Identification of Sequences in Apolipoprotein(a) that Maintain Its Closed Conformation:  A Novel Role for Apo(a) Isoform Size in Determining the Efficiency of Covalent Lp(a) Formation

Abstract

We have previously demonstrated that, in the presence of the lysine analogue epsilon-aminocaproic acid, apolipoprotein(a) [apo(a)] undergoes a conformational change from a closed to an open structure that is characterized by a change in tryptophan fluorescence, an increase in the radius of gyration, an alteration of domain stability, and an enhancement in the efficiency of covalent lipoprotein(a) [Lp(a)] formation. In the present study, to identify sequences within apo(a) that maintain its closed conformation, we used epsilon-aminocaproic acid to probe the conformational status of a variety of recombinant apo(a) isoforms using analytical ultracentrifugation, differential scanning calorimetry, intrinsic fluorescence, and in vitro covalent Lp(a) formation assays. We observed that the closed conformation of apo(a) is maintained by intramolecular interaction(s) between sequences within the amino- and carboxyl-terminal halves of the molecule. Using site-directed mutagenesis, we have identified the strong lysine-binding site present within apo(a) kringle IV type 10 as an important site within the C-terminal half of the molecule, which is involved in maintaining the closed conformation of apo(a). Apo(a) exhibits marked isoform size heterogeneity because of the presence of varying numbers of copies of the kringle IV type-2 domain located within the amino-terminal half of the molecule. Using recombinant apo(a) species containing either 1, 3, or 8 copies of kringle IV type 2, we observed that, while apo(a) isoform size does not alter the affinity of apo(a) for low-density lipoprotein, it affects the conformational status of the protein and therefore influences the efficiency of covalent Lp(a) assembly. The inverse relationship between apo(a) isoform size and the efficiency of covalent Lp(a) formation that we report in vitro may contribute to the inverse relationship between apo(a) isoform size and plasma Lp(a) concentrations that has been observed in vivo.

Apolipoprotein(a) Inhibits In Vitro Tube Formation in Endothelial Cells: Identification of Roles for Kringle V and the Plasminogen Activation System

“…1. The construction and expression of these r-apo(a) variants has been described previously [9], [10], [29]–[32]. All r-apo(a) variants were purified from the conditioned medium (CM) of stably expressing human embryonic kidney (293) cell lines by lysine-Sepharose affinity chromatography as previously described [29], with the exception of apo(a) KIV 1–4 [9].…”

Apolipoprotein(a), through Its Strong Lysine-binding Site in KIV10, Mediates Increased Endothelial Cell Contraction and Permeability via a Rho/Rho Kinase/MYPT1-dependent Pathway

“…An expression plasmid encoding the KIV 1-4 r-apo(a) variant was generated using the parental 17K r-apo(a) expression plasmid pRK5ha17 (26). Apo(a) KIV 1-4 is purified as previously described with some modifications (27). First, conditioned media containing the recombinant protein was passed over a 5-ml ConA-Sepharose (Amersham Biosciences) micro-column instead of a 20-ml lectin-Sepharose column.…”

Inhibition of plasminogen activation by apo(a): role of carboxyl-terminal lysines and identification of inhibitory domains in apo(a)

“…THP-1 cells were seeded at a density of 150,000-200,000 apo(a) and the C terminus of apoB-100 ( 21,22 ). Apo(a) KIV 10 contains a strong LBS that resembles the LBS present in plasminogen kringle 4 ( 23 ).…”