Determinants of binding of oxidized phospholipids on apolipoprotein (a) and lipoprotein (a)

Abstract

Oxidized phospholipids (OxPLs) are present on apolipoprotein (a) [apo(a)] and lipoprotein (a) [Lp(a)] but the determinants influencing their binding are not known. The presence of OxPLs on apo(a)/Lp(a) was evaluated in plasma from healthy humans, apes, monkeys, apo(a)/Lp(a) transgenic mice, lysine binding site (LBS) mutant apo(a)/Lp(a) mice with Asp(55/57)→Ala(55/57) substitution of kringle (K)IV10)], and a variety of recombinant apo(a) [r-apo(a)] constructs. Using antibody E06, which binds the phosphocholine (PC) headgroup of OxPLs, Western and ELISA formats revealed that OxPLs were only present in apo(a) with an intact KIV10 LBS. Lipid extracts of purified human Lp(a) contained both E06- and nonE06-detectable OxPLs by tandem liquid chromatography-mass spectrometry (LC-MS/MS). Trypsin digestion of 17K r-apo(a) showed PC-containing OxPLs covalently bound to apo(a) fragments by LC-MS/MS that could be saponified by ammonium hydroxide. Interestingly, PC-containing OxPLs were also present in 17K r-apo(a) with Asp(57)→Ala(57) substitution in KIV10 that lacked E06 immunoreactivity. In conclusion, E06- and nonE06-detectable OxPLs are present in the lipid phase of Lp(a) and covalently bound to apo(a). E06 immunoreactivity, reflecting pro-inflammatory OxPLs accessible to the immune system, is strongly influenced by KIV10 LBS and is unique to human apo(a), which may explain Lp(a)'s pro-atherogenic potential.

Keywords: kringles; lipoproteins; plasminogen.

Fig. 1.

Genetic architecture of PLG and apo(a). The illustration depicts chromosome 6q26 containing the genes for PLG and apo(a) (LPA), which is transcribed into apo(a) containing KIV₁, various repeats of KIV₂, KIV₃ to KIV₈, KIV₉ that contains an additional cysteine which covalently binds to apoB-100 of Lp(a) via a disulfide bond (S), KIV₁₀ that contains the LBS, and the inactive protease domain.

Fig. 2.

Description of the species differences in apo(a) at the various apo(a) Ks (A) and the various r-apo(a) peptides (B) used in this study. The domain structure of the full-length r-apo(a) construct comprising 17 kringles (17K) is presented at the top, where squares resemble individual Ks, subscripts 1 to 10 denote the subtypes of KIV sequences, KV denotes the KV-like sequence and P is the inactive protease domain. The T-bar over KIV₉ indicates the location of the free-cysteine by which apo(a) is covalently bound to apoB-100 in Lp(a) via a disulfide bond. The dot within KIV₁₀ indicates disruption of the LBS in this domain through mutagenesis and involved amino acids are indicated to the right. Variations include the 17K r-apo(a) without KV alone (17KΔV, baboon variant), also without the protease domain (17KΔVP), point mutations within the LBS (17KΔAsp⁵⁷, chimpanzee and gorilla variants), or deletions of key amino acids of KV (17KΔLys¹², 17KΔLys⁴², 17KΔLys^12/42), as well as fragments of the 17K construct including KIV₆-P, KIV₇-P, KIV₈-P, KIV₉-P, KIV₁₀-P, KIV₆-P, KIV_1–4, KIV_5–8, KIV_5–9, and KIV_5–10, and longer (23K, 27K, 30K, 33K) and shorter versions (6K, 12K, 14K) with variations of the KIV₂ repeats. 8K-IV indicates a short r-apo(a) version with a fusion of KIV₃ to KIV₅ that is identical to human apo(a) (WT) or has a disrupted LBS (LBS⁻).

Fig. 3.

Amino acid composition of human PLG KIV and KV₁₀ and human, ape, and monkey apo(a) KIV₁₀. One-letter symbols indicate amino acid changes and empty fields identical amino acids compared with KIV of human PLG. Blue: cysteine forming the disulfide bonds in the K structure. Green: amino acids of the LBS. Numbering according to KV of PLG. Note: KIV of PLG and IV₁₀ subtypes of apo(a) are missing two amino acids compared with KV (indicated by a minus sign).

Fig. 4.

ApoB-100 (A), OxPL/apoB (B), Lp(a) (C), and OxPL/apo(a) (D) of human (n = 5), chimpanzee (n = 7), bonobo (n = 4), gorilla (n = 6), baboon (n = 3), and cynomolgus monkey (n = 4) plasma. Bars indicate mean value and standard error of the mean. RLU, relative light units.

Fig. 5.

WT versus LBS-deficient 8K-IV r-apo(a) from HEK293 cell line. A: Western blot: LPA4 detects both the WT and mutant r-apo(a), which were loaded after purification by lysine-Sepharose binding and by size exclusion chromatography. Only the WT r-apo(a) has EO6 reactivity, unlike the LBS-deficient r-apo(a) construct. B: ELISA: both versions of the r-apo(a) are detectable with LPA4, however only the WT version shows EO6 reactivity. RLU, relative light units.

Fig. 6.

Lp(a) (A), OxPL/apoB (B), apo(a) (C), and OxPL/apo(a) (D) measurements of plasma from WT Lp(a) transgenic mice and LBS-deficient Lp(a) transgenic mice. Plasma from each of these mice was subjected to the various assays described in Methods, and the results presented as relative light units (RLU)/100 ms. Plasma from 15 WT and 15 LBS-deficient mice were studied.

Fig. 7.

Assessment of the presence of OxPL on various r-apo(a) constructs via ELISA as shown in Fig. 2. I4399M = LPA SNP rs3798220 (single nucleotide polymorphism of the LPA gene that is associated with increased Lp(a) levels and an increased risk for CAD). High Lp(a) (plasma) = plasma from a healthy subject with high Lp(a) plasma levels (70 mg/dl).

Fig. 8.

Immunoblotting of various r-apo(a) constructs and human PLG with E06 for PC-OxPL and an anti-apo(a) antibody. Individual constructs are explained in detail in Fig. 2. Arrows indicate where E06 activity is not present. In these immunoblots OxPLs are denoted to reflect only E06-detectable OxPLs and not all OxPLs. RLU, relative light units.

Fig. 9.

Identification of OxPLs in r-apo(a) constructs. LC-MS/MS experiments on 17K (A), 17KΔAsp⁵⁷ (B), and their hydrolyzed versions (C, D).

Fig. 10.

Determination of PC containing OxPLs in organic phase of isolated of Lp(a) following lipid extraction using LC-MS/MS. Data represent findings from three normal donors with Lp(a) levels ∼90 mg/dl.

Fig. 11.

Summary of differences in E06 immunoreactivity of various species. A: Depicts the presence of KIII, KIV variants, KV, and the protease domain of apo(a) from various species and human PLG. B: Depicts the key amino acids of the functionally intact LBS of KIV of PLG, the conservative substitution of KIV₁₀ of human apo(a), and two individual amino acid substitutions in monkeys, apes, and rare human mutations that render the LBS defective.