Quantitative evaluation of the contribution of weak lysine-binding sites present within apolipoprotein(a) kringle IV types 6-8 to lipoprotein(a) assembly

During lipoprotein(a) (Lp(a)) assembly, non-covalent interactions between apolipoprotein(a) (apo(a)) and low density lipoprotein precede specific disulfide bond formation. Studies have shown that the non-covalent step involves an interaction between the weak lysine-binding sites (WLBS) present within each of apo(a) kringle IV types 6, 7, and 8 (KIV6-8), and two lysine residues (Lys(680) and Lys(690)) within the NH2 terminus of the apolipoprotein B-100 (apoB) component of low density lipoprotein. In the present study, we introduced single point mutations (E56G) into each of the WLBS present in apo(a) KIV6-8 and expressed these mutations in the context of a 17-kringle (17K) recombinant apo(a) variant. Single mutations that disrupt the WLBS in KIV6, KIV7, and KIV8, as well as mutants that disrupt the WLBS in both KIV6 and KIV7, or both KIV7 and KIV8, were assessed for their ability to form non-covalent and covalent Lp(a) complexes. Our results demonstrate that both apo(a) KIV7 and KIV8, but not KIV6, are required for maximally efficient non-covalent and covalent Lp(a) assembly. Single mutations in the WLBS of KIV7 or KIV8 resulted in a 3-fold decrease in the affinity of 17K recombinant apo(a) for apoB, and a 20% reduction in the rate of covalent Lp(a) formation. Tandem mutations in the WLBS in both KIV7 and KIV8 resulted in a 13-fold reduction in the binding affinity between apo(a) and apoB, and a 75% reduction in the rate of the covalent step of Lp(a) formation. We also showed that KIV7 and KIV8 specifically bind with high affinity to apoB-derived peptides containing Lys690 or Lys680, respectively. Taken together, our data demonstrate that specific interactions between apo(a) KIV7 and KIV8 and Lys680 and Lys690 in apoB mediate a high affinity non-covalent interaction between apo(a) and low density lipoprotein, which dictates the efficiency of covalent Lp(a) formation.