Identification of the cysteine residue in apolipoprotein(a) that mediates extracellular coupling with apolipoprotein B-100

Abstract

We have utilized a recombinant expression system in order to study the assembly of lipoprotein(a) (Lp(a)) particles. Using a 17-kringle recombinant form of apolipoprotein(a) (apo(a)) to transiently transfect human hepatoma cells, we could not detect recombinant Lp(a) (r-Lp(a)) particles intracellularly, by analysis of postnuclear lysates. However, covalent r-Lp(a) complexes were observed in the transfected cell supernatants. Upon addition of [35S]Cys-labeled human embryonic kidney cell supernatants transfected with 9-kringle or 17-kringle recombinant apo(a) (r-apo(a)) variants to human plasma, covalent r-Lp(a) complexes were observed, which could be immunoprecipitated using antibodies specific for either apo(a) or apolipoprotein B-100 (apoB-100); r-Lp(a) complexes containing the 17-kringle r-apo(a) were shown to be in the 1.063 g/ml < d < 1.20 g/ml range by density gradient ultracentrifugation analysis. Complexes containing the 17-kringle r-apo(a) formed rapidly within 20 min, with a slow increase observed up to 90 min. Addition of increasing amounts of plasma, as well as increasing amounts of isolated human low density lipoprotein to cell culture supernatants containing [35S]Cys-labeled 17-kringle r-apo(a) led to enhanced r-Lp(a) complex formation. Blocking of free sulfhydryls in apo(a) with N-ethylmaleimide resulted in inhibition of r-Lp(a) complex formation in plasma, verifying the role of free sulfhydryls in Lp(a) particle assembly. Using site-directed mutagenesis, we demonstrated that Cys4057 in apo(a) is involved in disulfide linkage with apoB-100 in Lp(a) particles.