Formation and Stability of Pyrrole Adducts in the Reaction of Levuglandin E2 with Proteins

Abstract

Levuglandin (LG) E2 is rapidly sequestered by covalent binding with proteins. The reaction of LGE2 with a protein in neutral aqueous solution exhibits two phases. A metastable adduct rapidly accumulates initially. In the second phase, a protein-bound pyrrole is generated. Pyrrole formation and stability were monitored with an immunoassay using antibodies that were raised against a stable isostere. That LG-derived pyrroles are the major products (> 76%) of the LGE2-protein reaction is suggested by the level of antibody binding found for LG-protein adducts compared with that found for a pyrrole derived from LGE2 and 6-amino-1-hexanol. Because the initial metastable LG-protein adduct is a reactive electrophile, it can be trapped with amines, such as glycine, to give stable ternary adducts that do not cross-react with the antibodies. Although highly alkylated pyrroles are chemically sensitive compounds, the protein-bound LG-derived pyrrole appears to be stable in aqueous solution at pH 7.4. Thus, it shows no decrease in immunoreactivity over several weeks. This discovery leads to the expectation that such pyrroles will accumulate in vivo, especially in proteins that do not turn over rapidly. Thus, the LG-derived protein-bound pyrrole may be a useful marker of oxidative lipid damage, and an immunoassay for this post-translational protein modification can be exploited as a mild, sensitive method for detecting and quantifying the generation of LGs in chronic inflammatory states.

Protein Adducts of Iso[4]levuglandin E2, a Product of the Isoprostane Pathway, in Oxidized Low Density Lipoprotein

“…LGE 2 and Iso [4]LGE 2 Epitope Families-Owing to concerns that LG-derived protein-bound pyrroles would be readily modified by oxidation, our earliest efforts to detect LGE 2 -derived protein epitopes immunologically relied upon cross-reactivity of those epitopes with antibodies raised against a stable pyrazole isostere-derived antigen (53). Quite unexpectedly, the immunoreactivity generated by the reaction of LGE 2 with proteins showed no decrease over several weeks.…”

Posttranslational modification by an isolevuglandin diminishes activity of the mitochondrial cytochrome P450 27A1

“…Hence, we turned our attention to posttranslational modifi cation with isolevuglandins (isoLGs), a family of extremely reactive ␥ -ketoaldehyde derivatives, formed endogenously through enzyme or free radicalmediated oxidation of arachidonic acid ( 24,25 ). IsoLGs have been shown to posttranslationally modify free primary amino groups present in biomolecules with an avidity greatly exceeding other reactive oxidized lipids ( 26,27 ). Targets of isoLG modifi cation include proteins and phospholipids (PLs) in the serum, the vasculature, and the eye, forming stable covalent adducts with isoLGs (26)(27)(28)(29).…”

“…IsoLGs have been shown to posttranslationally modify free primary amino groups present in biomolecules with an avidity greatly exceeding other reactive oxidized lipids ( 26,27 ). Targets of isoLG modifi cation include proteins and phospholipids (PLs) in the serum, the vasculature, and the eye, forming stable covalent adducts with isoLGs (26)(27)(28)(29). IsoLG adduct levels have been shown to be a biomarker of oxidative stress, increased in patients with atherosclerosis, renal disease, and AMD ( 28,30,31 ).…”

Therapeutic Targets in Prostaglandin E2 Signaling for Neurologic Disease

“…LGs are highly chemically reactive compounds that form irreversible adducts with protein lysyl residues leading to protein-protein crosslinks [4]. An example of this potentially harmful activity is the ability of…”