Effect of phospholipase A2 digestion on the conformation and lysine/fibrinogen binding properties of human lipoprotein[a].

In vitro hydrolysis of human lipoprotein[a] (Lp[a]) by phospholipase A2 (PLA2) decreased the phosphatidylcholine (PC) content by 85%, but increased nonesterified fatty acids 3.2-fold and lysoPC 12.9-fold. PLA2-treated Lp[a] had a decreased molecular weight, increased density, and greater electronegativity on agarose gels. In solution, PLA2-Lp[a] was a monomer, and when assessed by sedimentation velocity it behaved like untreated Lp[a], in that it remained compact in NaCl solutions but assumed the extended form in the presence of 6-amino hexanoic acid, which was shown previously to have an affinity for the apo[a] lysine binding site II (LBS II) comprising kringles IV5-8. We interpreted our findings to indicate that PLA2 digestion had no effect on the reactivity of this site. This conclusion was supported by the results obtained from lysine Sepharose and fibrinogen binding experiments, in the presence and absence of Tween 20, showing that phospholipolysis had no effect on the reactivity of the LBS-II domain. A comparable binding behavior was also exhibited by the free apo[a] derived from each of the two forms of Lp[a]. We did observe a small increase in affinity of PLA2-Lp[a] to lysine Sepharose and attributed it to changes in reactivity of the LBS I domain (kringle IV10) induced by phospholipolysis. In conclusion, the extensive modification of Lp[a] caused by PLA2 digestion had no significant influence on the reactivity of LBS II, which is the domain involved in the binding of apo[a] to fibrinogen and apoB-100. These results also suggest that phospholipids do not play an important role in these interactions.

Specificity of ligand-induced conformational change of lipoprotein(a).

The conformation of Lp(a) was probed with a set of omega-aminocarboxylic acids and other analogs of 6-aminohexanoic acid (6-AHA). Using the viscosity-corrected sedimentation coefficient, six additional ligands were shown to induce a major conformational change in Lp(a), from a compact form to an extended form. These were trans-4-(aminomethyl)cyclohexanecarboxylic acid (t-AMCHA), proline, 4-aminobutyric acid, 8-aminooctanoic acid, Nalpha-acetyllysine, and glycine. Lysine, Nepsilon-acetyllysine, glutamic acid, and adipic acid were determined not to cause a conformational change. Urea and guanidine hydrochloride were ineffective at inducing this conformational change at concentrations at which the above ligands did unfold Lp(a). The conformational change was inhibited by 100 mM NaCl and to a lesser extent by 20 mM sodium glutamate. Despite the fact that these two salts have nearly the same ionic strengths, the greater inhibition of the unfolding by NaCl is consistent with a proposed stabilization of interkringle interactions by chloride ions. In 100 mM NaCl, which most closely resembles physiological conditions, only proline, 4-aminobutyric acid, 6-AHA, and t-AMCHA were effective ligands. By analyzing the dimensions of the conformation altering ligands, we propose that a critical variable in determining the effectiveness of a ligand in disrupting Lp(a) is the distance between the carboxyl and amine functions of the ligand. The optimal distance is approximately 6 A, which agrees with the observed 6.6-6.8 A separation of the cationic and anionic centers of known plasminogen and apo(a) lysine binding sites. These studies have implications for the mechanism of Lp(a) particle assembly.

Molecular weight determination of lipoprotein(a) [Lp(a)] in solutions containing either NaBr or D2O: relevance to the number of apolipoprotein(a) subunits in Lp(a).

Molecular weight determination of low-density lipoprotein (LDL) is usually performed in solutions containing high concentrations of salt (up to 13.4 M NaBr) by sedimentation velocity and diffusion experiments, because it does not preferentially bind salt or water. Considering that lipoprotein(a) [Lp(a)] is structurally similar to LDL, differing only by the presence of Apo(a), the molecular weight, M, of Lp(a) has also been measured in solutions containing high concentrations of NaBr. We questioned the suitability of this practice by comparing the apparent molecular weight, Mapp, and partial volume, phi', of Lp(a) determined by sedimentation and flotation equilibrium in a three-component system containing NaBr with the analogous parameters, M and partial specific volume, v, determined in a two-component system containing D2O. LDL served as a control. In agreement with previous findings obtained with different methods, our results indicate no significant differences in M and v of four different LDL samples and apparently no significant preferential binding of solvent components. In contrast, values of Mapp and phi' of Lp(a) evaluated in NaBr are significantly greater than M and v. Preferential binding of solvent components appeared to be a function of Apo(a) mass or the number of kringle IV domains, as expressed by increasing percentage differences between the two sets of parameters, ranging from 4 to 13% in M and 0.2 to 0.5% in v of Lp(a) species having Apo(a) with 15-27 kringle IV domains. Furthermore, our results indicate that the variable Apo(a) kringle IV domains are more involved in this process than the constant domain of Apo(a). These findings indicate that the Lp(a) molecular weight should be determined in D2O and that high concentrations of NaBr should be avoided as their use would lead to overestimated molecular weights and partial specific volumes. Application of this method to the question of how much Apo(a) is released upon the reduction of Lp(a) led to the conclusion that Lp(a) contains only one Apo(a) molecule.