Expression of a recombinant apolipoprotein(a) in HepG2 cells. Evidence for intracellular assembly of lipoprotein(a).
Apolipoprotein(a) (apo(a)), a large glycoprotein with extensive homology to plasminogen, forms a complex with apolipoprotein B100 (apoB100), which circulates in human plasma in the form of lipoprotein(a) (Lp(a)). Evidence indicates that the association of apo(a) with apoB100 occurs in the extracellular environment. We have reevaluated the possibility that apo(a)-B100 association can also occur as an intracellular event through studies with HepG2 cells stably transfected with an apo(a) minigene. Several lines of evidence support this possibility. First, continued Lp(a) production was demonstrated following incubation of transfected HepG2 cells with anti-apo(a) antisera, conditions that effectively block the fluid-phase association of apo(a) and apoB100 in vitro. Second, an apo(a)-B100 complex was detectable in Western blot analyses of transfected HepG2 lysates following immunoprecipitation with anti-apo(a) antisera. These studies incorporated precautions to eliminate cell-surface attachment of preformed apo(a)-B100 complexes to the low density lipoprotein receptor and were conducted in the presence of the lysine analog epsilon-aminocaproic acid, which precludes apo(a)-B100 association occurring during the isolation and analyses. Third, the presence of an intracellular apo(a)-B100 complex was demonstrated in lipoproteins isolated from microsomal contents. Of particular significance was the observation that this complex contained the precursor form of apo(a), which is not secreted, in addition to the mature, recombinant form. Finally, direct evidence was provided for the synthesis of a precursor form of apo(a) in a nascent intracellular complex with apoB100 following treatment of transfected HepG2 cells with brefeldin A plus N-acetyl-leucyl-leucyl-norleucinal. Taken together, these data suggest that apo(a)-B100 association can occur as an intracellular event in a human hepatoma-derived cell line, raising important implications for the regulation of Lp(a) secretion from human liver.
Bonen
D K
DK
Department of Medicine, University of Chicago, Chicago, Illinois 60637, USA.
Hausman
A M
AM
Hadjiagapiou
C
C
Skarosi
S F
SF
Davidson
N O
NO
D053299:Apolipoprotein B-100; D001054:Apolipoproteins A / Q000378:metabolism*; D001055:Apolipoproteins B / Q000378:metabolism; D006801:Humans; D007106:Immune Sera; D017270:Lipoprotein(a) / Q000737:chemistry / Q000378:metabolism*; D008861:Microsomes / Q000737:chemistry; D010449:Peptide Mapping; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D053299:Apolipoprotein B-100; D001054:Apolipoproteins A; D001055:Apolipoproteins B; D007106:Immune Sera; D017270:Lipoprotein(a); D011498:Protein Precursors; D011994:Recombinant Proteins