ApoA-I secretion from HepG2 cells: evidence for the secretion of both lipid-poor apoA-I and intracellularly assembled nascent HDL

Jeffrey W Chisholm,Ellen R Burleson,Gregory S Shelness,John S Parks

doi:10.1016/s0022-2275(20)30184-x

Abstract

The goal of this study was to determine whether apolipoprotein A-I (apoA-I) is lipidated before secretion by HepG2 cells. ApoA-I was extracted from microsomes after radiolabeling with [(35)S]Met/Cys. After ultracentrifugal flotation, d < 1.25 g/ml and d > 1.25 g/ml fractions were immunoprecipitated and analyzed by SDS-PAGE. Under steady state radiolabeling conditions, 20% of extracted microsomal apoA-I floated at d < 1.25 g/ml. Pulse-chase experiments demonstrated that the percentage of microsomal apoA-I associated with lipid peaked between 2 and 8 min postsynthesis. Density gradient ultracentrifugation, and nondenaturing gradient gel electrophoresis of HepG2 cell medium, indicated that 50% of secretory apoA-I existed as small HDL particles with a diameter of approximately 7.5 nm. These and additional data suggested that approximately 20% of newly secreted apoA-I is lipidated intracellularly and another 30% is secreted in lipid-free or lipid-poor form but acquires sufficient lipid to become small HDL within 1 h of secretion, with little further maturation over the time course of the incubation (2 h).These results indicate that a process exists for the presecretory intracellular assembly of apoA-I with lipid in HepG2 cells and that apoA-I is secreted in both lipid-poor and lipidated forms.

Highlights

The goal of this study was to determine whether apolipoprotein A-I is lipidated before secretion by HepG2 cells
Immunoprecipitation of apolipoprotein A-I (apoA-I) from the top (d Ͻ 1.25 g/ml) and bottom (d Ͼ 1.25 g/ml) fractions of chase medium subjected to ultracentrifugation demonstrated that ϳ50% of the radiolabeled apoA-I was associated with sufficient lipid to float by 120 min (Fig. 2)
Because ϳ25% of secreted apoA-I was lipidated at the earliest time point sampled (Fig. 2; 15 min), we hypothesized that some of the lipidation of apoA-I occurred in HepG2 cells before secretion and that the difference in lipidation between the early time points and 120 min was due to an extracellular lipidation process

Summary

Introduction

The goal of this study was to determine whether apolipoprotein A-I (apoA-I) is lipidated before secretion by HepG2 cells. Nondenaturing gradient gel electrophoresis of HepG2 cell medium, indicated that 50% of secretory apoA-I existed as small HDL particles with a diameter of ϳ7.5 nm. These and additional data suggested that ϳ20% of newly secreted apoA-I is lipidated intracellularly and another 30% is secreted in lipid-free or lipid-poor form but acquires sufficient lipid to become small HDL within 1 h of secretion, with little further maturation over the time course of the incubation (2 h).

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Conclusion