Molecular Structures of Isolevuglandin-Protein Cross-Links

Abstract

Isolevuglandins (isoLGs) are stereo and structurally isomeric γ-ketoaldehydes produced through free radical-induced oxidation of arachidonates. Some isoLG isomers are also generated through enzymatic cyclooxygenation. Post-translational modification of proteins by isoLG is associated with loss-of-function, cross-linking and aggregation. We now report that a low level of modification by one or two molecules of isoLG has a profound effect on the activity of a multi subunit protease, calpain-1. Modification of one or two key lysyl residues apparently suffices to abolish catalytic activity. Covalent modification of calpain-1 led to intersubunit cross-linking. Hetero- and homo-oligomers of the catalytic and regulatory subunits of calpain-1 were detected by SDS PAGE with Western blotting. N-Acetyl-glycyl-lysine methyl ester and β-amyloid(11-17) peptide EVHHQKL were used as models for characterizing the cross-linking of protein lysyl residues resulting from adduction of iso[4]LGE2. Aminal, bispyrrole and trispyrrole cross-links of these two peptides were identified and fully characterized by mass spectrometry. Aminal and bispyrrole dimers were both detected. Furthermore, a complex mixture of derivatives of the bispyrrole cross-link containing one or more additional atoms of oxygen was found. Interesting differences are evident in the predominant crosslink type generated in the reaction of iso[4]LGE2 with these peptides. More aminal cross-link versus bispyrrole is formed during reaction of the dipeptide with iso[4]LGE2. In contrast, more bispyrrole versus aminal cross-link is formed during the reaction of EVHHQKL with iso[4]LGE2. It is tempting to speculate that the EVHHQKL peptide-pyrrole modification forms noncovalent aggregates that favor the production of covalent bispyrrole cross-links because β-amyloid(11-17) tends to spontaneously oligomerize.

Chemistry and analysis of HNE and other prominent carbonyl-containing lipid oxidation compounds

“…There is mounting evidence that isolevuglandins form covalent adducts with proteins in various biological milieux, such as in normal and fibrotic lung tissue [69], human retina [70], and dendritic cells [71]. The mechanisms of protein crossing-linking by isolevuglandins have been investigated, and found to occur both by aminal and pyrrole-pyrrole cross-links [72]. Treatment of hepatic stellate cells with isolevuglandins caused their activation, and was thought to depend on formation of protein adducts, although this was not explicitly demonstrated [73].…”

Covalent modification of biological targets with natural products through Paal–Knorr pyrrole formation

“…39 Indeed, they have orders of magnitude higher reactivity in forming covalent adducts than other lipid peroxidation products, such as malondialdehyde. 40 …”

“…43 In the human eye, iso-LG-modified calpain-1 accumulates in the trabecular meshwork and loses its enzymatic activity. 40 Also, protein crosslinking by iso-LGs may interfere with aqueous outflow through the trabecular meshwork within the eye resulting in increased intraocular pressure in primary open angle glaucoma. 40 There is also evidence that a subfamily of iso-LGs produced through the cyclooxygenase pathway, such as LGE 2 and LGD 2 , may promote aggregation of the β-amyloid peptide and contribute to Alzheimer’s disease.…”

“…40 There is also evidence that a subfamily of iso-LGs produced through the cyclooxygenase pathway, such as LGE 2 and LGD 2 , may promote aggregation of the β-amyloid peptide and contribute to Alzheimer’s disease. 40 …”

“…40 The crude reaction product mixture produced ions at m / z 576, 592, 608, 853 and 1149, which were attributed to protonated pyrrole, lactam, hydroxylactam, aminal crosslink and bispyrrole, respectively, as well as adducts resulting from the loss of water from these products. The separation of this mixture using reverse phase HPLC with LC-ESI monitoring gave fractions corresponding to lactam, aminal crosslink and bispyrrole eluting in the order of decreasing polarity.…”