High-Level Expression of Various Apolipoprotein (a) Isoforms by "Transferrinfection". The Role of Kringle IV Sequences in the Extracellular Association with Low-Density Lipoprotein

Characterization of the assembly of lipoprotein(a) [Lp(a)] is of fundamental importance to understanding the biosynthesis and metabolism of this atherogenic lipoprotein. Since no established cell lines exist that express Lp(a) or apolipoprotein(a) [apo(a)], a "transferrinfection" system for apo(a) was developed utilizing adenovirus receptor- and transferrin receptor-mediated DNA uptake into cells. Using this method, different apo(a) cDNA constructions of variable length, due to the presence of 3, 5, 7, 9, 15, or 18 internal kringle IV sequences, were expressed in cos-7 cells or CHO cells. All constructions contained kringle IV-36, which includes the only unpaired cysteine residue (Cys-4057) in apo(a). r-Apo(a) was synthesized as a precursor and secreted as mature apolipoprotein into the medium. When medium containing r-apo(a) with 9, 15, or 18 kringle IV repeats was mixed with normal human plasma LDL, stable complexes formed that had a bouyant density typical of Lp(a). Association was substantially decreased if Cys-4057 on r-apo(a) was replaced by Arg by site-directed mutagenesis or if Cys-4057 was chemically modified. Lack of association was also observed with r-apo(a) containing only 3, 5, or 7 kringle IV repeats without "unique kringle IV sequences", although Cys-4057 was present in all of these constructions. Synthesis and secretion of r-apo(a) was not dependent on its sialic acid content. r-Apo(a) was expressed even more efficiently in sialylation-defective CHO cells than in wild-type CHO cells. In transfected CHO cells defective in the addition of N-acetylglucosamine, apo(a) secretion was found to be decreased by 50%. Extracellular association with LDL was not affected by the carbohydrate moiety of r-apo(a), indicating a protein-protein interaction between r-apo(a) and apoB. These results show that, besides kringle IV-36, other kringle IV sequences are necessary for the extracellular association of r-apo(a) with LDL. Changes in the carbohydrate moiety of apo(a), however, do not affect complex formation

Study of apo(a) length polymorphism and lipoprotein(a) concentrations in subjects with single or double apo(a) isoforms.

Cardiovascular risk is associated with high lipoprotein(a) (Lp(a)) concentrations and low molecular weight apolipoprotein(a) (apo(a)) isoforms. We studied the relationship between these two biological parameters, particularly in subjects expressing two apo(a) isoforms. Plasma Lp(a) was measured by immunonephelometry in 530 unrelated Caucasian patients at high cardiovascular risk, and apo(a) size determined by immunoblotting using a recombinant standard. Two, one, or no apo(a) isoforms were detected in 258, 270, and 2 subjects, respectively. Lp(a) concentrations showed a non-Gaussian distribution, being higher in the 'double band' than in the 'single band' group (median 0.42 vs. 0.11 g/l, p < 0.0005). Apo(a) size distribution was bimodal, with two frequency peaks at 18 kringles (K) and 27 K. Small size apo(a) isoforms were more frequently found in the 'double band' group, where major isoforms were of lower size than minor isoforms (median 20 vs. 27 K). Regression analysis showed that apo(a) gene length accounted for 33% of Lp(a) variation, with a threshold effect at 20 K, no correlation being found over this value. The minor apo(a) isoform did not significantly influence Lp(a) concentration. These data confirm the relationship between apo(a) size and Lp(a) concentration and suggest that the assessment of cardiovascular risk should take into account the threshold effect at 20 K and the absence of influence of the minor apo(a) isoform