Isolevuglandins and mitochondrial enzymes in the retina: mass spectrometry detection of post-translational modification of sterol-metabolizing CYP27A1

Abstract

We report the first peptide mapping and sequencing of an in vivo isolevuglandin-modified protein. Mitochondrial cytochrome P450 27A1 (CYP27A1) is a ubiquitous multifunctional sterol C27-hydroxylase that eliminates cholesterol and likely 7-ketocholesterol from the retina and many other tissues. We investigated the post-translational modification of this protein with isolevuglandins, arachidonate oxidation products. Treatment of purified recombinant CYP27A1 with authentic iso[4]levuglandin E(2) (iso[4]LGE(2)) in vitro diminished enzyme activity in a time- and phospholipid-dependent manner. A multiple reaction monitoring protocol was then developed to identify the sites and extent of iso[4]LGE(2) adduction. CYP27A1 exhibited only three Lys residues, Lys(134), Lys(358), and Lys(476), that readily interact with iso[4]LGE(2) in vitro. Such selective modification enabled the generation of an internal standard, (15)N-labeled CYP27A1 modified with iso[4]LGE(2), for the subsequent analysis of a human retinal sample. Two multiple reaction monitoring transitions arising from the peptide AVLK(358)(-C(20)H(26)O(3))ETLR in the retinal sample were observed that co-eluted with the corresponding two (15)N transitions from the supplemented standard. These data demonstrate that modified CYP27A1 is present in the retina. We suggest that such protein modification impairs sterol elimination and likely has other pathological sequelae. We also propose that the post-translational modifications identified in CYP27A1 exemplify a general mechanism whereby oxidative stress and inflammation deleteriously affect protein function, contributing, for example, to cholesterol-rich lesions associated with age-related macular degeneration and cardiovascular disease. The proteomic protocols developed in this study are generally applicable to characterization of lipid-derived oxidative protein modifications occurring in vivo, including proteins bound to membranes.

FIGURE 1.

Effect of iso[4]LGE₂ treatment (2-fold molar excess) on P450 content (dashed line) and enzyme activity (solid line) reconstituted into retinal mitochondrial PLs. Values are expressed as a percentage relative to control (CYP27A1 incubated without iso[4]LGE₂). Enzyme activities are the average of three experiments ± S.E.

FIGURE 2.

Effect of iso[4]LGE₂ treatment and PL content (retinal mitochondrial PLs) on the ¹⁵N/NIA-CYP27A1 ratios as assessed by MRM. Each tryptic peptide is represented by a separate color across all time points. Peptides from group I are represented in shades of blue, and the group II peptide is shown in shades of orange. Group III peptides are depicted in shades of green, and group IV peptides are shown in shades of brown. Data labels have been rounded to one digit after the decimal, and some labels have been omitted for clarity. For all data points and statistical treatment, see supplemental Table S2.

FIGURE 3.

Effect of iso[4]LGE₂ treatment and PL content (DLPC/DOPE mixture) on the ¹⁵N/NIA-CYP27A1 ratios as assessed by MRM. Each tryptic peptide is represented by a separate color across all time points. The color code for the peptides is the same as in Fig. 2. Data labels have been rounded to one digit after the decimal, and some labels have been omitted for clarity. For all data points and statistical treatment, see supplemental Table S3.

FIGURE 4.

Effect of iso[4]LGE₂ treatment and PL content (DLPC) on the ¹⁵N/NIA-CYP27A1 ratios as assessed by MRM. Each tryptic peptide is represented by a separate color across all time points. The color code for the peptides is the same as in Fig. 2. Data labels have been rounded to one digit after the decimal, and some labels have been omitted for clarity. For all data points and statistical treatment, see supplemental Table S4.

FIGURE 5.

Effect of iso[4]LGE₂ treatment and PL content (retinal mitochondrial PLs) on the ¹⁵N/NIA-CYP27A1 ratios as assessed by MRM with chymotryptic hydrolysis. Each peptide is represented by a separate color across all time points. Nonmodified peptides are shown in shades of gray, and Lys¹³⁴-containing peptides are shown in shades of yellow. The Lys³⁵⁸ and Lys⁴⁷⁶ peptides are shown in peach and orange, respectively. Data labels have been rounded to one digit after the decimal, and some labels have been omitted for clarity. For all data points and statistical treatment, see supplemental Table S5.

FIGURE 6.

Extracted ion chromatograms from human retina supplemented with modified ¹⁵N-CYP27A1. Transitions from [NIA] and [¹⁵N] AVLK(-C₂₀H₂₆O₃)ETLR were simultaneously monitored (shown in blue and red, respectively). Transitions arising from endogenous isoLG-adducts were observed at identical retention times as those arising from the iso[4]LGE₂-modified ¹⁵N-CYP27A1 internal standard.

FIGURE 7.

Putative cascade of events initiated by increased production of oxidized PUFAs.