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 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.
Koschinsky
M L
ML
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Côté
G P
GP
Gabel
B
B
van der Hoek
Y Y
YY
D000595:Amino Acid Sequence; D053299:Apolipoprotein B-100; D001053:Apolipoproteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D001055:Apolipoproteins B / Q000302:isolation & purification / Q000378:metabolism*; D053241:Apoprotein(a); D001483:Base Sequence; D002460:Cell Line; D003545:Cysteine / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007668:Kidney; D007700:Kinetics; D017270:Lipoprotein(a); D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013462:Sulfur Radioisotopes; D014162:Transfection; D014407:Tumor Cells, Cultured
D053299:Apolipoprotein B-100; D001053:Apolipoproteins; D001055:Apolipoproteins B; D017270:Lipoprotein(a); D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D053241:Apoprotein(a); D003545:Cysteine