LIPOPROTEIN(A) ASSEMBLY - QUANTITATIVE ASSESSMENT OF THE ROLE OF APO(A) KRINGLE-IV TYPE-2-10 IN PARTICLE FORMATION

We have developed a system for the quantitative assessment of the effi ciency of lipoprotein(a) [Lp(a)] formation in vitro. Amino-terminally truncated derivatives of a 17-kringle form of recombinant apo(a) [r-ap o(a)] were transiently expressed in human embryonic kidney cells. Equi molar amounts of r-apo(a) derivatives were incubated with a fourfold m olar excess of purified human low density lipoprotein, and r-Lp(a) for mation was assessed by densitometric analysis of Western blots. Althou gh r-Lp(a) formation was observed with each r-apo(a) derivative, both the rate and extent of particle formation were greatly lower on remova l of kringle IV type 7. Additional substantial decreases in these para meters were observed on removal of kringle IV type 8, thereby suggesti ng a major role for these two kringles in Lp(a) assembly. We directly demonstrated that the lysine-binding sites (LBSs) within kringle IV ty pes 5-9 are ''masked'' in the context of the Lp(a) particle and are co nsequently unavailable for interaction with lysine-Sepharose. Using si te-directed mutagenesis, we also demonstrated that the previously desc ribed LBS in kringle IV type 10 is not required for r-Lp(a) formation: r-Lp(a) formation using a mutated form of apo(a) that lacks this LBS is comparable in efficiency to that of wild-type r-apo(a) and can be i nhibited to a similar extent by epsilon-amino-n-caproic acid. In summa ry, the results of our study indicate that apo(a) kringle IV types 7 a nd 8 are required for maximal efficiency of Lp(a) formation, likely by virtue of their ability to mediate lysine-dependent noncovalent inter actions with apoB-100 that precede disulfide bond formation.