Isolevuglandin adducts in disease

Abstract

Significance: A diverse family of lipid-derived levulinaldehydes, isolevuglandins (isoLGs), is produced by rearrangement of endoperoxide intermediates generated through both cyclooxygenase (COX) and free radical-induced cyclooxygenation of polyunsaturated fatty acids and their phospholipid esters. The formation and reactions of isoLGs with other biomolecules has been linked to alcoholic liver disease, Alzheimer's disease, age-related macular degeneration, atherosclerosis, cardiac arythmias, cancer, end-stage renal disease, glaucoma, inflammation of allergies and infection, mitochondrial dysfunction, multiple sclerosis, and thrombosis. This review chronicles progress in understanding the chemistry of isoLGs, detecting their production in vivo and understanding their biological consequences.

Critical issues: IsoLGs have never been isolated from biological sources, because they form adducts with primary amino groups of other biomolecules within seconds. Chemical synthesis enabled investigation of isoLG chemistry and detection of isoLG adducts present in vivo.

Recent advances: The first peptide mapping and sequencing of an isoLG-modified protein present in human retina identified the modification of a specific lysyl residue of the sterol C27-hydroxylase Cyp27A1. This residue is preferentially modified by iso[4]LGE2 in vitro, causing loss of function. Adduction of less than one equivalent of isoLG can induce COX-associated oligomerization of the amyloid peptide Aβ1-42. Adduction of isoLGE2 to phosphatidylethanolamines causes gain of function, converting them into proinflammatory isoLGE2-PE agonists that foster monocyte adhesion to endothelial cells.

Future directions: Among the remaining questions on the biochemistry of isoLGs are the dependence of biological activity on isoLG isomer structure, the structures and mechanism of isoLG-derived protein-protein and DNA-protein cross-link formation, and its biological consequences.

FIG. 1.

The cyclooxygenase (COX) and radical-induced cyclooxygenation of arachidonate leads to the formation of isoLGs. The COX pathway generates LGE₂ as a single stereoisomer whereas the radical-induced pathway also generates LGE₂ as well as seven other stereoisomers (referred to collectively as isoLGE₂). Protein adduction generates a pyrrole adduct as a single allylic hydroxyl S-epimer from LGE₂ but both the R- and S-epimers of the allylic hydroxyl are produced from the eight stereoisomers of LGE₂. Only the radical-induced pathway generates structural isomers of LGE₂, for example, iso[4]LGE₂. Thus, protein and aminophospholipid adducts of iso[n]LGs are biomarkers of radical-induced lipid oxidation.

FIG. 2.

A model study in 1977 led to our discovery that the prostaglandin endoperoxide PGH₂ rearranges nonenzymatically to levulinaldehyde derivatives with prostanoid side chains (levuglandins, LGs). Detecting the formation of LGs in vivo was complicated by their proclivity to rapidly form covalent adducts with primary amines. Finally, after two decades of research, the first evidence for the formation of LG-adducts in vivo emerged. We detected elevated levels of LGE₂-protein adduct immunoreactivity in blood from patients with atherosclerosis (AS) or end-stage renal disease (RD). That same year, we showed that LGs and a vast array of stereo and structural isomers, referred to collectively as isolevuglandins (isoLGs), are also generated in vivo through free radical-induced cyclooxygenation. Since then, our studies and those of others continue to expand the list of diseases associated with isoLGs, as well as understanding the mechanisms by which isoLGs contribute to pathology.

FIG. 3.

Cyclooxygenation of polyunsaturated fatty acyls with 3, 4, 5, or 6 C=C bonds alternating with CH₂ groups (doubly allylic methylenes) leads to the formation of a vast array of structurally diverse isoLGs.

FIG. 4.

The reactive functionality of all isoLGs is located in a “common core” structure (highlighted). This ensures that the chemistry of all isoLGs will be virtually identical.

FIG. 5.

IsoLG adducts that incorporate the amino group of phosphatidylethanolamines (PEs) complex into isoLG-hydroxylactams are generated in vivo. Analysis of the adducts of phospholipids with a mixture of acyl groups is simplified by phospholipase catalyzed conversion into 2-isoLG adducts of lyso phospholipids or ethanolamine. Treatment with methylamine or sodium hydroxide converts them into isoLG adducts of glycerophosphate ethanolamine.

FIG. 6.

Conversion of histone lysyl primary amino groups into pyrroles or lactams, neither of which is protonated at physiological pH, in conjunction with the addition of a negatively charged carboxyl group, could disrupt histone-DNA binding.

FIG. 7.

Hypothetical endogenously generated suicide substrate mechanism for irreversible inhibition of the proteasome.

FIG. 8.

Structures postulated for protein–protein crosslinks caused by isoLGs.