IDENTIFICATION OF THE CYSTEINE RESIDUE IN APOLIPOPROTEIN(A) THAT MEDIATES EXTRACELLULAR COUPLING WITH APOLIPOPROTEIN-B-100

We have utilized a recombinant expression system in order to study the assembly of lipoprotein(a) (Lp(a)) particles. Using a 17-kringle reco mbinant form of apolipoprotein(a) (apo(a)) to transiently transfect hu man hepatoma cells, we could not detect recombinant Lp(a) (r-Lp(a)) pa rticles intracellularly, by analysis of post-nuclear lysates. However, covalent r-Lp(a) complexes were observed in the transfected cell supe rnatants. Upon addition of [S-35]Cys-labeled human embryonic kidney ce ll supernatants transfected with 9-kringle or 17-kringle recombinant a po(a) (r-apo(a)) variants to human plasma, covalent r-Lp(a) complexes were observed, which could be immunoprecipitated using antibodies spec ific for either apo(a) or apolipoprotein B-100 (apoB-100); r-Lp(a) com plexes containing the 17-kringle r-apo(a) were shown to be in the 1.06 3 g/ml < d < 1.20 g/ml range by density gradient ultracentrifugation a nalysis. Complexes containing the 17-kringle r-apo(a) formed rapidly w ithin 20 min, with a slow increase observed up to 90 min. Addition of increasing amounts of plasma, as well as increasing amounts of isolate d human low density lipoprotein to cell culture supernatants containin g [S-35]Cys-labeled 17-kringle r-apo(a) led to enhanced r-Lp(a) comple x formation. Blocking of free sulfhydryls in apo(a) with N-ethylmaleim ide resulted in inhibition of r-Lp(a) complex formation in plasma, ver ifying the role of free sulfhydryls in Lp(a) particle assembly. Using site-directed mutagenesis, we demonstrated that Cys4057 in apo(a) is i nvolved in disulfide linkage with apoB-100 in Lp(a) particles.