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 homolo gy 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 occu rs in the extracellular environment, We have reevaluated the possibili ty that apo(a)-B100 association can also occur as an intracellular eve nt through studies with HepG2 cells stably transfected with an apo(a) minigene. Several lines of evidence support this possibility, First, c ontinued Lp(a) production was demonstrated following incubation of tra nsfected HepG2 cells with anti-apo(a) antisera, conditions that effect ively block the fluid-phase association of apo(a) and apoB100 in vitro , Second, an apo(a)-B100 complex was detectable in Western blot analys es of transfected HepG2 lysates following immunoprecipitation with ant i-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 th e lysine analog E-aminocaproic acid, which precludes apo(a)-B100 assoc iation occurring during the isolation and analyses, Third, the presenc e of an intracellular apo(a)-B100 complex was demonstrated in lipoprot eins 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 for m, Finally, direct evidence was provided for the synthesis of a precur sor form of apo(a) in a nascent intracellular complex with apoB100 fol lowing treatment of transfected HepG2 cells with brefeldin A plus N-ac etyl-leucyl-leucyl-norleucinal, Taken together, these data suggest tha t apo(a)B100 association can occur as an intracellular event in a huma n hepatoma-derived cell line, raising important implications for the r egulation of Lp(a) secretion from human liver.