Immunological detection of N-formylkynurenine in oxidized proteins

Laboratory of Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Free Radical Biology and Medicine (Impact Factor: 5.74). 06/2009; 46(9):1260-6. DOI: 10.1016/j.freeradbiomed.2009.01.020
Source: PubMed


Reactions of tryptophan residues in proteins with radical and other oxidative species frequently lead to cleavage of the indole ring, modifying tryptophan residues into N-formylkynurenine (NFK) and kynurenine. Tryptophan modification has been detected in physiologically important proteins and has been associated with a number of human disease conditions. Modified residues have been identified through various combinations of proteomic analyses, tryptic digestion, HPLC, and mass spectrometry. Here we present a novel, immunological approach using polyclonal antiserum for detection of NFK. The specificity of our antiserum is confirmed using photooxidation and radical-mediated oxidation of proteins with and without tryptophan residues. The sensitivity of our antiserum is validated through detection of NFK in photooxidized myoglobin (two tryptophan residues) and in carbonate radical-oxidized human SOD1, which contains a single tryptophan residue. Analysis of photooxidized milk also shows that our antiserum can detect NFK residues in a mixture of proteins. Results from mass spectrometric analysis of photooxidized myoglobin samples corroborate the immunological data, detecting an increase in NFK content as the extent of photooxidation increases.

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    • "The previously described anti-NFK antiserum [26] [27] [28] was developed in our laboratory, and mouse monoclonal anti-αA and anti-αB crystallin and goat polyclonal anti-actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The (goat) anti-mouse IRdye 800 used in Western analysis was from Li-Cor Biosciences (Lincoln, NE, USA) and the DAPI and all secondary antibodies used in the confocal experiments were purchased from Life Technologies ( "
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    ABSTRACT: St. John's wort (Hypericum perforatum), a perennial herb native to Europe, is widely used and appears to be effective in treatment of mild to moderate depression. Hypericin, a singlet oxygen-generating photosensitizer that absorbs in both the visible and UVA range, is considered to be one of the bioactive ingredients, and commercial preparations are frequently calibrated to contain a standard concentration. Hypericin can accumulate in ocular tissues, including lenses, and can bind in vitro to α-crystallin, a major lens protein. Alpha-crystallin is required for lens transparency and also acts as a chaperone to ensure its own integrity and the integrity of all lens proteins. Because there is no crystallin turnover, damage to α-crystallin is cumulative over the lifetime of the lens, and can lead to cataracts, the principal cause of blindness worldwide. In this work we study hypericin photosensitization of α-crystallin and detect extensive polymerization of bovine α-crystallin exposed in vitro to hypericin and UVA. We use fluorescent confocal microscopy to visualize binding between hypericin and α-crystallin in a human lens epithelial (HLE) cell line. Further, we show that UVA irradiation of hypericin-treated HLE cells results in a dramatic decrease in α-crystallin detection concurrent with a dramatic accumulation of the tryptophan oxidation product N-formylkynurenine (NFK). Examination of actin in HLE cells indicates that this cytoskeleton protein accumulates NFK resulting from hypericin-mediated photosensitization. This work also shows that filtration of wavelengths <400nm provides incomplete protection against α-crystallin modifications and NFK accumulation, suggesting that even by wearing UV blocking sunglasses, routine users of St. John's wort cannot adequately shield their lenses from hypericin-mediated photosensitized damage.
    Free Radical Biology and Medicine 02/2013; 60. DOI:10.1016/j.freeradbiomed.2013.02.023 · 5.74 Impact Factor
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    • "Post-translational oxidation of Trp to form N-formylkynurenine (NFK) (Figure 1A) plays a role in oxidative stress responses in some proteins (see for example [13]). NFK has been identified in mitochondrial ATP synthase [14], spinach LHCII [15], milk proteins [16], skeletal muscle proteins [17], apolipoprotein B-100 [13], and Methylococcus capsulatus-secreted MopE protein [18]. "
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    ABSTRACT: Light stress in plants results in damage to the water oxidizing reaction center, photosystem II (PSII). Redox signaling, through oxidative modification of amino acid side chains, has been proposed to participate in this process, but the oxidative signals have not yet been identified. Previously, we described an oxidative modification, N-formylkynurenine (NFK), of W365 in the CP43 subunit. The yield of this modification increases under light stress conditions, in parallel with the decrease in oxygen evolving activity. In this work, we show that this modification, NFK365-CP43, is present in thylakoid membranes and may be formed by reactive oxygen species produced at the Mn(4)CaO(5) cluster in the oxygen-evolving complex. NFK accumulation correlates with the extent of photoinhibition in PSII and thylakoid membranes. A modest increase in ionic strength inhibits NFK365-CP43 formation, and leads to accumulation of a new, light-induced NFK modification (NFK317) in the D1 polypeptide. Western analysis shows that D1 degradation and oligomerization occur under both sets of conditions. The NFK modifications in CP43 and D1 are found 17 and 14 Angstrom from the Mn(4)CaO(5) cluster, respectively. Based on these results, we propose that NFK is an oxidative modification that signals for damage and repair in PSII. The data suggest a two pathway model for light stress responses. These pathways involve differential, specific, oxidative modification of the CP43 or D1 polypeptides.
    PLoS ONE 07/2012; 7(7):e42220. DOI:10.1371/journal.pone.0042220 · 3.23 Impact Factor
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    • "The highest complexity of oxidation products was observed for W432 in both HisRS* and recHisRS* (Fig. S1). 1 The oxidation of tryptophan is known to consist of 3 sequential steps resulting in the formation of oxidized tryptophan, N′-formylkunurenine, and kynurenine, respectively (Fig. S2) 1 (Taylor et al. 2003). The presence of (formyl)kynurenine has been described in mitochondrial proteins, but has so far never been linked to an autoantigen or to autoimmunity (Ehrenshaft et al. 2009). The oxidative HisRS modifications might generate epitopes recognized by the immune system. "
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    ABSTRACT: The aminoacyl-tRNA synthetases are ubiquitously expressed enzymes that catalyze the esterification of amino acids to their cognate tRNAs. Autoantibodies against several aminoacyl-tRNA synthetases are found in autoimmune polymyositis and dermatomyositis patients. Because necrosis is often found in skeletal muscle biopsies of these patients, we hypothesized that cell-death-induced protein modifications may help in breaking immunological tolerance. Since cell death is associated with oxidative stress, the effect of oxidative stress on the main myositis-specific autoantibody target Jo-1 (histidyl-tRNA synthetase; HisRS) was studied in detail. The exposure of Jurkat cells to hydrogen peroxide resulted in the detection of several oxidized methionines and one oxidized tryptophan residue in the HisRS protein, as demonstrated by mass spectrometry. Unexpectedly, the tRNA aminoacylation activity of HisRS appeared to be increased upon oxidative modification. The analysis of myositis patient sera did not lead to the detection of autoantibodies that are specifically reactive with the modified HisRS protein. The results of this study demonstrate that the Jo-1/HisRS autoantigen is modified under oxidative stress conditions. The consequences of these modifications for the function of HisRS and its autoantigenicity are discussed.
    Biochemistry and Cell Biology 11/2011; 89(6):545-53. DOI:10.1139/o11-055 · 2.15 Impact Factor
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