads were from Pierce (Rockford, IL). To examine binding of apoE to the glycosaminoglycans, apoVLDL prepared from VLDL as described [17] was added to the wells (2.5 ␮g/100 ␮l) and incubated in the absence or presence of heparin (1 mg/ml), suramin (0.5 mg/ml), or chondroitin sulfates A, B, or C (1 mg/ml) for 2 h at 37ЊC. Displacement assays were conducted as follows: apoVLDL was pre-incubated with the immobilized glycosaminoglycans as described above; unbound material was removed with three washes with PBS; and Eagle’s minimum essential medium (EMEM)-BSA containing heparin, suramin, or chondroitin sulfates A, B, or C was added and incubated at 37ЊC for 1 h. The wells were washed three times with PBS, and 125I-7C9 in PBS containing 2 mg/ml BSA was added and incubated with the adsorbed lipoproteins for 1 h at 20ЊC. The wells were washed three times with PBS and counted in a gamma counter

Miscellaneous procedures

RESULTS

DISCUSSION