Abstract 530: Identification Of The Specific Residue In Apolipoprotein(a) Responsible For Mediating Covalent Attachment Of Oxidized Phospholipids

Oxidized phospholipids (oxPL) are highly inflammatory molecules that are linked to atherogenesis. Previously, Lp(a) was identified as the major carrier of oxPL in human plasma, with the majority of the oxPL being covalently associated to its apolipoprotein(a) (apo(a)) component. Earlier work by our group identified the Kringle (K) IV 10 domain of apo(a) as the site of oxPL binding; additionally, mutation of the strong lysine binding site (sLBS) in this domain eliminated oxPL addition. However, the specific residue(s) in apo(a) that bind oxPL remain elusive. In this study, we used site-directed mutagenesis of KIV 10 to identify the key residue of apo(a) involved in covalent attachment of oxPL and induction of inflammatory responses in vitro . Specifically, two residues in KIV 10 capable of binding oxPL by a Michael addition reaction (His 31 and 33) were identified as the probable sites of attachment. We mutagenized the residues to aspartic acid (Asp) and asparagine (Asn), respectively, based on the presence of these residues at these positions in plasminogen kringles. Six-kringle (6K) apo(a) variants harbouring these mutations were expressed in human embryonic kidney 293 cells and purified using lysine-Sepharose chromatography. Covalent oxPL modification of each apo(a) variant was assessed by E06 immunoblotting. Wild-type (WT) 6K and 6KHis31Asp were detected by E06 whereas 6KHis33Asn and 6KAsp56Ala (the LBS mutant) were not. Next, THP-1 macrophages were treated for 4 hours with 300 nmol/L of WT 6K apo(a), 6KHis33Asn, or 6KAsp56Ala. Expression of mRNA encoding the pro-inflammatory cytokine IL-8 by THP-1 cells was measured with quantitative real-time PCR. Relative to control, WT 6K apo(a) treatment of THP-1 cells significantly increased expression of IL8 (2.32-fold; p<0.001). Treatment of THP-1 cells with 6KHis33Asn or 6KAsp56Ala apo(a) did not significantly affect IL8 expression. We have thus identified the site of covalent attachment of oxPL to apo(a) as His33 in KIV 10 and we demonstrate that apo(a)-induced inflammatory responses in macrophages are abolished when oxPL binding is prevented through mutation of this residue. These findings provide avenues for development of therapeutics to potentially mitigate Lp(a)-mediated atherogenesis.