Bacterial expression and characterization of human recombinant apolipoprotein(a) kringle IV type 9

Elevated plasma lipoprotein(a) [Lp(a)] is an independent risk factor for several vascular diseases. Lp(a) particles are generated through the formation of a disulfide bond between Cys(4057) of kringle TV type 9, (KIVt9), of the multikringle apolipoprotein(a) [apo(a)] and a cysteine in apoB-100 low-density Lipoprotein (LDL). To better understand this interaction, we have expressed and purified KIVt9 from Escherichia coli as a His-Tag fusion protein. Dithiothreitol (DTT)-treated purified KIVt9 migrated as a single similar to 17.3-kDa band on SDS-PAGE gels. Without DTT, an additional band twice the molecular weight of KIVt9 was observed. The double-size band presumably resulted hom dimerization of individual kringles, through their unpaired cysteine residues, since a mutation Cys(4057) -->Ser ([Ser(4057)]KIVt9) abolished dimer formation. Using a gel-shift assay, we showed that KIVt9 could couple to 14-amino-acid apoB-100 synthetic peptides (apoB(3732-3745) and apoB(4319-4332)) containing Cys(3734) or cys(4326). Both Of these apoB-100 cysteines have been reported to associate with apo(a) to generate Lp(a). In the presence of either apoB-100 peptide, KIVt9 was shifted to a higher molecular weight that was consistent with the covalent addition of a 1.2-kDa apoE-100 peptide. Identical apoB-100 peptides in which the cysteine residues were replaced by alanine ([Ala(3734)]apoB(3732)-(3745) and [Ala(4326)]apo(B4319-4332)) had no effect in the gel-shift assay. Furthermore, [Ser(4057)]KIVt9 did not covalently interact with apoB(3732-3745) or apoB(4319-4332) These results indicated that KIVt9 couples to the Cys-apoB-100 peptides through a disulfide linkage. This system may be suitable for further investigating the apo(a)/apoB-100 coupling reaction and the structure of KIVt9 through X-ray crystallographic studies. (C) 1998 Academic Press.