Lipid peroxidation of lipoprotein(a) [Lp(a)] by defined oxygen-centred free radicals (O2-· /OH·, O2-·, O2-· /HO2·) produced by γ radiolysis was compared with that of paired samples of low-density lipoprotein (LDL). Lp(a) appeared to be more resistant to oxidation than LDL, as indicated by the kinetic study of four markers of lipid peroxidation: decrease in vitamin E, formation of conjugated dienes and aldehydic products, and modification of electrophoretic mobility. In contrast, similar kinetics of lipid peroxidation were obtained for LDL and Lp(a-), which is the lipoparticle issued following the reductive cleavage of apolipoprotein(a) from Lp(a), thus suggesting that the greater resistance of Lp(a) to lipid peroxidation was due to the presence of apolipoprotein(a). Lipid peroxidation of Lp(a) and LDL induced by peroxyl radicals, which were produced by an azo compound [2,2′-azobis-(2-amidinopropane)dihydrochloride], confirmed both the resistance of Lp(a) to lipid peroxidation and the propensity of Lp(a-) to exhibit a greater susceptibility to oxidation than intact Lp(a). Our findings also indicated that the high content of apolipoprotein(a) in N-acetylneuraminic acid residues was only partly responsible for the resistance of Lp(a) to oxidation.
Elevated levels of lipoprotein(a) (Lp(a)) have been strongly correlated with the development of atherosclerosis in human populations. Lp(a) is distinguishable from low density lipoprotein by the presence of the unique protein component apolipoprotein(a) (apo(a)), which contains repeated domains that closely resemble that of plasminogen kringle IV. Using human embryonic kidney cells, we have expressed a recombinant form of apo(a) (r-apo(a)) containing 17 kringle IV-like domains. We have utilized this recombinant expression system to study the assembly of Lp(a) particles. We have demonstrated that Lp(a) particles containing r-apo(a) can be assembled extracellularly in plasma by covalent linkage to low density lipoprotein. Using site-directed mutagenesis, we have demonstrated that a cysteine residue present at position 4057 of the apo(a) protein (i.e., in the penultimate kringle IV repeat) mediates this covalent linkage. Using polymerase chain reaction amplification of liver apo(a) complementary DNA, we have demonstrated the presence of a polymorphism in apo(a) kringle IV type 10, which results in the substitution of a threonine for a methionine. Preliminary studies indicate that the presence of a threonine at this position may enhance the interaction of Lp(a) with lysine–Sepharose.Key words: apolipoprotein(a), lipoprotein(a), kringles, lipoprotein(a) assembly, polymorphism.
Abstract
Lipoprotein(a) [Lp(a)], a variant of low-density lipoprotein, is heterogeneous in density because of variability in the content and composition of its core lipids and size polymorphism of its specific glycoprotein component, apolipoprotein(a) [apo(a)]. In some individuals, density polymorphism may also derive from the fact that Lp(a) contains 2 mol of apo(a) per mole of apoB100, contrary to the more common 1:1 molar stoichiometry. Moreover, the size of apo(a) is polymorphic because of variations in the number of kringle 4 type 2 repeats. Another type of apo(a) polymorphism is related to sequence mutations at the kringle level. Two mutations can occur in kringle 4 type 10: one, Trp72-->Arg, is affiliated with an Lp(a) that is lysine-binding defective; the other, Met66-->Thr, with a normal lysine-binding function. Thus, Lp(a) is structurally and functionally polymorphic, a notion that must be considered in assessing the cardiovascular pathogenicity of this lipoprotein variant and in immunoquantification assays.
Abstract
A quantitative sandwich ELISA for lipoprotein(a) [Lp(a)], utilizing a monoclonal capture antibody that recognizes human and rhesus monkey apolipoprotein(a) [apo(a)] isoforms in combination with a polyclonal anti-apolipoprotein B-peroxidase conjugate was developed. This assay generates a linear calibration curve from 31.2 to 1000 mg/L, is highly reproducible (intra- and interassay CV of < 5% and < or = 12%, respectively), and shows no interference from plasminogen (1 g/L), low-density lipoprotein (6.00 g/L), triglycerides (27.00 g/L from chylomicrons and 10.00 g/L from very-low-density lipoprotein), hemoglobin (5 g/L), or bilirubin (30 mg/L). This assay format quantifies the concentration of Lp(a) on an equal molar basis regardless of apo(a) isoform. In contrast, a commercially available ELISA [Macra Lp(a)] method with a monoclonal anti-apo(a) capture antibody and a polyclonal anti-apo(a) conjugate was found to underestimate the Lp(a) concentrations of individuals with lower-M(r) apo(a) isoforms--whether quantifying the Lp(a) in plasma or the purified lipoprotein. This demonstrates the importance of assay format selection in quantifying Lp(a).
Abstract
Lipoprotein(a) is composed of low density lipoprotein
and apolipoprotein(a). Apolipoprotein(a) has evolved
from plasminogen and contains 10 different plasminogen
kringle 4 homologous domains [KIV(1 110)]. Previous
studies indicated that lipoprotein(a) noncovalently
binds the Nterminal region of lipoprotein B100
and the plasminogen kringle 4 binding plasma protein
tetranectin. In this study recombinant KIV(2), KIV(7)
and KIV(10) derived from apolipoprotein(a) were produced
in E. coli and the binding to tetranectin and low
density lipoprotein was examined. Only KIV(10) bound
to tetranectin and binding was similar to that of plasminogen
kringle 4 to tetranectin. Only KIV(7) bound to
LDL. In order to identify the residues responsible for
the difference in specificity between KIV(7) and
KIV(10), a number of surfaceexposed residues located
around the lysine binding clefts were exchanged.
Ligand binding analysis of these derivatives showed
that Y62, and to a minor extent W32 and E56, of KIV(7)
are important for LDL binding to KIV(7), whereas R32
and D56 of KIV(10) are required for tetranectin binding
of KIV(10).
Resistance of lipoprotein(a) to lipid peroxidation induced by oxygenated free radicals produced by γ radiolysis: a comparison with low-density lipoprotein