Abstract 530: Identification Of The Specific Residue In Apolipoprotein(a) Responsible For Mediating Covalent Attachment Of Oxidized Phospholipids
Bibliographic record
Abstract
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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".