Abstract
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 (KIV(6-8)), and two lysine residues (Lys(680) and Lys(690)) within the NH(2) 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) KIV(6-8) and expressed these mutations in the context of a 17-kringle (17K) recombinant apo(a) variant. Single mutations that disrupt the WLBS in KIV(6), KIV(7), and KIV(8), as well as mutants that disrupt the WLBS in both KIV(6) and KIV(7), or both KIV(7) and KIV(8), were assessed for their ability to form non-covalent and covalent Lp(a) complexes. Our results demonstrate that both apo(a) KIV(7) and KIV(8), but not KIV(6), are required for maximally efficient non-covalent and covalent Lp(a) assembly. Single mutations in the WLBS of KIV(7) or KIV(8) 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 KIV(7) and KIV(8) 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 KIV(7) and KIV(8) specifically bind with high affinity to apoB-derived peptides containing Lys(690) or Lys(680), respectively. Taken together, our data demonstrate that specific interactions between apo(a) KIV(7) and KIV(8) and Lys(680) and Lys(690) 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.
Highlights
Lp(a) assembly is a two-step process in which non-covalent interactions between apo(a) and the apolipoprotein B-100 (apoB) component of low density lipoprotein (LDL) precede the formation of a single disulfide bond; the affinity of these non-covalent interactions is thought to be a determinant of the rate of covalent Lp(a) assembly
We have previously established that the initial non-covalent interaction is mediated by the interaction of lysine residues in the NH2 terminus of apoB (11) with weak lysine-binding sites (WLBS) present in apo(a) KIV6–8 (14)
To quantitatively define a role for each of the WLBS in apo(a) KIV6–8, we created single point mutations in each of the WLBS in these kringle domains in the context of 17K r-apo(a), and studied non-covalent and covalent binding to apoB in LDL
Summary
Lp(a), lipoprotein(a); LDL, low density lipoprotein; apo(a), apolipoprotein(a); apoB, apolipoprotein B; ⑀-ACA, ⑀-aminocaproic acid; WLBS, weak lysine-binding sites; 17K, 17-kringle; KIV, kringle IV; flu-LDL, fluorescein-labeled low density lipoprotein. Apo(a) adopts a closed conformation that inhibits covalent Lp(a) assembly, possibly by restricting access to the free sulfhydryl group in apo(a) KIV9 (15) In this regard, we have previously shown that the addition of the lysine analog ⑀-aminocaproic acid (⑀-ACA) elicits a conformational change in apo(a) to a more open structure, which results in a 6-fold enhancement in its ability to form covalent Lp(a) particles. With respect to sequence requirements in apo(a) that mediate its non-covalent interaction with apoB, several groups have shown that the weak lysine-binding sites (WLBS) present within apo(a) KIV5–8 are likely involved in mediating this interaction (11, 14, 19 –21). Our data identify a critical role for the WLBS in both apo(a) KIV7 and KIV8 in mediating non-covalent association with the Lys[690] and Lys[680] residues in apoB, respectively, and demonstrate that these interactions are required for the covalent step of Lp(a) formation
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