Lysine-Phosphatidylcholine Adducts in Kringle V Impart Unique Immunological and Potential Pro-inflammatory Properties to Human Apolipoprotein(a)

Abstract

Lipoprotein(a), Lp(a), an athero-thrombotic risk factor, reacts with EO6, a natural monoclonal autoantibody that recognizes the phophorylcholine (PC) group of oxidized phosphatidylcholine (oxPtdPC) either as a lipid or linked by a Schiff base to lysine residues of peptides/proteins. Here we show that EO6 reacts with free apolipoprotein(a) apo(a), its C-terminal domain, F2 (but not the N-terminal F1), kringle V-containing fragments obtained by the enzymatic digestion of apo(a) and also kringle V-containing apo(a) recombinants. The evidence that kringle V is critical for EO6 reactivity is supported by the finding that apo(a) of rhesus monkeys lacking kringle V did not react with EO6. Based on the previously established EO6 specificity requirements, we hypothesized that all or some of the six lysines in human kringle V are involved in Schiff base linkage with oxPtdPC. To test this hypothesis, we made use of a recombinant lysine-containing apo(a) fragment, rIII, containing kringle V but not the protease domain. EO6 reacted with rIII before and after reduction to stabilize the Schiff base and also after extensive ethanol/ ether extraction that yielded no lipids. On the other hand, delipidation of the saponified product yielded an average of two mol of phospholipids/mol of protein consistent with direct analysis of inorganic phosphorous on the nonsaponified rIII. Moreover, only two of the six theoretical free lysine amino groups per mol of rIII were unavailable to chemical modification by 2,4,6-trinitrobenzene sulfonic acid. Finally, rIII, like human apo(a), stimulated the production of interleukin 8 in THP-1 macrophages in culture. Together, our studies provide evidence that in human apo(a), kringle V is the site that reacts with EO6 via lysine-oxPtdPC adducts that may also be involved in the previously reported pro-inflammatory effect of apo(a) in cultured human macrophages.

Lipoprotein(a) Oxidation, Autoimmune and Atherosclerosis

“…The above results are also supported by the studies about ox-PL, which have demonstrated convincingly that a key ox-PL is preferentially associated with Lp(a) [52] and correlates with both the presence and extent of angiographically documented CAD [40,53], and their concentrations increase after ACS [51] and immediately after PCI [54].…”

Percutaneous Coronary Intervention Results in Acute Increases in Oxidized Phospholipids and Lipoprotein(a)

“…26 We have recently shown that kringle V of apo(a) covalently binds Ϸ2 moles of OxPL detected by antibody E06 and that this portion of apo(a) induced IL-8 production by macrophages. 26 We recently demonstrated that both OxLDL-E06 and Lp(a) rise concurrently after acute coronary syndromes, but unlike the present study, in which levels increased only after PCI in otherwise uncomplicated procedures, levels remained elevated up to 3 to 6 months.…”

“…26 We have recently shown that kringle V of apo(a) covalently binds Ϸ2 moles of OxPL detected by antibody E06 and that this portion of apo(a) induced IL-8 production by macrophages. 26 We recently demonstrated that both OxLDL-E06 and Lp(a) rise concurrently after acute coronary syndromes, but unlike the present study, in which levels increased only after PCI in otherwise uncomplicated procedures, levels remained elevated up to 3 to 6 months. 11 In the present study, we documented for the first time a transient disassociation of OxLDL-E06 and Lp(a) in the post-PCI samples, suggesting that these two compounds are independently generated to some extent but later reassociate.…”

Lipoprotein(a)

“…In a series of studies, Witztum et al have demonstrated convincingly that a key oxidized phospholipid is preferentially associated with Lp(a). [117][118][119] Proinflammatory, oxidized phospholipids are covalently bound to kringle V in apo(a), a portion of apo(a) associated with macrophage IL-8 production. 117 These results suggest that Lp(a) may act as a preferential acceptor that tightly binds oxidized phospholipids transferred from tissues or from other lipoproteins.…”

“…[117][118][119] Proinflammatory, oxidized phospholipids are covalently bound to kringle V in apo(a), a portion of apo(a) associated with macrophage IL-8 production. 117 These results suggest that Lp(a) may act as a preferential acceptor that tightly binds oxidized phospholipids transferred from tissues or from other lipoproteins. 119 This could imply that Lp(a) functions as a scavenger absorbing potentially deleterious oxidized lipids, preventing an increased uptake in the vessel wall of other lipoproteins, primarily LDL, containing this factor.…”